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NORMAL GENOMIC

• Feng J, Roberts MF, Drin G, Scarlata S
• Dissection of the steps of phospholipase C beta 2 activity that are enhanced by G beta gamma subunits.
• Biochemistry. 2005; 44: 2577-84
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• Phosphatidylinositol-specific phospholipase C (PLC) enzymes catalyze the hydrolysis of phosphatidylinositol 4,5 bisphosphate in a two step reaction that involves a cyclic intermediate. The PLCbetafamily are activated by both the alpha and betagamma subunits of heterotrimeric G proteins. To determine which catalytic step is affected by Gbetagamma subunits, we compared the change in PLCbeta(2) activity catalysis toward monomeric short-chain phosphatidylinositol (PI) substrates and monomeric water-soluble cyclic inositol phosphates as well as long-chain PI in bilayer and micellar interfaces in the absence and presence of Gbetagammasubunits. Unlike other PLC enzymes, no cyclic products were detected for either wild-type PLCbeta(2) or a chimeric protein composed of the PH domain of PLCbeta(2) and the catalytic domain of PLCdelta(1). Using cIP as a substrate to examine the second step of the reaction, we found that the presence of Gbetagamma subunits stimulated this step by a higher level than that for the overall reaction (k(cat) 1.5-fold (cIP) as opposed to 1.20-fold for soluble diC(4)PI). Detergents above their CMC can generate the same kinetic activation of PLCbeta(2) as Gbetagamma, suggesting that hydrophobic compounds stabilize the activated state of the enzyme. The most pronounced effect of Gbetagamma is that it relieves competitive product inhibition. Taken together, our results show that activation of PLCbeta(2) occurs through enhancement in the catalytic rate of hydrolysis of the cyclic intermediate and increased product release, and that hydrophobic interactions play a key role.

• Apiyo D, Zhao L, Tsai MD, Selby TL
• X-ray structure of the R69D phosphatidylinositol-specific phospholipase C enzyme: insight into the role of calcium and surrounding amino acids in active site geometry and catalysis.
• Biochemistry. 2005; 44: 9980-9
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• Phosphatidylinositol-specific phospholipase Cs (PLCs) are a family of phosphodiesterases that catalyze the cleavage of the P-O bond via transesterification using the internal hydroxyl group of the substrate as a nucleophile, generating the five-membered cyclic inositol phosphate as an intermediate or product. To better understand the role of calcium in the catalytic mechanism of PLCs, we have determined the X-ray crystal structure of an engineered PLC enzyme from Bacillus thuringiensis to 2.1 A resolution. The active site of this enzyme has been altered by substituting the catalytic arginine with an aspartate at position 69 (R69D). This single-amino acid substitution converted a metal-independent, low-molecular weight enzyme into a metal ion-dependent bacterial PLC with an active site architecture similar to that of the larger metal ion-dependent mammalian PLC. The Ca(2+) ion shows a distorted square planar geometry in the active site that allows for efficient substrate binding and transition state stabilization during catalysis. Additional changes in the positions of the catalytic general acid/general base (GA/GB) were also observed, indicating the interrelation of the intricate hydrogen bonding network involved in stabilizing the active site amino acids. The functional information provided by this X-ray structure now allows for a better understanding of the catalytic mechanism, including stereochemical effects and substrate interactions, which facilitates better inhibitor design and sheds light on the possibilities of understanding how protein evolution might have occurred across this enzyme family.

• Stieglitz KA, Yang H, Roberts MF, Stec B
• Reaching for mechanistic consensus across life kingdoms: structure and insights into catalysis of the myo-inositol-1-phosphate synthase (mIPS) from Archaeoglobus fulgidus.
• Biochemistry. 2005; 44: 213-24
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• myo-Inositol-1-phosphate synthase (mIPS) catalyzes the first step in the synthesis of l-myo-inositol-1-phosphate. We have solved and refined the structure of the mIPS from the hyperthermophilic sulfate reducer Archaeoglobus fulgidus at 1.9 A resolution. The enzyme crystallized from poly(ethylene glycol) in the P1 space group with one tetramer in the asymmetric unit and provided a view of the entire biologically active oligomer. Despite significant changes in sequence length and amino acid composition, the general architecture of the archaeal enzyme is similar to that of the eukaryotic mIPS from Saccharomyces cerevisiae and bacterial mIPS from Mycobacterium tuberculosis. The enhanced thermostability of the archaeal enzyme as compared to that from yeast is consistent with deletion of a number of surface loops that results in a significantly smaller protein. In the structure of the A. fulgidus mIPS, the active sites of all four subunits were fully ordered and contained NAD(+) and inorganic phosphate. The structure also contained a single metal ion (identified as K(+)) in two of the four subunits. The analysis of the electrostatic potential maps of the protein suggested the presence of a second metal-ion-binding site in close proximity to the first metal ion and NAD(+). The modeling of the substrate and known inhibitors suggests a critical role for the second metal ion in catalysis and provides insights into the common elements of the catalytic cycle in enzymes from different life kingdoms.

• Sakamoto Y, Tanaka N, Ichimiya T, Kurihara T, Nakamura KT
• Crystal structure of the catalytic fragment of human brain 2',3'-cyclic-nucleotide 3'-phosphodiesterase.
• J Mol Biol. 2005; 346: 789-800
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• 2',3'-Cyclic-nucleotide 3'-phosphodiesterase (CNP), a member of the 2H phosphoesterase superfamily, is firmly bound to brain white matter and found mainly in the central nervous system of vertebrates, and it catalyzes the hydrolysis of 2',3'-cyclic nucleotide to produce 2'-nucleotide. Recent studies on CNP-knockout mice have revealed that the absence of CNP causes axonal swelling and neuronal degeneration. Here, the crystal structure of the catalytic fragment (CF) of human CNP (hCNP-CF) is solved at 1.8A resolution. It is an alpha+beta type structure consisting of three alpha-helices and nine beta-strands. The structural core of the molecule is comprised of two topologically equivalent three-stranded antiparallel beta-sheets that are related by a pseudo 2-fold symmetry. Each beta-sheet contains an H-X-T-X motif, which is strictly conserved among members of the 2H phosphoesterase superfamily. The phosphate ion is bound to the side-chains of His and Thr from each of the two motifs. Structural comparison of hCNP-CF with plant 1'',2''-cyclic nucleotide phosphodiesterase (CPDase) and bacterial 2'-5' RNA ligase reveals that the H-X-T-X motifs are structurally conserved among these enzymes, but the surface properties of the active site are quite different among the enzymes, reflecting the differences in their substrates. On the basis of the present crystal structure of the hCNP-CF/phosphate complex, the available structure of the CPDase/cyclic-nucleotide analogue complex, and the recent functional studies of rat CNP-CF, we propose a possible substrate-binding mode and catalytic mechanism of CNP, which employs the nucleophilic water molecule activated by His310. The proposed mechanism is basically equivalent to the second step of the well-accepted reaction mechanism of RNase A. Since the overall structure of hCNP-CF differs considerably from that of RNase A, it is likely that the similar active sites with two catalytic histidine residues in these enzymes arose through convergent evolution.

• Flesch FM, Yu JW, Lemmon MA, Burger KN
• Membrane activity of the phospholipase C-delta1 pleckstrin homology (PH) domain.
• Biochem J. 2005; 389: 435-41
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• PH-PLCdelta1 [the PH domain (pleckstrin homology domain) of PLCdelta1 (phospholipase C-delta1)] is among the best-characterized phosphoinositide-binding domains. PH-PLCdelta1 binds with high specificity to the headgroup of PtdIns(4,5)P2, but little is known about its interfacial properties. In the present study, we show that PH-PLCdelta1 is also membrane-active and can insert significantly into PtdIns(4,5)P2-containing monolayers at physiological (bilayer-equivalent) surface pressures. However, this membrane activity appears to involve interactions distinct from those that target PH-PLCdelta1 to the PtdIns(4,5)P2 headgroup. Whereas the majority of PtdIns(4,5)P2-bound PH-PLCdelta1 can be displaced by adding excess of soluble headgroup [Ins(1,4,5)P3], membrane activity of PH-PLCdelta1 cannot. PH-PLCdelta1 differs from other phosphoinositide-binding domains in that its membrane insertion does not require that the phosphoinositide-binding site be occupied. Significant monolayer insertion remains when the phosphoinositide-binding site is mutated, and PH-PLCdelta1 can insert into monolayers that contain no PtdIns(4,5)P2 at all. Our results suggest a model in which reversible membrane binding of PH-PLCdelta1, mediated by PtdIns(4,5)P2 or other acidic phospholipids, occurs without membrane insertion. Accumulation of the PH domain at the membrane surface enhances the efficiency of insertion, but does not significantly affect its extent, whereas the presence of phosphatidylethanolamine and cholesterol in the lipid mixture promotes the extent of insertion. This is the first report of membrane activity in an isolated PH domain and has implications for understanding the membrane targeting by this common type of domain.

• Nakahara M et al.
• A novel phospholipase C, PLC(eta)2, is a neuron-specific isozyme.
• J Biol Chem. 2005; 280: 29128-34
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• Twelve phospholipase C (PLC) isozymes have been cloned so far, and they are divided into six classes, beta-, gamma-, delta-, epsilon-, zeta-, and eta-type, on the basis of structure and activation mechanisms. Here we report the identification of a novel PLC isozyme, PLC(eta)2. PLC(eta)2 is composed of conserved domains including pleckstrin homology, EF-hand, X and Y catalytic, and C2 domains and the isozyme-specific C-terminal region. PLC(eta)2 consists of 1164 amino acids with a molecular mass of 125 kDa. The PLC activity of PLC(eta)2 was more sensitive to calcium concentration than the PLC activity of the PLCdelta-type enzyme, which is thought to be the most calcium-sensitive PLC. Immunofluorescence analysis showed that PLC(eta)2 was localized predominantly to the plasma membrane at resting state via the pleckstrin homology domain. This observation was supported by Western blot analysis of cytosol and membrane fractions. In addition, expression of PLC(eta)2 was detected after birth and showed a restricted distribution in the brain; it was particularly abundant in the hippocampus, cerebral cortex, and olfactory bulb. The pattern was similar to that of the neuronal marker microtubule-associated protein 2 by Western blot. Furthermore, in situ hybridization showed positive signals for PLC(eta)2 in pyramidal cells of the hippocampus. Finally, we found that PLC(eta)2 was expressed abundantly in neuron-containing primary culture but not in astrocyte-enriched culture. These results indicate that PLC(eta)2 is a neuron-specific isozyme that may be important for the formation and/or maintenance of the neuronal network in the postnatal brain.

• Kobayashi M et al.
• Spectroscopic characterization of the EF-hand domain of phospholipase C delta1: identification of a lipid interacting domain.
• Arch Biochem Biophys. 2005; 440: 191-203
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• The interaction of the isolated EF-hand domain of phospholipase C delta1 with arachidonic acid (AA) was characterized using circular dichroism (CD) and fluorescence spectroscopy. The far-UV CD spectral changes indicate that AA binds to the EF domain. The near-UV CD spectra suggest that the orientations of aromatic residues in the peptide are affected when AA binds to the protein. The fluorescence of the single intrinsic tryptophan located in EF1 was enhanced by the addition of dodecylmaltoside (DDM) and AA suggesting that this region of the protein is involved in hydrophobic interactions. In the presence of a low concentration of DDM it was found that AA induced a change in fluorescence resonance energy transfer, which is indicative of a conformational change. The lipid induced conformational change may play a role in calcium binding because the isolated EF-hand domain did not bind Ca2+ in the absence of lipids, but Ca2+-dependent changes in the intrinsic tryptophan emission were observed when free fatty acids were present. These studies identify specific EF-hand domains as allosteric regulatory domains that require hydrophobic ligands such as lipids.

• Stewart AJ, Mukherjee J, Roberts SJ, Lester D, Farquharson C
• Identification of a novel class of mammalian phosphoinositol-specific phospholipase C enzymes.
• Int J Mol Med. 2005; 15: 117-21
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• Phosphoinositol (PhoIns)-specific phospholipase C enzymes (PLCs) are central to the inositol lipid signaling pathways and contribute to intracellular Ca2+ release and protein kinase C activation. Five distinct classes of PhoIns-specific PLCs are known to exist in mammals, which are activated by membrane receptor-mediated events. Here we have identified a sixth class of PhoIns-specific PLC with a novel domain structure, which we have termed PLC-eta. Two putative PLC-eta enzymes were identified in humans and in mice. Sequence analysis revealed that residues implicated in substrate binding and catalysis from other PhoIns-specific PLCs are conserved in the novel enzymes. PLC-eta enzymes are most closely related to the PLC-delta class and share a close evolutionary relationship with other PLC isozymes. EST analysis and RT-PCR data suggest that PLC-eta enzymes are expressed in several cell types and, by analogy with other mammalian PhoIns-specific PLCs, are likely to be involved in signal transduction pathways.

• Nomikos M et al.
• Role of phospholipase C-zeta domains in Ca2+-dependent phosphatidylinositol 4,5-bisphosphate hydrolysis and cytoplasmic Ca2+ oscillations.
• J Biol Chem. 2005; 280: 31011-8
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• The sperm-specific phospholipase C-zeta (PLCzeta) elicits fertilization-like Ca2+ oscillations and activation of embryo development when microinjected into mammalian eggs (Saunders, C. M., Larman, M. G., Parrington, J., Cox, L. J., Royse, J., Blayney, L. M., Swann, K., and Lai, F. A. (2002) Development (Camb.) 129, 3533-3544; Cox, L. J., Larman, M. G., Saunders, C. M., Hashimoto, K., Swann, K., and Lai, F. A. (2002) Reproduction 124, 611-623). PLCzeta may represent the physiological stimulus for egg activation and development at mammalian fertilization. PLCzeta is the smallest known mammalian PLC isozyme, comprising two EF hand domains, a C2 domain, and the catalytic X and Y core domains. To gain insight into PLCzeta structure-function, we assessed the ability of PLCzeta and a series of domain-deletion constructs to cause phosphatidylinositol 4,5-bisphosphate hydrolysis in vitro and also to generate cytoplasmic Ca2+ changes in intact mouse eggs. PLCzeta and the closely related PLCdelta1 had similar K(m) values for phosphatidylinositol 4,5-bisphosphate, but PLCzeta was around 100 times more sensitive to Ca2+ than was PLCdelta1. Notably, specific phosphatidylinositol 4,5-bisphosphate hydrolysis activity was retained in PLCzeta constructs that had either EF hand domains or the C2 domain removed, or both. In contrast, Ca2+ sensitivity was greatly reduced when either one, or both, of the EF hand domains were absent, and the Hill coefficient was reduced upon deletion of the C2 domain. Microinjection into intact mouse eggs revealed that all domain-deletion constructs were ineffective at initiating Ca2+ oscillations. These data suggest that the exquisite Ca2+-dependent features of PLCzeta regulation are essential for it to generate inositol 1,4,5-trisphosphate and Ca2+ oscillations in intact mouse eggs.

• Lee SB, Varnai P, Balla A, Jalink K, Rhee SG, Balla T
• The pleckstrin homology domain of phosphoinositide-specific phospholipase Cdelta4 is not a critical determinant of the membrane localization of the enzyme.
• J Biol Chem. 2004; 279: 24362-71
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• The inositol lipid and phosphate binding properties and the cellular localization of phospholipase Cdelta(4) (PLCdelta(4)) and its isolated pleckstrin homology (PH) domain were analyzed in comparison with the similar features of the PLCdelta(1) protein. The isolated PH domains of both proteins showed plasma membrane localization when expressed in the form of a green fluorescent protein fusion construct in various cells, although a significantly lower proportion of the PLCdelta(4) PH domain was membrane-bound than in the case of PLCdelta(1)PH-GFP. Both PH domains selectively recognized phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)), but a lower binding of PLCdelta(4)PH to lipid vesicles containing PI(4,5)P(2) was observed. Also, higher concentrations of inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) were required to displace the PLCdelta(4)PH from the lipid vesicles, and a lower Ins(1,4,5)P(3) affinity of PLCdelta(4)PH was found in direct Ins(1,4,5)P(3) binding assays. In sharp contrast to the localization of its PH domain, the full-length PLCdelta(4) protein localized primarily to intracellular membranes mostly to the endoplasmic reticulum (ER). This ER localization was in striking contrast to the well documented PH domain-dependent plasma membrane localization of PLCdelta(1). A truncated PLCdelta(4) protein lacking the entire PH domain still showed the same ER localization as the full-length protein, indicating that the PH domain is not a critical determinant of the localization of this protein. Most important, the full-length PLCdelta(4) enzyme still showed binding to PI(4,5)P(2)-containing micelles, but Ins(1,4,5)P(3) was significantly less potent in displacing the enzyme from the lipid than with the PLCdelta(1) protein. These data suggest that although structurally related, PLCdelta(1) and PLCdelta(4) are probably differentially regulated in distinct cellular compartments by PI(4,5)P(2) and that the PH domain of PLCdelta(4) does not act as a localization signal.

• Kim SK et al.
• Point mutations in the split PLC-gamma1 PH domain modulate phosphoinositide binding.
• J Biochem Mol Biol. 2004; 37: 720-5
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• A number of signaling molecules contain small pleckstrin homology (PH) domains capable of binding phosphoinositides or proteins. Phospholipase C (PLC)-gamma1 has two putative PH domains, an NH(2)-terminal (PH(1)) and a split PH domain (nPH(2) and cPH(2)). We previously reported that the split PH domain of PLC-gamma1 binds to phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) (Chang et al., 2002). To identify the amino acid residues responsible for binding with PI(4)P and PI(4,5)P(2), we used site-directed mutagenesis to replace each amino acid in the variable loop-1 (VL-1) region of the PLC-gamma1 nPH(2) domain with alanine (a neutral amino acid). The phosphoinositide-binding affinity of these mutant molecules was analyzed by Dot-blot assay followed by ECL detection. We found that two PLC-gamma1 nPH2 domain mutants, P500A and H503A, showed reduced affinities for phosphoinositide binding. Furthermore, these mutant PLC-gamma1 molecules showed reduced PI(4,5)P(2) hydrolysis. Using green fluorescent protein (GFP) fusion protein system, we showed that both PH(1) and nPH(2) domains are responsible for membrane-targeted translocation of PLC-gamma1 upon serum stimulation. Together, our data reveal that the amino acid residues Pro(500) and His(503) are critical for binding of PLC-gamma1 to one of its substrates, PI(4,5)P(2) in the membrane.

• Venkataraman G, Goswami M, Tuteja N, Reddy MK, Sopory SK
• Isolation and characterization of a phospholipase C delta isoform from pea that is regulated by light in a tissue specific manner.
• Mol Genet Genomics. 2003; 270: 378-86
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• Phosphoinositide-specific phospholipases C (PLCs) play an important role in many cellular responses and are involved in the production of secondary messengers. We report the cloning and characterization of a cDNA encoding a PLC-delta from Pisum sativum (PsPLC). The amino acid sequence deduced from the cDNA sequence showed 75-80% identity to other plant PLCs and contained the characteristic X, Y and C2 domains. The genomic PLC clone from pea was also characterized and found to contain eight introns. The protein was expressed in Escherichia coli, but the recombinant product did not show any phosphoinositide (PI)- or phosphatidylinositol-4, 5-bisphosphate (PIP2)-specific activity, despite having all known residues required for such activity, and in spite of the fact that its C2 domain was shown to bind calcium. Under similar in vitro assay conditions the recombinant tobacco PLC used as a control showed calcium-dependent PI- and PIP2-specific activity. Though PsPLC did not show enzyme activity in vitro, and may represent an inactive form of PLC, such as those reported in some mammalian systems, analysis of the transcription of PsPLC showed that the gene is expressed in all pea tissues, and is regulated by light in a tissue-specific manner. Roots showed higher expression of PsPLC than shoots. A putative PsPLC promoter region (792 bp) was also cloned and found to contain root-specific and light-responsive cis elements, suggesting that this form of PLC may be involved in important functions in plants.

• Wing MR, Snyder JT, Sondek J, Harden TK
• Direct activation of phospholipase C-epsilon by Rho.
• J Biol Chem. 2003; 278: 41253-8
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• Unique among the phospholipase C isozymes, the recently identified phospholipase C-epsilon (PLC-epsilon) contains an amino-terminal CDC25 domain capable of catalyzing nucleotide exchange on Ras family GTPases as well as a tandem array of Ras-associating (RA) domains near its carboxyl terminus that are effector binding sites for activated H-Ras and Rap. To determine whether other small GTPases activate PLC-epsilon, we measured inositol phosphate accumulation in COS-7 cells expressing a broad range of GTPase-deficient mutants of Ras superfamily proteins. RhoA, RhoB, and RhoC all markedly stimulated inositol phosphate accumulation in PLC-epsilon-expressing cells. This stimulation matched or exceeded phospholipase activation promoted by co-expression of PLC-epsilon with the known regulators Ras, Galpha12/13, or Gbeta1gamma2. In contrast, little effect was observed with the other Rho family members Rac1, Rac2, Rac3, and Cdc42. Truncation of the two carboxyl-terminal RA domains caused loss of responsiveness to H-Ras but not to Rho. Truncation of PLC-epsilon to remove the CDC25 and pleckstrin homology (PH) domains also did not cause loss of responsiveness to Rho, Galpha12/13, or Gbeta1gamma2. Comparative sequence analysis of mammalian phospholipase C isozymes revealed a unique approximately 65 amino acid insert within the catalytic core of PLC-epsilon not present in PLC-beta, gamma, delta, or zeta. A PLC-epsilon construct lacking this region was no longer activated by Rho or Galpha12/13 but retained regulation by Gbetagamma and H-Ras. GTP-dependent interaction of Rho with PLC-epsilon was illustrated in pull-down experiments with GST-Rho, and this interaction was retained in the PLC-epsilon construct lacking the unique insert within the catalytic core. These results are consistent with the conclusion that Rho family GTPases directly interact with PLC-epsilon by a mechanism independent of the CDC25 or RA domains. A unique insert within the catalytic core of PLC-epsilon imparts responsiveness to Rho, which may signal downstream of Galpha12/13 in the regulation of PLC-epsilon, because activation by both Rho and Galpha12/13 is lost in the absence of this sequence.

• Guo Y, Philip F, Scarlata S
• The Pleckstrin homology domains of phospholipases C-beta and -delta confer activation through a common site.
• J Biol Chem. 2003; 278: 29995-30004
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• Mammalian inositol-specific phospholipase C-beta2 (PLC beta 2) and PLC delta 1 differ in their cellular activators. PLC beta 2 can be activated by G beta gamma subunits, whereas PLC delta 1 can be activated by phosphatidylinositol 4,5 bisphosphate (PI(4,5)P2). For both proteins, the N-terminal pleckstrin homology (PH) domain appears to mediate activation. Here, we have constructed a chimera in which we placed the N-terminal PH domain of PLC delta 1 into remaining C-terminal regions of PLC beta 2. The PH delta PLC beta chimera showed PI(4,5)P2-dependent membrane binding similar to PLC delta 1 and a G beta gamma interaction energy close to that of PLC delta 1. Like PLC delta 1, the chimera was activated by PI(4,5)P2 through the PH domain but not by G beta gamma. Because these and previous results indicate a common site of contact between the PH and catalytic domains in these two enzymes, we computationally docked the known structures of the PH and catalytic domains of PLC delta 1. A synthetic peptide whose sequence matches a potential interaction site between the two domains inhibited the basal activity of PLC beta 2, PLC delta 1, and a G beta gamma-activable PH beta 2-PLC delta 1 chimera. Also, the peptide was able to inhibit PI(4,5)P2 and G beta gamma activation of the PH-PLC delta 1 PH-PLC beta 2 enzymes in a concentration-dependent manner, suggesting that this is the region responsible for PH domain-mediated activation of the catalytic core.

• Kravchuk AV, Zhao L, Bruzik KS, Tsai MD
• Engineering a catalytic metal binding site into a calcium-independent phosphatidylinositol-specific phospholipase C leads to enhanced stereoselectivity.
• Biochemistry. 2003; 42: 2422-30
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• Eukaryotic phosphatidylinositol-specific phospholipase Cs (PI-PLCs) utilize calcium as a cofactor during catalysis, whereas prokaryotic PI-PLCs use a spatially conserved guanidinium group from Arg69. In this study, we aimed to construct a metal-dependent mutant of a bacterial PI-PLC and characterize the catalytic role of the metal ion, seeking an enhanced understanding of the functional differences between these two positively charged moieties. The following results indicate that a bona fide catalytic metal binding site was created by the single arginine-to-aspartate mutation at position 69: (1) The R69D mutant was activated by Ca(2+), and the activation was specific for R69D, not for other mutants at this position. (2) Titration of R69D with Ca(2+), monitored by (15)N-(1)H HSQC (heteronuclear single quantum coherence) NMR, showed that addition of Ca(2+) to R69D restores the environment of the catalytic site analogous to that attained by the WT enzyme. (3) Upon Ca(2+) activation, the thio effect of the S(P)-isomer of the phosphorothioate analogue (k(O)/k(Sp) = 4.4 x 10(5)) approached a value similar to that of the WT enzyme, suggesting a structural and functional similarity between the two positively charged moieties (Arg69 and Asp69-Ca(2+)). The R(P)-thio effect (k(O)/k(Rp) = 9.4) is smaller than that of the WT enzyme by a factor of 5. Consequently, R69D-Ca(2+) displays higher stereoselectivity (k(Rp)/k(Sp) = 47,000) than WT (k(Rp)/k(Sp) = 7600). (4) Results from additional mutagenesis analyses suggest that the Ca(2+) binding site is comprised of side chains from Asp33, Asp67, Asp69, and Glu117. Our studies provide new insight into the mechanism of metal-dependent and metal-independent PI-PLCs.

• Ochocka AM, Pawelczyk T
• Isozymes delta of phosphoinositide-specific phospholipase C and their role in signal transduction in the cell.
• Acta Biochim Pol. 2003; 50: 1097-110
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• Phospholipase C (PLC, EC 3.1.4.11) is an enzyme crucial for the phosphoinositol pathway and whose activity is involved in eukaryotic signal transduction as it generates two second messengers: diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). There are four major types of phospholipase C named: beta, gamma, delta and the recently discovered epsilon, but this review will focus only on the recent advances for the delta isozymes of PLC. So far, four delta isozymes (named delta1-4) have been discovered and examined. They differ with regard to cellular distribution, activities, biochemical features and involvement in human ailments.

• Wehbi H, Feng J, Roberts MF
• Water-miscible organic cosolvents enhance phosphatidylinositol-specific phospholipase C phosphotransferase as well as phosphodiesterase activity.
• Biochim Biophys Acta. 2003; 1613: 15-27
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• Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis catalyzes the hydrolysis of phosphatidylinositol (PI) in a Ca(2+)-independent two-step mechanism: (i) an intramolecular phosphotransferase reaction to form inositol 1,2-(cyclic)-phosphate (cIP), followed by (ii) a cyclic phosphodiesterase activity that converts cIP to inositol 1-phosphate (I-1-P). Moderate amounts of water-miscible organic solvents have previously been shown to dramatically enhance the cyclic phosphodiesterase activity, that is, hydrolysis of cIP. Cosolvents [isopropanol (iPrOH), dimethylsufoxide (DMSO), and dimethylformamide (DMF)] also enhance the phosphotransferase activity of PI-PLC toward PI initially presented in vesicles, monomers, or micelles. Although these water-miscible organic cosolvents caused large changes in PI particle size and distribution (monitored with pyrene-labeled PI fluorescence, 31P NMR spectroscopy, gel filtration, and electron microscopy) that differed with the activating solvent, the change in PI substrate structure in different cosolvents was not correlated with the enhanced catalytic efficiency of PI-PLC toward its substrates. PI-PLC stability was decreased in water/organic cosolvent mixtures (e.g., the T(m) for PI-PLC thermal denaturation decreased linearly with added iPrOH). However, the addition of myo-inositol, a water-soluble inhibitor of PI-PLC, helped stabilize the protein. At 30% iPrOH and 4 degrees C (well below the T(m) for PI-PLC in the presence of iPrOH), cosolvent-induced changes in protein secondary structure were minimal. iPrOH and diheptanoylphosphatidylcholine, each of which activates PI-PLC for cIP hydrolysis, exhibited a synergistic effect for cIP hydrolysis that was not observed with PI as substrate. This behavior is consistent with a mechanism for cosolvent activation that involves changes in active site polarity along with small conformational changes involving the barrel rim tryptophan side chains that have little effect on protein secondary structure.

• Varnai P et al.
• Inositol lipid binding and membrane localization of isolated pleckstrin homology (PH) domains. Studies on the PH domains of phospholipase C delta 1 and p130.
• J Biol Chem. 2002; 277: 27412-22
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• The relationship between the ability of isolated pleckstrin homology (PH) domains to bind inositol lipids or soluble inositol phosphates in vitro and to localize to cellular membranes in live cells was examined by comparing the PH domains of phospholipase Cdelta(1) (PLCdelta(1)) and the recently cloned PLC-like protein p130 fused to the green fluorescent protein (GFP). The prominent membrane localization of PLCdelta(1)PH-GFP was paralleled with high affinity binding to inositol 1,4,5-trisphosphate (InsP(3)) as well as to phosphatidylinositol 4,5-bisphosphate-containing lipid vesicles or nitrocellulose membrane strips. In contrast, no membrane localization was observed with p130PH-GFP despite its InsP(3) and phosphatidylinositol 4,5-bisphosphate-binding properties being comparable with those of PLCdelta(1)PH-GFP. The N-terminal ligand binding domain of the type I InsP(3) receptor also failed to localize to the plasma membrane despite its 5-fold higher affinity to InsP(3) than the PH domains. By using a chimeric approach and cassette mutagenesis, the C-terminal alpha-helix and the short loop between the beta6-beta7 sheets of the PLCdelta(1)PH domain, in addition to its InsP(3)-binding region, were identified as critical components for membrane localization in intact cells. These data indicate that binding to the inositol phosphate head group is necessary but may not be sufficient for membrane localization of the PLCdelta(1)PH-GFP fusion protein, and motifs located within the C-terminal half of the PH domain provide auxiliary contacts with additional membrane components.

• Buitrago C, Gonzalez Pardo V, de Boland AR
• Nongenomic action of 1 alpha,25(OH)(2)-vitamin D3. Activation of muscle cell PLC gamma through the tyrosine kinase c-Src and PtdIns 3-kinase.
• Eur J Biochem. 2002; 269: 2506-15
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• We have previously demonstrated that the steroid hormone 1 alpha,25(OH)(2)-vitamin D(3)[1 alpha,25(OH)(2)D(3)] stimulates the production of inositol trisphosphate (InsP(3)), the breakdown product of phosphatidylinositol 4,5-biphosphate (PtdInsP(2)) by phospholipase C (PtdIns-PLC), and activates the cytosolic tyrosine kinase c-Src in skeletal muscle cells. In the present study we examined whether 1 alpha,25(OH)(2)D(3) induces the phosphorylation and membrane translocation of PLC gamma and the mechanism involved in this isozyme activation. We found that the steroid hormone triggers a significant phosphorylation on tyrosine residues of PLC gamma and induces a rapid increase in membrane-associated PLC gamma immunoreactivity with a time course that correlates with that of phosphorylation in muscle cells. Genistein, a tyrosine kinase inhibitor, blocked the phosphorylation of PLC gamma. Inhibition of 1 alpha,25(OH)(2)D(3)-induced c-Src activity by its specific inhibitor PP1 or muscle cell transfection with an antisense oligodeoxynucleotide directed against c-Src mRNA, prevented hormone stimulation of PLC gamma tyrosine phosphorylation. The isozyme phosphorylation is also blocked by both wortmannin and LY294002, two structurally different inhibitors of phosphatidyl inositol 3-kinase (PtdIns3K), the enzyme that produces PtdInsP(3) known to activate PLC gamma isozymes specifically by interacting with their SH2 and pleckstrin homology domains. The hormone also increases the physical association of c-Src and PtdIns3K with PLC gamma and induces a c-Src-dependent tyrosine phosphorylation of the p85 regulatory subunit of PtdIns3K. The time course of hormone-dependent PLC gamma phosphorylation closely correlates with the time course of its redistribution to the membrane, suggesting that phosphorylation and redistribution to the membrane of PLC gamma are two interdependent events. 1 alpha,25(OH)(2)D(3)-induced membrane translocation of PLC gamma was prevented to a great extent by c-Src and PtdIns3K inhibitors, PP1 and LY294002. Taken together, the present data indicates that the cytosolic tyrosine kinase c-Src and PtdIns 3-kinase play indispensable roles in 1 alpha,25(OH)(2)D(3) signal transduction cascades leading to PLC gamma activation.

• Singer AU, Waldo GL, Harden TK, Sondek J
• A unique fold of phospholipase C-beta mediates dimerization and interaction with G alpha q.
• Nat Struct Biol. 2002; 9: 32-6
• Display abstract

• GTP-bound subunits of the Gq family of G alpha subunits directly activate phospholipase C-beta (PLC-beta) isozymes to produce the second messengers inositol 1,4,5-trisphosphate and diacylglycerol. PLC-betas are GTPase activating proteins (GAPs) that also promote the formation of GDP-bound, inactive G beta subunits. Both phospholipase activation by G alpha-GTP subunits and GAP activity require a C-terminal region unique to PLC-beta isozymes. The crystal structure of the C-terminal region from an avian PLC-beta, determined at 2.4 A resolution, reveals a novel fold composed almost entirely of three long helices forming a coiled-coil that dimerizes along its long axis in an antiparallel orientation. The dimer interface is extensive ( approximately 3,200 A(2)), and, based on gel exclusion chromatography, full length PLC-betas are dimeric, indicating that PLC-betas likely function as dimers. Sequence conservation, mutational data and molecular modeling show that an electrostatically positive surface of the dimer contains the major determinants for binding G beta q. Effector dimerization, as highlighted by PLC-betas, provides a viable mechanism for regulating signaling cascades linked to heterotrimeric G proteins.

• Piccolo E, Innominato PF, Mariggio MA, Maffucci T, Iacobelli S, Falasca M
• The mechanism involved in the regulation of phospholipase Cgamma1 activity in cell migration.
• Oncogene. 2002; 21: 6520-9
• Display abstract

• Activation of the enzyme phospholipase C (PLC) leads to the formation of second messengers inositol 1,4,5-trisphosphate and diacylglycerol. Tyrosine kinase receptors activate this reaction through PLCgamma isoenzymes. PLCgamma activity involves its activation with, and phosphorylation by, receptor tyrosine kinases. Recently, it has been shown that phosphoinositide 3-kinase (PI 3-K) may regulate PLCgamma activity through the interaction of the PI 3-K product phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P(3)) and the PLCgamma pleckstrin homology (PH) domain. In an effort to understand the signalling pathway that involves PI 3-K regulation of PLCgamma, we found that EGF induces a PI 3-K-dependent translocation of PLCgamma1 at the leading edge of migrating cells in a wound healing assay. Similarly, the isolated PH, but not the Src-homology (SH) domains, N-SH2 or SH3, of PLCgamma1, translocates at the leading edge. Our experiments also showed that stable PH PLCgamma1 expression blocks epidermal growth factor (EGF)- and serum-induced cell motility and increases cell adhesion in MDA-MB-231 cells. This may suggest that influence of PI 3-K on PLCgamma1 could be relevant in cell migration, where PLCgamma1 seems to play a key role by modulating a series of events involved in actin polymerization.

• Davies DR, Interthal H, Champoux JJ, Hol WG
• The crystal structure of human tyrosyl-DNA phosphodiesterase, Tdp1.
• Structure. 2002; 10: 237-48
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• Tyrosyl-DNA phosphodiesterase (Tdp1) catalyzes the hydrolysis of a phosphodiester bond between a tyrosine residue and a DNA 3' phosphate. The enzyme appears to be responsible for repairing the unique protein-DNA linkage that occurs when eukaryotic topoisomerase I becomes stalled on the DNA in the cell. The 1.69 A crystal structure reveals that human Tdp1 is a monomer composed of two similar domains that are related by a pseudo-2-fold axis of symmetry. Each domain contributes conserved histidine, lysine, and asparagine residues to form a single active site. The structure of Tdp1 confirms that the protein has many similarities to the members of the phospholipase D (PLD) superfamily and indicates a similar catalytic mechanism. The structure also suggests how the unusual protein-DNA substrate binds and provides insights about the nature of the substrate in vivo.

• Mueller-Roeber B, Pical C
• Inositol phospholipid metabolism in Arabidopsis. Characterized and putative isoforms of inositol phospholipid kinase and phosphoinositide-specific phospholipase C.
• Plant Physiol. 2002; 130: 22-46
• Display abstract

• Phosphoinositides (PIs) constitute a minor fraction of total cellular lipids in all eukaryotic cells. They fulfill many important functions through interaction with a wide range of cellular proteins. Members of distinct inositol lipid kinase families catalyze the synthesis of these phospholipids from phosphatidylinositol. The hydrolysis of PIs involves phosphatases and isoforms of PI-specific phospholipase C. Although our knowledge of the roles played by plant PIs is clearly limited at present, there is no doubt that they are involved in many physiological processes during plant growth and development. In this review, we concentrate on inositol lipid-metabolizing enzymes from the model plant Arabidopsis for which biochemical characterization data are available, namely the inositol lipid kinases and PI-specific phospholipase Cs. The biochemical properties and structure of characterized and genome-predicted isoforms are presented and compared with those of the animal enzymes to show that the plant enzymes have some features clearly unique to this kingdom.

• Litosch I
• Novel mechanisms for feedback regulation of phospholipase C-beta activity.
• IUBMB Life. 2002; 54: 253-60
• Display abstract

• The receptor-regulated phospholipase C-beta (PLC-beta) signaling pathway is an important component in a network of signaling cascades that regulate cell function. PLC-beta signaling has been implicated in the regulation of cardiovascular function and neuronal plasticity. The Gq family of G proteins mediate receptor stimulation of PLC-beta activity at the plasma membrane. Mitogens stimulate the activity of a nuclear pool of PLC-beta. Stimulation of PLC-beta activity results in the rapid hydrolysis of phosphatidylinositol-4,5-bisphosphate, with production of inositol-1,4,5-trisphosphate and diacylglycerol, intracellular mediators that increase intracellular Ca2+ levels and activate protein kinase C activity, respectively. Diacylglycerol kinase converts diacylglycerol to phosphatidic acid, a newly emerging intracellular mediator of hormone action that targets a number of signaling proteins. Activation of the Gq linked PLC-beta signaling pathway can also generate additional signaling lipids, including phosphatidylinositol-3-phosphate and phosphatidylinositol-3,4,5-trisphosphate, which regulate the activity and/or localization of a number of proteins. Novel feedback mechanisms, directed at the level of Gq and PLC-beta, have been identified. PLC-beta and regulators of G protein signaling (RGS) function as GTPase-activating proteins on Gq to control the amplitude and duration of stimulation. Protein kinases phosphorylate and regulate the activation of specific PLC-beta isoforms. Phosphatidic acid regulates PLC-beta1 activity and stimulation of PLC-beta1 activity by G proteins. These feedback mechanisms coordinate receptor signaling and cell activation. Feedback mechanisms constitute possible targets for pharmacological intervention in the treatment of disease.

• Chang JS et al.
• Interaction of elongation factor-1alpha and pleckstrin homology domain of phospholipase C-gamma 1 with activating its activity.
• J Biol Chem. 2002; 277: 19697-702
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• The pleckstrin homology (PH) domain is a small motif for membrane targeting in the signaling molecules. Phospholipase C (PLC)-gamma1 has two putative PH domains, an NH(2)-terminal and a split PH domain. Here we report studies on the interaction of the PH domain of PLC-gamma1 with translational elongation factor (EF)-1alpha, which has been shown to be a phosphatidylinositol 4-kinase activator. By pull-down of cell extract with the glutathione S-transferase (GST) fusion proteins with various domains of PLC-gamma1 followed by peptide sequence analysis, we identified EF-1alpha as a binding partner of a split PH domain of PLC-gamma1. Analysis by site-directed mutagenesis of the PH domain revealed that the beta2-sheet of a split PH domain is critical for the interaction with EF-1alpha. Moreover, Dot-blot assay shows that a split PH domain specifically binds to phosphoinositides including phosphatidylinositol 4-phosphate and phosphatidylinositol 4, 5-bisphosphate (PIP(2)). So the PH domain of PLC-gamma1 binds to both EF-1alpha and PIP(2). The binding affinity of EF-1alpha to the GST.PH domain fusion protein increased in the presence of PIP(2), although PIP(2) does not bind to EF-1alpha directly. This suggests that EF-1alpha may control the binding affinity between the PH domain and PIP(2). PLC-gamma1 is substantially activated in the presence of EF-1alpha with a bell-shaped curve in relation to the molar ratio between them, whereas a double point mutant PLC-gamma1 (Y509A/F510A) that lost its binding affinity to EF-1alpha shows basal level activity. Taken together, our data show that EF-1alpha plays a direct role in phosphoinositide metabolism of cellular signaling by regulating PLC-gamma1 activity via a split PH domain.

• Otterhag L, Sommarin M, Pical C
• N-terminal EF-hand-like domain is required for phosphoinositide-specific phospholipase C activity in Arabidopsis thaliana.
• FEBS Lett. 2001; 497: 165-70
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• Phosphoinositide-specific phospholipase C's (PI-PLCs) are ubiquitous in eukaryotes, from plants to animals, and catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate into the two second messengers inositol 1,4,5-trisphosphate and diacylglycerol. In animals, four distinct subfamilies of PI-PLCs have been identified, and the three-dimensional structure of one rat isozyme, PLC-delta1, determined. Plants appear to contain only one gene family encoding PI-PLCs. The catalytic properties of plant PI-PLCs are very similar to those of animal enzymes. However, very little is known about the regulation of plant PI-PLCs. All plant PI-PLCs comprise three domains, X, Y and C2, which are also conserved in isoforms from animals and yeast. We here show that one PI-PLC isozyme from Arabidopsis thaliana, AtPLC2, is predominantly localized in the plasma membrane, and that the conserved N-terminal domain may represent an EF-hand domain that is required for catalytic activity but not for lipid binding.

• Ryan M, Liu T, Dahlquist FW, Griffith OH
• A catalytic diad involved in substrate-assisted catalysis: NMR study of hydrogen bonding and dynamics at the active site of phosphatidylinositol-specific phospholipase C.
• Biochemistry. 2001; 40: 9743-50
• Display abstract

• Phosphatidylinositol-specific phospholipase Cs (PI-PLCs, EC 3.1.4.10) are ubiquitous enzymes that cleave phosphatidylinositol or phosphorylated derivatives, generating second messengers in eukaryotic cells. A catalytic diad at the active site of Bacillus cereus PI-PLC composed of aspartate-274 and histidine-32 was postulated from the crystal structure to form a catalytic triad with the 2-OH group of the substrate [Heinz, D. W., et al. (1995) EMBO J. 14, 3855-3863]. This catalytic diad has been observed directly by proton NMR. The single low-field line in the 1H NMR spectrum is assigned by site-directed mutagenesis: The peak is present in the wild type but absent in the mutants H32A and D274A, and arises from the histidine Hdelta1 forming the Asp274-His32 hydrogen bond. This hydrogen is solvent-accessible, and exchanges slowly with H2O on the NMR time scale. The position of the low-field peak shifts from 16.3 to 13.8 ppm as the pH is varied from 4 to 9, reflecting a pKa of 8.0 at 6 degrees C, which is identified with the pKa of His32. The Hdelta1 signal is modulated by rapid exchange of the Hepsilon2 with the solvent. Estimates of the exchange rate as a function of pH and protection factors are derived from a line shape analysis. The NMR behavior is remarkably similar to that of the serine proteases. The postulated function of the Asp274-His32 diad is to hydrogen-bond with the 2-OH of phosphatidylinositol (PI) substrate to form a catalytic triad analogous to Asp-His-Ser of serine proteases. This is an example of substrate-assisted catalysis where the substrate provides the catalytic nucleophile of the triad. This hydrogen bond becomes shorter as the imidazole is protonated, suggesting it is stronger in the transition state, contributing further to the catalytic efficiency. The hydrogen bond fits the NMR criteria for a short, strong hydrogen bond, i.e., a highly deshielded proton resonance, bond length of 2.64 +/- 0.04 A at pH 6 measured by NMR, a D/H fractionation factor significantly lower than 1.0, and a protection factor > or = 100.

• Santagata S et al.
• G-protein signaling through tubby proteins.
• Science. 2001; 292: 2041-50
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• Dysfunction of the tubby protein results in maturity-onset obesity in mice. Tubby has been implicated as a transcription regulator, but details of the molecular mechanism underlying its function remain unclear. Here we show that tubby functions in signal transduction from heterotrimeric GTP-binding protein (G protein)-coupled receptors. Tubby localizes to the plasma membrane by binding phosphatidylinositol 4,5-bisphosphate through its carboxyl terminal "tubby domain." X-ray crystallography reveals the atomic-level basis of this interaction and implicates tubby domains as phosphorylated-phosphatidyl- inositol binding factors. Receptor-mediated activation of G protein alphaq (Galphaq) releases tubby from the plasma membrane through the action of phospholipase C-beta, triggering translocation of tubby to the cell nucleus. The localization of tubby-like protein 3 (TULP3) is similarly regulated. These data suggest that tubby proteins function as membrane-bound transcription regulators that translocate to the nucleus in response to phosphoinositide hydrolysis, providing a direct link between G-protein signaling and the regulation of gene expression.

• Kravchuk AV, Zhao L, Kubiak RJ, Bruzik KS, Tsai MD
• Mechanism of phosphatidylinositol-specific phospholipase C: origin of unusually high nonbridging thio effects.
• Biochemistry. 2001; 40: 5433-9
• Display abstract

• Phosphatidylinositol-specific phospholipase C (PI-PLC) has been proposed previously to employ a catalytic mechanism highly reminiscent of that of ribonuclease A (RNase A). Both catalytic sites are comprised of two histidine side chains acting as a general base-general acid pair and a phosphate-activating residue: an arginine in the case of PI-PLC and a lysine in RNase A. Despite these structural similarities, the PI-PLC reaction is slowed 10(5)-fold upon substitution of one of the phosphate nonbridging oxygen atoms with sulfur, whereas a much smaller effect is observed in the analogous RNase A reaction. Here, we report a systematic study of this property in PI-PLC, conducted by means of site-directed chemical modification of a cysteine residue replacing the arginine at position 69. The results show that mutant enzymes featuring bidentate side chains at this position display significantly higher activity, higher thio effects, and greater stereoselectivity than do those with monodentate side chains. The results suggest that the bidentate nature of Arg69 is the origin of the large thio effects and stereoselectivity in PI-PLC. We propose that in addition to binding the phosphate, the function of arginine 69 is to bring the phosphate group and the 2-OH group of inositol into proximity and to induce proper alignment for nucleophilic attack, and possibly to lower the pK(a) of the 2-OH. The results presented here could be important to mechanisms of phosphoryl transfer enzymes in general, suggesting that a major part of thio effects observed in enzymatic phosphoryl transfer reactions can originate from factors other than direct interaction between a side chain and a phosphate group, and caution the use of the absolute magnitude of the thio effect as an indicator of the strength of such interactions.

• Montero-Moran GM, Lara-Gonzalez S, Alvarez-Anorve LI, Plumbridge JA, Calcagno ML
• On the multiple functional roles of the active site histidine in catalysis and allosteric regulation of Escherichia coli glucosamine 6-phosphate deaminase.
• Biochemistry. 2001; 40: 10187-96
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• The active site of glucosamine-6-phosphate deaminase (EC 3.5.99.6, formerly 5.3.1.10) from Escherichia coli was first characterized on the basis of the crystallographic structure of the enzyme bound to the competitive inhibitor 2-amino-2-deoxy-glucitol 6-phosphate. The structure corresponds to the R allosteric state of the enzyme; it shows the side-chain of His143 in close proximity to the O5 atom of the inhibitor. This arrangement suggests that His143 could have a role in the catalysis of the ring-opening step of glucosamine 6-phosphate whose alpha-anomer is the true substrate. The imidazole group of this active-site histidine contacts the carboxy groups from Glu148 and Asp141, via its Ndelta1 atom [Oliva et al. (1995) Structure 3, 1323-1332]. These interactions change in the T state because the side chain of Glu148 moves toward the allosteric site, leaving at the active site the dyad Asp141-His143 [Horjales et al. (1999) Structure 7, 527-536]. In this research, a dual approach using site-directed mutagenesis and controlled chemical modification of histidine residues has been used to investigate the role of the active-site histidine. Our results support a multifunctional role of His143; in the forward reaction, it is involved in the catalysis of the ring-opening step of the substrate, glucosamine 6-P. In the reverse reaction, the substrate fructose 6-P binds in its open chain, carbonylic form. The role of His143 in the binding of both glucosamine 6-P and reaction intermediates in their extended-chain forms was demonstrated by binding experiments using the reaction intermediate analogue, 2-amino-2-deoxy-D-glucitol 6-phosphate. His143 was also shown to be a critical residue for the conformational coupling between active and allosteric sites. From the pH dependence of the reactivity of the active site histidine to diethyl dicarbonate, we observed a pK(a) change of 1.2 units to the acid side when the enzyme undergoes the allosteric T to R transition during which the side chain of Glu148 moves toward the active site. The kinetic study of the Glu148-Gln mutant deaminase shows that the loss of the carboxy group and its replacement with the corresponding amide modifies the k(cat) versus pH profile of the enzyme, suggesting that the catalytic step requiring the participation of His143 has become rate-limiting. This, in turn, indicates that the interaction Glu148-His143 in the wild-type enzyme in the R state contributes to make the enzyme functional over a wide pH range.

• Hedberg KK, Cogan EB, Birrell GB, Griffith OH
• Sensitive fluorescent quantitation of myo-inositol 1,2-cyclic phosphate and myo-inositol 1-phosphate by high-performance thin-layer chromatography.
• J Chromatogr B Biomed Sci Appl. 2001; 757: 317-24
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• A non-radioactive micro-assay for the cyclic phosphodiesterase reaction catalyzed by Bacillus cereus phosphatidylinositol-specific phospholipase C is described. The assay involves high-performance thin-layer chromatography on silica gel to resolve the substrate (myo-inositol 1,2-cyclic phosphate) and the product (myo-inositol 1-phosphate), followed by detection with a lead tetraacetate-fluorescein stain. The quantitation of these inositol phosphates in sample spots relative to a series of standards is accomplished by analysis of the fluorescent plate image with a commercial phosphoimager and associated software. The experimental considerations for reliable quantitation of inositol monophosphates in the range of 0.1 to 50 nmol are presented.

• Kubiak RJ, Yue X, Hondal RJ, Mihai C, Tsai MD, Bruzik KS
• Involvement of the Arg-Asp-His catalytic triad in enzymatic cleavage of the phosphodiester bond.
• Biochemistry. 2001; 40: 5422-32
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• Phosphatidylinositol-specific phospholipase C (PI-PLC) catalyzes the cleavage of the P-O bond in phosphatidylinositol via intramolecular nucleophilic attack of the 2-hydroxyl group of inositol on the phosphorus atom. Our earlier stereochemical and site-directed mutagenesis studies indicated that this reaction proceeds by a mechanism similar to that of RNase A, and that the catalytic site of PI-PLC consists of three major components analogous to those observed in RNase A, the His32 general base, the His82 general acid, and Arg69 acting as a phosphate-activating residue. In addition, His32 is associated with Asp274 in forming a catalytic triad with inositol 2-hydroxyl, and His82 is associated with Asp33 in forming a catalytic diad. The focus of this work is to provide a global view of the mechanism, assess cooperation between various catalytic residues, and determine the origin of enzyme activation by the hydrophobic leaving group. To this end, we have investigated kinetic properties of Arg69, Asp33, and His82 mutants with phosphorothioate substrate analogues which feature leaving groups of varying hydrophobicity and pK(a). Our results indicate that interaction of the nonbridging pro-S oxygen atom of the phosphate group with Arg69 is strongly affected by Asp33, and to a smaller extent by His82. This result in conjunction with those obtained earlier can be rationalized in terms of a novel, dual-function triad comprised of Arg69, Asp33, and His82 residues. The function of this triad is to both activate the phosphate group toward the nucleophilic attack and to protonate the leaving group. In addition, Asp33 and His82 mutants displayed much smaller degrees of activation by the fatty acid-containing leaving group as compared to the wild-type (WT) enzyme, and the level of activation was significantly reduced for substrates featuring the leaving group with low pK(a) values. These results strongly suggest that the assembly of the above three residues into the fully catalytically competent triad is controlled by the hydrophobic interactions of the enzyme with the substrate leaving group.

• Shin S, Ha NC, Oh BC, Oh TK, Oh BH
• Enzyme mechanism and catalytic property of beta propeller phytase.
• Structure. 2001; 9: 851-8
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• BACKGROUND: Phytases hydrolyze phytic acid (myo-inositol-hexakisphosphate) to less-phosphorylated myo-inositol derivatives and inorganic phosphate. Phytases are used in animal feed to reduce phosphate pollution in the environment. Recently, a thermostable, calcium-dependent Bacillus phytase was identified that represents the first example of the beta propeller fold exhibiting phosphatase activity. We sought to delineate the catalytic mechanism and property of this enzyme. RESULTS: The crystal structure of the enzyme in complex with inorganic phosphate reveals that two phosphates and four calcium ions are tightly bound at the active site. Mutation of the residues involved in the calcium chelation results in severe defects in the enzyme's activity. One phosphate ion, chelating all of the four calcium ions, is close to a water molecule bridging two of the bound calcium ions. Fluoride ion, which is expected to replace this water molecule, is an uncompetitive inhibitor of the enzyme. The enzyme is able to hydrolyze any of the six phosphate groups of phytate. CONCLUSIONS: The enzyme reaction is likely to proceed through a direct attack of the metal-bridging water molecule on the phosphorous atom of a substrate and the subsequent stabilization of the pentavalent transition state by the bound calcium ions. The enzyme has two phosphate binding sites, the "cleavage site", which is responsible for the hydrolysis of a substrate, and the "affinity site", which increases the binding affinity for substrates containing adjacent phosphate groups. The existence of the two nonequivalent phosphate binding sites explains the puzzling formation of the alternately dephosphorylated myo-inositol triphosphates from phytate and the hydrolysis of myo-inositol monophosphates.

• Zambrzycka A, Strosznajder RP, Strosznajder JB
• Aggregated beta amyloid peptide 1-40 decreases Ca2+- and cholinergic receptor-mediated phosphoinositide degradation by alteration of membrane and cytosolic phospholipase C in brain cortex.
• Neurochem Res. 2000; 25: 189-96
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• The effects of full-length amyloid beta protein, A(beta) (1-40), on phosphoinositide-specific phospholipase C (PLC) were investigated in synaptic plasma membranes (SPM) and cytosol prepared from the cerebral cortex of adult rats. Moreover, the role of A(beta) (1-40) on the activation of lipid peroxidation was evaluated. The activity of phospholipase C (PLC) acting on phosphatidylinositol (PI) and phosphatidylinositol-4,5-bisphosphate (PIP2) was determined using exogenous labeled substrates. The subcellular fractions were the source of enzyme(s). The radioactivity of lipid messengers derived from degradation of [14C- arachidonoyl] PI was also determined. The stable aggregated form of beta-amyloid peptide (1-40) at 25 microM concentration exerted reproducible effects. The aggregated form of A(beta) (1-40) inhibited Ca(2+)-regulated PI and PIP2 degradation by SPM and cytosolic enzymes. Aggregated A(beta) also decreased significantly the level of diacylglycerol, the product of PLC. This additionally supports the inhibitory effect of A(beta) on membrane-bound and cytosolic PLC. Moreover, A(beta) (1-40) significantly decreased the basal activity of the PIP2-PLC in SPM and the enzyme activity regulated through cholinergic receptors. However, in spite of the lower enzyme activity, the percentage distribution of inositol (1,4,5) P3 radioactivity (IP3) in the total pool of inositol metabolites was not significantly changed. The aggregated neurotoxic fragment, A(beta) (25-35), mimicked the effect of full-length A(beta) (1-40). A(beta) (1-40) enhanced the level of malondialdehyde indicating an activation of free radical stimulated membrane lipid peroxidation that may be involved in alteration of phospholipase(s) activity. Our results indicated that aggregated A(beta) (1-40) alters Ca(2+)-dependent phosphoinositide degradation affecting synaptic plasma membrane and cytosolic phospholipase(s) activity. Moreover, this peptide significantly decreased the phosphoinositide-dependent signal transduction mediated by cholinergic receptors. The effect of aggregated A(beta) (1-40) is more pronounced than that of the neurotoxic fragment A(beta) (25-35). Our study suggests that the deposition of aggregated A(beta) may alter phosphoinositide signaling in brain.

• Sternfeld L, Th inverted question markevenod F, Schulz I
• fMLP-induced arachidonic acid release in db-cAMP-differentiated HL-60 cells is independent of phosphatidylinositol-4, 5-bisphosphate-specific phospholipase C activation and cytosolic phospholipase A(2) activation.
• Arch Biochem Biophys. 2000; 378: 246-58
• Display abstract

• In inflammatory cells, agonist-stimulated arachidonic acid (AA) release is thought to be induced by activation of group IV Ca(2+)-dependent cytosolic phospholipase A(2) (cPLA(2)) through mitogen-activated protein kinase (MAP kinase)- and/or protein kinase C (PKC)-mediated phosphorylation and Ca(2+)-dependent translocation of the enzyme to the membrane. Here we investigated the role of phospholipases in N-formylmethionyl-l-leucyl-l-phenylalanine (fMLP; 1 nM-10 microM)-induced AA release from neutrophil-like db-cAMP-differentiated HL-60 cells. U 73122 (1 microM), an inhibitor of phosphatidyl-inositol-4,5-biphosphate-specific phospholipase C, or the membrane-permeant Ca(2+)-chelator 1, 2-bis inverted question mark2-aminophenoxyethane-N,N,N',N'-tetraacetic acid (10 microM) abolished fMLP-mediated Ca(2+) signaling, but had no effect on fMLP-induced AA release. The protein kinase C-inhibitor Ro 318220 (5 microM) or the inhibitor of cPLA(2) arachidonyl trifluoromethyl ketone (AACOCF(3); 10-30 microM) did not inhibit fMLP-induced AA release. In contrast, AA release was stimulated by the Ca(2+) ionophore A23187 (10 microM) plus the PKC activator phorbol myristate acetate (PMA) (0.2 microM). This effect was inhibited by either Ro 318220 or AACOCF(3). Accordingly, a translocation of cPLA(2) from the cytosol to the membrane fraction was observed with A23187 + PMA, but not with fMLP. fMLP-mediated AA release therefore appeared to be independent of Ca(2+) signaling and PKC and MAP kinase activation. However, fMLP-mediated AA release was reduced by approximately 45% by Clostridium difficile toxin B (10 ng/ml) or by 1-butanol; both block phospholipase D (PLD) activity. The inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), D609 (100 microM), decreased fMLP-mediated AA release by approximately 35%. The effect of D609 + 1-butanol on fMLP-induced AA release was additive and of a magnitude similar to that of propranolol (0.2 mM), an inhibitor of phosphatidic acid phosphohydrolase. This suggests that the bulk of AA generated by fMLP stimulation of db-cAMP-differentiated HL-60 cells is independent of the cPLA(2) pathway, but may originate from activation of PC-PLC and PLD.

• Coursol S, Giglioli-Guivarc'h N, Vidal J, Pierre JN
• An increase in phosphoinositide-specific phospholipase C activity precedes induction of C4 phosphoenolpyruvate carboxylase phosphorylation in illuminated and NH4Cl-treated protoplasts from Digitaria sanguinalis.
• Plant J. 2000; 23: 497-506
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• A Ca2+-dependent phosphoinositide-specific phospholipase C (PI-PLC) activity has been characterized in the microsomal fraction of Digitaria sanguinalis mesophyll cell protoplasts. Microsomal PI-PLC was found to be inhibited in vitro by a mammalian anti-PLC-delta1 antibody and by the aminosteroide U-73122, an inhibitor of PI-PLC activity in animal cells. In Western blot experiments, the antibody recognized an 85 kDa protein in both microsomal protein extracts from mesophyll protoplasts and rat brain protein extracts containing the authentic enzyme. The involvement of the microsomal PI-PLC in the light-dependent transduction pathway leading to the phosphorylation of C4 phosphoenolpyruvate carboxylase (PEPC) was investigated in D. sanguinalis protoplasts. A transient increase in the PI-PLC reaction product inositol-1,4,5-trisphosphate (Ins(1,4, 5)P3) was observed in situ during early induction of the C4 PEPC phosphorylation cascade. U-73122, but not the inactive analogue U-73343, efficiently blocked the transient accumulation of Ins(1,4, 5)P3, and both the increase in C4 PEPC kinase activity and C4 PEPC phosphorylation in illuminated and weak base-treated protoplasts. Taken together, these data suggest that PI-PLC-based signalling is a committed step in the cascade controlling the regulation of C4 PEPC phosphorylation in C4 leaves.

• Furuya T, Kashuba C, Docampo R, Moreno SN
• A novel phosphatidylinositol-phospholipase C of Trypanosoma cruzi that is lipid modified and activated during trypomastigote to amastigote differentiation.
• J Biol Chem. 2000; 275: 6428-38
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• The phosphoinositide (PI)-specific phospholipase C gene (TcPI-PLC) of the protozoan parasite Trypanosoma cruzi was cloned, sequenced, expressed in Escherichia coli, and the protein product (TcPI-PLC) was shown to have enzymatic characteristics similar to those of mammalian delta-type PI-PLCs. The TcPI-PLC gene is expressed at high levels in the epimastigote and amastigote stages of the parasite, and its expression is induced during the differentiation of trypomastigotes into amastigotes, where TcPI-PLC associates with the plasma membrane and increases its catalytic activity. In contrast to other PI-PLCs described so far, the deduced amino acid sequence of TcPI-PLC revealed some unique features such as an N-myristoylation consensus sequence at its amino-terminal end, lack of an apparent pleckstrin homology domain and a highly charged linker region between the catalytic X and Y domains. TcPI-PLC is lipid modified in vivo, as demonstrated by metabolic labeling with [(3)H]myristate and [(3)H]palmitate and fatty acid analysis of the immunoprecipitated protein, and may constitute the first example of a new group of PI-PLCs.

• Morais MC, Zhang W, Baker AS, Zhang G, Dunaway-Mariano D, Allen KN
• The crystal structure of bacillus cereus phosphonoacetaldehyde hydrolase: insight into catalysis of phosphorus bond cleavage and catalytic diversification within the HAD enzyme superfamily.
• Biochemistry. 2000; 39: 10385-96
• Display abstract

• Phosphonoacetaldehyde hydrolase (phosphonatase) catalyzes the hydrolysis of phosphonoacetaldehyde to acetaldehyde and phosphate using Mg(II) as cofactor. The reaction proceeds via a novel bicovalent catalytic mechanism in which an active-site nucleophile abstracts the phosphoryl group from the Schiff-base intermediate formed from Lys53 and phosphonoacetaldehyde. In this study, the X-ray crystal structure of the Bacillus cereus phosphonatase homodimer complexed with the phosphate (product) analogue tungstate (K(i) = 50 microM) and the Mg(II) cofactor was determined to 3.0 A resolution with an R(cryst) = 0.248 and R(free) = 0.284. Each monomer is made up of an alpha/beta core domain consisting of a centrally located six-stranded parallel beta-sheet surrounded by six alpha-helices. Two flexible, solvated linkers connect to a small cap domain (residues 21-99) that consists of an antiparallel, five-helix bundle. The subunit-subunit interface, formed by the symmetrical packing of the two alpha8 helices from the respective core domains, is stabilized through the hydrophobic effect derived from the desolvation of paired Met171, Trp164, Tyr162, Tyr167, and Tyr176 side chains. The active site is located at the domain-domain interface of each subunit. The Schiff base forming Lys53 is positioned on the cap domain while tungstate and Mg(II) are bound to the core domain. Mg(II) ligands include two oxygens of the tungstate ligand, one oxygen of the carboxylates of Asp12 and Asp186, the backbone carbonyl oxygen of Ala14, and a water that forms a hydrogen bond with the carboxylate of Asp190 and Thr187. The guanidinium group of Arg160 binds tungstate and the proposed nucleophile Asp12, which is suitably positioned for in-line attack at the tungsten atom. The side chains of the core domain residue Tyr128 and the cap domain residues Cys22 and Lys53 are located nearby. The identity of Asp12 as the active-site nucleophile was further evidenced by the observed removal of catalytic activity resulting from Asp12Ala substitution. The similarity of backbone folds observed in phosphonatase and the 2-haloacid dehalogenase of the HAD enzyme superfamily indicated common ancestry. Superposition of the two structures revealed a conserved active-site scaffold having distinct catalytic stations. Analysis of the usage of polar amino acid residues at these stations by the dehalogenases, phosphonatases, phosphatases, and phosphomutases of the HAD superfamily suggests possible ways in which the active site of an ancient enzyme ancestor might have been diversified for catalysis of C-X, P-C, and P-O bond cleavage reactions.

• Kim MJ, Kim E, Ryu SH, Suh PG
• The mechanism of phospholipase C-gamma1 regulation.
• Exp Mol Med. 2000; 32: 101-9
• Display abstract

• Phospholipase C (PLC)1 hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate the second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 induces a transient increase in intracellular free Ca2+, while DAG directly activates protein kinase C. Upon stimulation of cells with growth factors, PLC-gamma1 is activated upon their association with and phosphorylation by receptor and non-receptor tyrosine kinases. In this review, we will focus on the activation mechanism and regulatory function of PLC-gamma1.

• Rebecchi MJ, Pentyala SN
• Structure, function, and control of phosphoinositide-specific phospholipase C.
• Physiol Rev. 2000; 80: 1291-335
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• Phosphoinositide-specific phospholipase C (PLC) subtypes beta, gamma, and delta comprise a related group of multidomain phosphodiesterases that cleave the polar head groups from inositol lipids. Activated by all classes of cell surface receptor, these enzymes generate the ubiquitous second messengers inositol 1,4, 5-trisphosphate and diacylglycerol. The last 5 years have seen remarkable advances in our understanding of the molecular and biological facets of PLCs. New insights into their multidomain arrangement and catalytic mechanism have been gained from crystallographic studies of PLC-delta(1), while new modes of controlling PLC activity have been uncovered in cellular studies. Most notable is the realization that PLC-beta, -gamma, and -delta isoforms act in concert, each contributing to a specific aspect of the cellular response. Clues to their true biological roles were also obtained. Long assumed to function broadly in calcium-regulated processes, genetic studies in yeast, slime molds, plants, flies, and mammals point to specific and conditional roles for each PLC isoform in cell signaling and development. In this review we consider each subtype of PLC in organisms ranging from yeast to mammals and discuss their molecular regulation and biological function.

• Martin SF, Follows BC, Hergenrother PJ, Trotter BK
• The choline binding site of phospholipase C (Bacillus cereus): insights into substrate specificity.
• Biochemistry. 2000; 39: 3410-5
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• The phosphatidylcholine-preferring phospholipase C from Bacillus cereus (PLC(Bc)) is a 28.5 kDa enzyme with three zinc ions in its active site. The roles that a number of amino acid residues play as zinc ligands and in binding and catalysis have been elucidated. Recent mechanistic studies indicate that the rate of the reaction is limited by a proton-transfer step during chemical hydrolysis and not substrate binding or product release. An X-ray structure of PLC(Bc) complexed with a phosphonate inhibitor related to phosphatidylcholine revealed that the three amino acid residues Glu4, Tyr56, and Phe66 comprise the choline binding pocket. However, because the contributions that these three residues make to substrate recognition and specificity were unknown, a series of site-specific mutants for Glu4, Tyr56, and Phe66 were constructed by PCR mutagenesis. On the basis of a comparison of their respective CD spectra and melting temperatures, it appears that the mutants adopt folded structures in solution that are virtually identical to that of wild-type PLC(Bc). The kinetic parameters k(cat) and K(m) for the hydrolysis of the three soluble substrates 1, 2-dihexanoyl-sn-glycero-3-phosphocholine (C6PC), 1, 2-dihexanoyl-sn-glycero-3-phosphoethanolamine (C6PE), and 1, 2-dihexanoyl-sn-glycero-3-phospho-L-serine (C6PS) at concentrations below their corresponding critical micelle concentration (cmc) values were determined for each mutant. Replacement of Phe66 with a nonaromatic residue dramatically decreased k(cat) (approximately 200-fold) and reduced PLC(Bc) activity toward C6PC, C6PE, and C6PS, whereas changes to Glu4 and Tyr56 typically led to much more modest losses in catalytic efficiencies. Mutations of Glu4 had relatively little effect upon k(cat) and K(m) for C6PS, but they significantly influenced K(m) for C6PC and C6PE. Replacing Tyr56 with nonaromatic residues also affects catalytic efficiency, albeit to a much lesser degree than the corresponding changes at position 66. However, the presence of an aromatic residue at position 56 seems to confer some substrate selectivity for C6PC and C6PE, which bear a positive charge on the headgroup, relative to C6PS, which has no net charge on the headgroup; this increase in specificity arises largely from a reduced k(cat) for C6PS.

• Zgiby SM, Thomson GJ, Qamar S, Berry A
• Exploring substrate binding and discrimination in fructose1, 6-bisphosphate and tagatose 1,6-bisphosphate aldolases.
• Eur J Biochem. 2000; 267: 1858-68
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• Fructose 1,6-bisphosphate aldolase catalyses the reversible condensation of glycerone-P and glyceraldehyde 3-phosphate into fructose 1,6-bisphosphate. A recent structure of the Escherichia coli Class II fructose 1,6-bisphosphate aldolase [Hall, D.R., Leonard, G.A., Reed, C.D., Watt, C.I., Berry, A. & Hunter, W.N. (1999) J. Mol. Biol. 287, 383-394] in the presence of the transition state analogue phosphoglycolohydroxamate delineated the roles of individual amino acids in binding glycerone-P and in the initial proton abstraction steps of the mechanism. The X-ray structure has now been used, together with sequence alignments, site-directed mutagenesis and steady-state enzyme kinetics to extend these studies to map important residues in the binding of glyceraldehyde 3-phosphate. From these studies three residues (Asn35, Ser61 and Lys325) have been identified as important in catalysis. We show that mutation of Ser61 to alanine increases the Km value for fructose 1, 6-bisphosphate 16-fold and product inhibition studies indicate that this effect is manifested most strongly in the glyceraldehyde 3-phosphate binding pocket of the active site, demonstrating that Ser61 is involved in binding glyceraldehyde 3-phosphate. In contrast a S61T mutant had no effect on catalysis emphasizing the importance of an hydroxyl group for this role. Mutation of Asn35 (N35A) resulted in an enzyme with only 1.5% of the activity of the wild-type enzyme and different partial reactions indicate that this residue effects the binding of both triose substrates. Finally, mutation of Lys325 has a greater effect on catalysis than on binding, however, given the magnitude of the effects it is likely that it plays an indirect role in maintaining other critical residues in a catalytically competent conformation. Interestingly, despite its proximity to the active site and high sequence conservation, replacement of a fourth residue, Gln59 (Q59A) had no significant effect on the function of the enzyme. In a separate study to characterize the molecular basis of aldolase specificity, the agaY-encoded tagatose 1,6-bisphosphate aldolase of E. coli was cloned, expressed and kinetically characterized. Our studies showed that the two aldolases are highly discriminating between the diastereoisomers fructose bisphosphate and tagatose bisphosphate, each enzyme preferring its cognate substrate by a factor of 300-1500-fold. This produces an overall discrimination factor of almost 5 x 105 between the two enzymes. Using the X-ray structure of the fructose 1,6-bisphosphate aldolase and multiple sequence alignments, several residues were identified, which are highly conserved and are in the vicinity of the active site. These residues might potentially be important in substrate recognition. As a consequence, nine mutations were made in attempts to switch the specificity of the fructose 1,6-bisphosphate aldolase to that of the tagatose 1,6-bisphosphate aldolase and the effect on substrate discrimination was evaluated. Surprisingly, despite making multiple changes in the active site, many of which abolished fructose 1, 6-bisphosphate aldolase activity, no switch in specificity was observed. This highlights the complexity of enzyme catalysis in this family of enzymes, and points to the need for further structural studies before we fully understand the subtleties of the shaping of the active site for complementarity to the cognate substrate.

• Dwivedi Y, Pandey GN
• Repeated administration of dexamethasone increases phosphoinositide-specific phospholipase C activity and mRNA and protein expression of the phospholipase C beta 1 isozyme in rat brain.
• J Neurochem. 1999; 73: 780-90
• Display abstract

• Altered hypothalamic-pituitary-adrenal (HPA) function has been shown to be associated with changes in mood and behavior. The enzyme phosphoinositide-specific phospholipase C (PI-PLC), an important component of the PI signal transduction system, plays a major role in mediating various physiological functions. In the present study, we investigated the effects of a single dose and of repeated administration (0.5 or 1.0 mg/kg for 10 days) of dexamethasone (DEX), a synthetic glucocorticoid, on PI-PLC activity and on expression of PLC isozymes (beta1, delta1, and gamma1) in rat brain. Repeated administration of DEX (1.0 mg/kg) caused a significant increase in PI-PLC activity and in protein expression of the PLC beta1 isozyme in both membrane and cytosol fractions of cortex and hippocampus; however, the repeated administration of a smaller dose of DEX (0.5 mg/kg) caused these changes only in hippocampus but not in cortex. The increase in PLC beta1 protein was associated with an increase in its mRNA level, as measured by competitive RT-PCR. A single administration of DEX (0.5 or 1.0 mg/kg) to rats had no significant effects on PI-PLC activity or on the protein expression of PLC isozymes. These results suggest that DEX up-regulates PI-PLC in rat brain, which presumably is due to a selective increase in expression of the PLC beta1 isozyme, and that these changes in PI-PLC may be related to HPA axis-mediated changes in mood and behavior.

• Griffith OH, Ryan M
• Bacterial phosphatidylinositol-specific phospholipase C: structure, function, and interaction with lipids.
• Biochim Biophys Acta. 1999; 1441: 237-54
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• The bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) is a small, water-soluble enzyme that cleaves the natural membrane lipids PI, lyso-PI, and glycosyl-PI. The crystal structure, NMR and enzymatic mechanism of bacterial PI-PLCs are reviewed. These enzymes consist of a single domain folded as a (betaalpha)(8)-barrel (TIM barrel), are calcium-independent, and interact weakly with membranes. Sequence similarity among PI-PLCs from different bacterial species is extensive, and includes the residues involved in catalysis. Bacterial PI-PLCs are structurally similar to the catalytic domain of mammalian PI-PLCs. Comparative studies of both prokaryotic and eukaryotic isozymes have proved useful for the identification of distinct regions of the proteins that are structurally and functionally important.

• Williams BL et al.
• Phosphorylation of Tyr319 in ZAP-70 is required for T-cell antigen receptor-dependent phospholipase C-gamma1 and Ras activation.
• EMBO J. 1999; 18: 1832-44
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• Accumulating evidence indicates that the interdomain B regions of ZAP-70 and Syk play pivotal roles in the coupling of T-cell antigen receptor (TCR) stimulation to the activation of downstream signaling pathways. The interdomain B region of ZAP-70 contains at least three candidate sites of tyrosine phosphorylation. In this report, we identify Tyr319 as a functionally important phosphorylation site in the ZAP-70 interdomain B region. TCR crosslinkage triggered a rapid increase in the phosphorylation of Tyr319 in Jurkat T cells. Although mutation of Tyr319 to Phe had no effect on the tyrosine kinase activity of ZAP-70, the resulting ZAP(Y319-->F) mutant failed to reconstitute TCR-dependent Ca2+ mobilization, Ras activation, CD69 expression and NFAT-dependent transcription in ZAP-70-deficient Jurkat cells. These defects were correlated with reduced tyrosine phosphorylation of phospholipase C (PLC)-gamma1 and the LAT adapter protein in the ZAP(Y319-->F)-expressing cells. On the other hand, ZAP(Y319-->F)-expressing cells displayed normal increases in SLP-76 phosphorylation and ERK activation during TCR stimulation. Phosphorylation of Tyr319 promoted the association of ZAP-70 with the SH2 domains of two key signaling molecules, Lck and PLC-gamma1. These studies suggest that Tyr319 phosphorylation is required for the assembly of a ZAP-70-containing signaling complex that leads to the activation of the PLC-gamma1- and Ras-dependent signaling cascades in antigen-stimulated T cells.

• Harris TK et al.
• Kinetic, stereochemical, and structural effects of mutations of the active site arginine residues in 4-oxalocrotonate tautomerase.
• Biochemistry. 1999; 38: 12343-57
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• Three arginine residues (Arg-11, Arg-39, Arg-61) are found at the active site of 4-oxalocrotonate tautomerase in the X-ray structure of the affinity-labeled enzyme [Taylor, A. B., Czerwinski, R. M., Johnson, R. M., Jr., Whitman, C. P., and Hackert, M. L. (1998) Biochemistry 37, 14692-14700]. The catalytic roles of these arginines were examined by mutagenesis, kinetic, and heteronuclear NMR studies. With a 1,6-dicarboxylate substrate (2-hydroxymuconate), the R61A mutation showed no kinetic effects, while the R11A mutation decreased k(cat) 88-fold and increased K(m) 8.6-fold, suggesting both binding and catalytic roles for Arg-11. With a 1-monocarboxylate substrate (2-hydroxy-2,4-pentadienoate), no kinetic effects of the R11A mutation were found, indicating that Arg-11 interacts with the 6-carboxylate of the substrate. The stereoselectivity of the R11A-catalyzed protonation at C-5 of the dicarboxylate substrate decreased, while the stereoselectivity of protonation at C-3 of the monocarboxylate substrate increased in comparison with wild-type 4-OT, indicating the importance of Arg-11 in properly orienting the dicarboxylate substrate by interacting with the charged 6-carboxylate group. With 2-hydroxymuconate, the R39A and R39Q mutations decreased k(cat) by 125- and 389-fold and increased K(m) by 1.5- and 2.6-fold, respectively, suggesting a largely catalytic role for Arg-39. The activity of the R11A/R39A double mutant was at least 10(4)-fold lower than that of the wild-type enzyme, indicating approximate additivity of the effects of the two arginine mutants on k(cat). For both R11A and R39Q, 2D (1)H-(15)N HSQC and 3D (1)H-(15)N NOESY-HSQC spectra showed chemical shift changes mainly near the mutated residues, indicating otherwise intact protein structures. The changes in the R39Q mutant were mainly in the beta-hairpin from residues 50 to 57 which covers the active site. HSQC titration of R11A with the substrate analogue cis, cis-muconate yielded a K(d) of 22 mM, 37-fold greater than the K(d) found with wild-type 4-OT (0.6 mM). With the R39Q mutant, cis, cis-muconate showed negative cooperativity in active site binding with two K(d) values, 3.5 and 29 mM. This observation together with the low K(m) of 2-hydroxymuconate (0.47 mM) suggests that only the tight binding sites function catalytically in the R39Q mutant. The (15)Nepsilon resonances of all six Arg residues of 4-OT were assigned, and the assignments of Arg-11, -39, and -61 were confirmed by mutagenesis. The binding of cis,cis-muconate to wild-type 4-OT upshifts Arg-11 Nepsilon (by 0.05 ppm) and downshifts Arg-39 Nepsilon (by 1.19 ppm), indicating differing electronic delocalizations in the guanidinium groups. A mechanism is proposed in which Arg-11 interacts with the 6-carboxylate of the substrate to facilitate both substrate binding and catalysis and Arg-39 interacts with the 1-carboxylate and the 2-keto group of the substrate to promote carbonyl polarization and catalysis, while Pro-1 transfers protons from C-3 to C-5. This mechanism, together with the effects of mutations of catalytic residues on k(cat), provides a quantitative explanation of the 10(7)-fold catalytic power of 4-OT. Despite its presence in the active site in the crystal structure of the affinity-labeled enzyme, Arg-61 does not play a significant role in either substrate binding or catalysis.

• Pawelczyk T
• [Signal transduction in the cell by phosphoinositide-specific phospholipase C isozymes]
• Postepy Hig Med Dosw. 1999; 53: 173-82
• Display abstract

• This article reviews recent studies on phosphoinositide-specific phospholipase C species named beta, gamma and delta. The localization of PLC isozymes in the cell, structural organization, regulation of PLC activity and the interaction with various receptors is presented.

• Ronnstrand L, Siegbahn A, Rorsman C, Johnell M, Hansen K, Heldin CH
• Overactivation of phospholipase C-gamma1 renders platelet-derived growth factor beta-receptor-expressing cells independent of the phosphatidylinositol 3-kinase pathway for chemotaxis.
• J Biol Chem. 1999; 274: 22089-94
• Display abstract

• We have previously shown that porcine aortic endothelial cells expressing the Y934F platelet-derived growth factor (PDGF) beta-receptor mutant respond to PDGF-BB in a chemotaxis assay at about 100-fold lower concentration than do wild-type PDGF beta-receptor-expressing cells (Hansen, K., Johnell, M., Siegbahn, A. , Rorsman, C., Engstrom, U., Wernstedt, C., Heldin, C.-H., and Ronnstrand, L. (1996) EMBO J. 15, 5299-5313). Here we show that the increased chemotaxis correlates with increased activation of phospholipase C-gamma1 (PLC-gamma1), measured as inositol-1,4, 5-trisphosphate release. By two-dimensional phosphopeptide mapping, the increase in phosphorylation of PLC-gamma1 was shown not to be selective for any site, rather a general increase in phosphorylation of PLC-gamma1 was seen. Specific inhibitors of protein kinase C, bisindolylmaleimide (GF109203X), and phosphatidylinositol 3-kinase (PI3-kinase), LY294002, did not affect the activation of PLC-gamma1. To assess whether increased activation of PLC-gamma1 is the cause of the hyperchemotactic behavior of the Y934F mutant cell line, we constructed cell lines expressing either wild-type or a catalytically compromised version of PLC-gamma1 under a tetracycline-inducible promoter. Overexpression and concomitant increased activation of wild-type PLC-gamma1 in response to PDGF-BB led to a hyperchemotactic behavior of the cells, while the catalytically compromised PLC-gamma1 mutant had no effect on PDGF-BB-induced chemotaxis. Furthermore, in cells expressing normal levels of PLC-gamma1, chemotaxis was inhibited by LY294002. In contrast, the increase in chemotactic response seen upon overexpression of PLC-gamma1 was not inhibited by the PI3-kinase inhibitor LY294002. These observations suggest the existence of two different pathways which mediate PDGF-induced chemotaxis; depending on the cellular context, the PI3-kinase pathway or the PLC-gamma1 pathway may dominate.

• Sekiya F, Bae YS, Rhee SG
• Regulation of phospholipase C isozymes: activation of phospholipase C-gamma in the absence of tyrosine-phosphorylation.
• Chem Phys Lipids. 1999; 98: 3-11
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• Activation of PLC-gamma isozymes in response to various agonists involves tyrosine phosphorylation of the effector enzymes. Recent evidence indicates that PLC-gamma isozymes are additionally activated by phosphatidic acid, phosphatidylinositol 3,4,5-trisphosphate and arachidonic acid in the absence of PLC-gamma tyrosine phosphorylation. These lipid-derived messengers are the immediate products of phospholipase D, phosphatidylinositol 3-kinase, and phospholipase A2, enzymes which are often stimulated along with PLC-gamma in response to an agonist. Furthermore, phosphatidylinositol 4,5-bisphosphate acts as a substrate for both PLC-gamma and phosphatidylinositol 3-kinase and as an activator for phospholipase D and phospholipase A2. These results reveal an elaborate mechanism of cross-talk and mutual regulation between four effector enzymes that participate in receptor signaling by acting on phospholipids.

• Dwivedi Y, Pandey GN
• Effects of treatment with haloperidol, chlorpromazine, and clozapine on protein kinase C (PKC) and phosphoinositide-specific phospholipase C (PI-PLC) activity and on mRNA and protein expression of PKC and PLC isozymes in rat brain.
• J Pharmacol Exp Ther. 1999; 291: 688-704
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• The effects of acute (single) and chronic (21-day) administration of haloperidol (HAL), chlorpromazine (CPZ), or clozapine (CLOZ) on components of the phosphoinositide (PI)-signaling pathway were studied in rat brain. Chronic administration of HAL decreased protein kinase C (PKC) activity and mRNA and protein levels of PKC alpha and epsilon isozymes in both membrane and cytosol fractions of cortex, hippocampus, and striatum. Chronic administration of CPZ, however, decreased PKC activity only in the membrane fraction of cortex, hippocampus, and striatum, and had no effect on the levels of any PKC isozymes. On the other hand, chronic administration of CLOZ decreased PKC activity and mRNA and protein levels of PKC alpha, gamma, and epsilon isozymes in membrane and cytosol fractions of cortex, hippocampus, and cerebellum. Studies of the effects on phospholipase C (PLC) revealed that only chronic administration of CPZ significantly decreased PI-PLC activity and mRNA and protein levels of the specific PLC beta(1) isozyme in membrane and cytosol fractions of cortex, hippocampus, cerebellum, and striatum. Acute-treatment data suggest that CPZ or CLOZ had no significant effects on PI-PLC or PKC; however, HAL translocated PKC, as evidenced from increased PKC activity and protein levels of PKC alpha and epsilon isozymes in the membrane fraction and the decrease in these parameters in the cytosol fraction of cortex, hippocampus, and striatum. Our results thus suggest that the interaction of antipsychotic drugs with PKC and PLC may be associated with their mechanisms of action.

• Wang T, Pentyala S, Rebecchi MJ, Scarlata S
• Differential association of the pleckstrin homology domains of phospholipases C-beta 1, C-beta 2, and C-delta 1 with lipid bilayers and the beta gamma subunits of heterotrimeric G proteins.
• Biochemistry. 1999; 38: 1517-24
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• Pleckstrin homology (PH) domains are recognized in more than 100 different proteins, including mammalian phosphoinositide-specific phospholipase C (PLC) isozymes (isotypes beta, gamma, and delta). These structural motifs are thought to function as tethering devices linking their host proteins to membranes containing phosphoinositides or beta gamma subunits of heterotrimeric GTP binding (G) proteins. Although the PH domains of PLC-delta and PLC-gamma have been studied, the comparable domains of the beta isotypes have not. Here, we have measured the affinities of the isolated PH domains of PLC-beta 1 and -beta 2 (PH-beta 1 and PH-beta 2, respectively) for lipid bilayers and G-beta gamma subunits. Like the intact enzymes, these PH domains bind to membrane surfaces composed of zwitterionic phosphatidylcholine with moderate affinity. Inclusion of the anionic lipid phosphatidylserine or phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and inclusion of G-beta gamma subunits had little affect on their membrane affinity. In contrast, binding of PLC-delta 1 or its PH domain was highly dependent on PI(4,5)P2. We also determined whether these domains laterally associate with G-beta gamma subunits bound to membrane surfaces using fluorescence resonance energy transfer. Affinities for G-beta gamma were in the following order: PH-beta 2 >/= PH-beta 1 > PH-delta 1; the affinities of the native enzyme were as follows: PLC-beta 2 >> PLC-delta 1 > PLC-beta 1. Thus, the PH domain of PLC-beta 1 interacts with G-beta gamma in isolation, but not in the context of the native enzyme. By contrast, docking of the PH domain of PLC-beta2 with G-beta gamma is comparable to that of the full-length protein and may play a key role in G-beta gamma recognition.

• Williams RL
• Mammalian phosphoinositide-specific phospholipase C.
• Biochim Biophys Acta. 1999; 1441: 255-67
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• Mammalian phosphoinositide-specific phospholipases C (PI-PLCs) are involved in most receptor-mediated signal transduction pathways. The mammalian isozymes employ a modular arrangement of domains to achieve a regulated production of two key second messengers. The roles of the PH, EF hand, C2, SH2 and SH3 modules in regulation of these enzymes and in interactions with membranes and other proteins is becoming apparent from recent structural and functional studies.

• Lomasney JW, Cheng HF, Roffler SR, King K
• Activation of phospholipase C delta1 through C2 domain by a Ca(2+)-enzyme-phosphatidylserine ternary complex.
• J Biol Chem. 1999; 274: 21995-2001
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• The concentration of free Ca(2+) and the composition of nonsubstrate phospholipids profoundly affect the activity of phospholipase C delta1 (PLCdelta1). The rate of PLCdelta1 hydrolysis of phosphatidylinositol 4,5-bisphosphate was stimulated 20-fold by phosphatidylserine (PS), 4-fold by phosphatidic acid (PA), and not at all by phosphatidylethanolamine or phosphatidylcholine (PC). PS reduced the Ca(2+) concentration required for half-maximal activation of PLCdelta1 from 5.4 to 0.5 microM. In the presence of Ca(2+), PLCdelta1 specifically bound to PS/PC but not to PA/PC vesicles in a dose-dependent and saturable manner. Ca(2+) also bound to PLCdelta1 and required the presence of PS/PC vesicles but not PA/PC vesicles. The free Ca(2+) concentration required for half-maximal Ca(2+) binding was estimated to be 8 microM. Surface dilution kinetic analysis revealed that the K(m) was reduced 20-fold by the presence of 25 mol % PS, whereas V(max) and K(d) were unaffected. Deletion of amino acid residues 646-654 from the C2 domain of PLCdelta1 impaired Ca(2+) binding and reduced its stimulation and binding by PS. Taken together, the results suggest that the formation of an enzyme-Ca(2+)-PS ternary complex through the C2 domain increases the affinity for substrate and consequently leads to enzyme activation.

• Yamamoto T et al.
• Involvement of EF hand motifs in the Ca(2+)-dependent binding of the pleckstrin homology domain to phosphoinositides.
• Eur J Biochem. 1999; 265: 481-90
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• The pleckstrin homology (PH) domains of phospholipase C (PLC)-delta1 and a related catalytically inactive protein, p130, both bind inositol phosphates and inositol lipids. The binding to phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] by PLC-delta1 is proposed to be the critical interaction required for membrane localization to where the substrate resides; it is also required for the Ca(2+)-dependent activation of PLC-delta1 observed in the permeabilized cells. In the proximity of the PH domain, both PLC-delta1 and p130 possess the EF-hand domain, containing classical motifs implicated in calcium binding. Therefore, in the present study we examined whether the binding of the PH domain to PtdIns(4,5)P2 is regulated by changes in free Ca2+ concentration within the physiological range. A Ca2+ dependent increase in the binding to PtdIns(4,5)P2 was observed with a full-length PLC-delta1, while the isolated PH domain did not show any Ca2+ dependence. However, the connection of the EF-hand motifs to the PH domain restored the Ca2+ dependent increase in binding, even in the absence of the C2 domain. The p130 protein showed similar properties to PLC-delta1, and the EF-hand motifs were again required for the PH domain to exhibit a Ca2+ dependent increase in the binding to PtdIns(4,5)P2. The isolated PH domains from several other proteins which have been demonstrated to bind PtdIns(4,5)P2 showed no Ca2+ dependent enhancement of binding. However, when present within a chimera also containing PLC-delta1 EF-hand motifs, the Ca2+ dependent binding was again observed. These results suggest that the binding of Ca2+ to the EF-hand motifs can modulate binding to PtdIns(4,5)P2 mediated by the PH domain.

• Strosznajder JB, Zambrzycka A, Kacprzak MD, Strosznajder RP
• Amyloid beta peptide 25-35 modulates hydrolysis of phosphoinositides by membrane phospholipase(s) C of adult brain cortex.
• J Mol Neurosci. 1999; 12: 101-9
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• Phosphoinositide-specific phospholipase C (PLC) is a key enzyme in signal transduction. A subset of muscarinic cholinergic receptors are linked to G-proteins that activate phospholipase C. Cholinergic pathways are important in learning and memory, and deficits in cholinergic transmission have been implicated in Alzheimer's disease (AD). AD is also associated with increased beta-amyloid plaques. In the present study, we have investigated the effect of the amyloid beta (A beta) synthetic peptide homologous to residue 25-35 of A beta in nonaggregated and aggregated forms on the degradation of inositol phospholipids. Synaptic plasma membranes (SPM) and the cytosolic fraction from rat brain cortex served as a source of enzymes. The studies were carried out with radioactive inositol phospholipids in the presence of endogenous and 2 mM CaCl2. The enzyme(s) activity was evaluated by determination of the product formation of [3H]inositol-1-phosphate (IP1) or [3H]inositol-1,4,5-trisphosphate (IP3). Results show that the PI-PLC activity was significantly higher in cytosol compared to SPM, and this enzyme was stimulated by 2 mM CaCl2, but not by GTPgammaS or carbachol, a cholinergic receptor agonist. Activity of the SPM-bound PIP2-PLC was similar to that in cytosol and was not activated by 2 mM CaCl2. The SPM PIP2-PLC was significantly stimulated by GTPgammaS together with the cholinergic agonist, carbachol. Fresh-water-soluble A beta 25-35 activated PI-PLC in SPM markedly by two- to threefold, but this effect was absent in the presence of 2 mM CaCl2. Moreover, A beta 25-35 had no effect on basal PIP2-PLC activity and cytosolic PI-PLC and PIP2-PLC. The aggregated form of A beta 25-35 significantly inhibited PIP2-PLC only in the presence of endogenous CaCl2. It also inhibited the carbachol and GTP(gamma)S-stimulated PIP2-PLC. Our findings show that depending on the aggregation state and Ca2+ concentration, A beta modulates phosphoinositide degradation differently and exclusively in brain synaptic plasma membranes. Our data suggested that aggregated A beta peptide may be responsible for the significant impairment of phosphoinositide signaling found in brain membranes during AD.

• Zhou C, Horstman D, Carpenter G, Roberts MF
• Action of phosphatidylinositol-specific phospholipase Cgamma1 on soluble and micellar substrates. Separating effects on catalysis from modulation of the surface.
• J Biol Chem. 1999; 274: 2786-93
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• The kinetics of PI-PLCgamma1 toward a water-soluble substrate (inositol 1,2-cyclic phosphate, cIP) and phosphatidylinositol (PI) in detergent mixed micelles were monitored by 31P NMR spectroscopy. That cIP is also a substrate (Km = approximately 15 mM) implies a two-step mechanism (intramolecular phosphotransferase reaction to form cIP followed by cyclic phosphodiesterase activity to form inositol-1-phosphate (I-1-P)). PI is cleaved by PI-PLCgamma1 to form cIP and I-1-P with the enzyme specific activity and ratio of products (cIP/I-1-P) regulated by assay temperature, pH, Ca2+, and other amphiphilic additives. Cleavage of both cIP and PI by the enzyme is optimal at pH 5. The effect of Ca2+ on PI-PLCgamma1 activity is unique compared with other isozymes enzymes: Ca2+ is necessary for the activity and low Ca2+ activates the enzyme; however, high Ca2+ inhibits PI-PLCgamma1 hydrolysis of phosphoinositides (but not cIP) with the extent of inhibition dependent on pH, substrate identity (cIP or PI), substrate presentation (e.g. detergent matrix), and substrate surface concentration. This inhibition of PI-PLCgamma1 by high Ca2+ is proposed to derive from the divalent metal ion-inducing clustering of the PI and reducing its accessibility to the enzyme. Amphiphilic additives such as phosphatidic acid, fatty acid, and sodium dodecylsulfate enhance PI cleavage in micelles at pH 7.5 but not at pH 5.0; they have no effect on cIP hydrolysis at either pH value. These different kinetic patterns are used to propose a model for regulation of the enzyme. A key hypothesis is that there is a pH-dependent conformational change in the enzyme that controls accessibility of the active site to both water-soluble cIP and interfacially organized PI. The low activity enzyme at pH 7.5 can be activated by PA (or phosphorylation by tyrosine kinase). However, this activation requires lipophilic substrate (PI) present because cIP hydrolysis is not enhanced in the presence of PA.

• Pandey GN et al.
• Low phosphoinositide-specific phospholipase C activity and expression of phospholipase C beta1 protein in the prefrontal cortex of teenage suicide subjects.
• Am J Psychiatry. 1999; 156: 1895-901
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• OBJECTIVE: The enzyme phosphoinositide-specific phospholipase C (PI-PLC) is a component of the phosphoinositide signal transduction system. Other components of this system have been found to be abnormal in adults and adolescents who have committed suicide, and so the authors examined whether PI-PLC activity and protein expression of PLC isozymes are abnormal in postmortem brains of teenage suicide subjects. METHOD: PI-PLC activity and protein expression of the PLC beta1, delta1, and gamma1 isozymes were examined in Brodmann's areas 8 and 9 of postmortem brains obtained from 18 teenage suicide subjects and 18 matched comparison subjects. PI-PLC activity was determined by enzymatic assay, and protein expression of the PLC isozymes was determined by the Western blot technique. RESULTS: Compared with the normal subjects, the teenage suicide subjects had significantly lower PI-PLC activity and immunolabeling of the specific PLC beta1 isozyme in both membrane and cytosol fractions of Brodmann's areas 8 and 9 combined (prefrontal cortex). There was also a significant correlation between PI-PLC activity and protein levels of the PLC beta1 isozyme in the brains of the teenage suicide subjects. There was no significant difference in PI-PLC activity or level of PLC beta1 protein between the suicide subjects with a history of mental disorders and those with no history of mental disorders; however, both groups had significantly lower PI-PLC activity and expression of PLC beta1 protein than the normal subjects. CONCLUSIONS: Low PI-PLC activity and expressed levels of the PLC beta1 isozyme in postmortem brains of suicide subjects may have clinical relevance in the pathophysiology of suicidal behavior.

• Pawelczyk T
• Isozymes delta of phosphoinositide-specific phospholipase C.
• Acta Biochim Pol. 1999; 46: 91-8
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• Phospholipase C (PLC, EC 3.1.4.11) is the major starting point in the phosphatidylinositol pathway, which generates intracellular signals that regulate protein kinase C and intracellular calcium concentration. To date, three major types of phosphoinositide-specific PLC species named beta, gamma and delta, have been characterized. This article reviews recent studies on isozymes delta of PLC. Four such isozymes have been cloned and termed delta1-4. Their structural organization, regulation of activity and the interaction with membrane lipid are considered. The intracellular localization of delta isozymes and distribution in various tissues are presented. Attention is given to the pathological conditions in which an abnormal protein level of PLC delta or its activity have been observed.

• Ching TT, Wang DS, Hsu AL, Lu PJ, Chen CS
• Identification of multiple phosphoinositide-specific phospholipases D as new regulatory enzymes for phosphatidylinositol 3,4, 5-trisphosphate.
• J Biol Chem. 1999; 274: 8611-7
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• In the course of delineating the regulatory mechanism underlying phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) metabolism, we have discovered three distinct phosphoinositide-specific phospholipase D (PI-PLD) isozymes from rat brain, tentatively designated as PI-PLDa, PI-PLDb, and PI-PLDc. These enzymes convert [3H]PI(3,4,5)P3 to generate a novel inositol phosphate, D-myo-[3H]inositol 3,4,5-trisphosphate ([3H]Ins(3,4,5)P3) and phosphatidic acid. These isozymes are predominantly associated with the cytosol, a notable difference from phosphatidylcholine PLDs. They are partially purified by a three-step procedure consisting of DEAE, heparin, and Sephacryl S-200 chromatography. PI-PLDa and PI-PLDb display a high degree of substrate specificity for PI(3,4, 5)P3, with a relative potency of PI(3,4,5)P3 >> phosphatidylinositol 3-phosphate (PI(3)P) or phosphatidylinositol 4-phosphate (PI(4)P) > phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) > phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2). In contrast, PI-PLDc preferentially utilizes PI(3)P as substrate, followed by, in sequence, PI(3,4,5)P3, PI(4)P, PI(3,4)P2, and PI(4,5)P2. Both PI(3, 4)P2 and PI(4,5)P2 are poor substrates for all three isozymes, indicating that the regulatory mechanisms underlying these phosphoinositides are different from that of PI(3,4,5)P3. None of these enzymes reacts with phosphatidylcholine, phosphatidylserine, or phosphatidylethanolamine. All three PI-PLDs are Ca2+-dependent. Among them, PI-PLDb and PI-PLDc show maximum activities within a sub-microM range (0.3 and 0.9 microM Ca2+, respectively), whereas PI-PLDa exhibits an optimal [Ca2+] at 20 microM. In contrast to PC-PLD, Mg2+ has no significant effect on the enzyme activity. All three enzymes require sodium deoxycholate for optimal activities; other detergents examined including Triton X-100 and Nonidet P-40 are, however, inhibitory. In addition, PI(4,5)P2 stimulates these isozymes in a dose-dependent manner. Enhancement in the enzyme activity is noted only when the molar ratio of PI(4,5)P2 to PI(3,4, 5)P3 is between 1:1 and 2:1.

• Iwasaki Y et al.
• Two distinct phosphatidylinositol-specific phospholipase Cs from Streptomyces antibioticus.
• Biochim Biophys Acta. 1998; 1391: 52-66
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• Two phosphatidylinositol-specific phospholipase C (PI-PLC) genes from Streptomyces antibioticus were cloned by a shotgun method using Streptomyces lividans TK24 as a host. The genes of the two PI-PLCs (named as PLC1 and PLC2) were adjoined and opposite in the direction of transcription/translation. Both of them were confirmed to be expressed in S. antibioticus. The two enzymes were different in the following properties. (i) PLC2 had considerable sequence similarity to other bacterial PI-PLCs, while PLC1 had a short stretch that was similar to PI-PLCs of eukaryotes rather than the other bacterial enzymes. (ii) PLC1 was Ca2+-dependent, whereas PLC2 was not. (iii) PLC1 generated myo-inositol-1-phosphate and myo-inositol-1:2-cyclic phosphate simultaneously from PI, but PLC2 showed sequential formation of them. (iv) PLC2 has GPI-anchor-degrading activity while PLC1 does not have. Both enzymes did not hydrolyze phosphatidylcholine, phosphatidylinositol-4-monophosphate and phosphatidylinositol-4,5-bisphosphate. Both PLC1 and PLC2 contained two histidine residues that might be catalytic residues. PLC1 has residues that possibly form a Ca2+-binding site. Then it was suggested that both PLC1 and PLC2 act according to the catalytic mechanism using the two histidine residues as proposed in both eukaryotic and prokaryotic enzymes, but that PLC1 has a more 'eukaryotic' mechanism in which Ca2+ participates than that of the Ca2+-independent bacterial enzymes. Thus, we propose that PLC2 is a conventional 'bacteria-type' enzyme, while PLC1 is more closely related to the eukaryotic enzymes rather than the bacterial enzymes.

• Kopka J, Pical C, Gray JE, Muller-Rober B
• Molecular and enzymatic characterization of three phosphoinositide-specific phospholipase C isoforms from potato.
• Plant Physiol. 1998; 116: 239-50
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• Many cellular responses to stimulation of cell-surface receptors by extracellular signals are transmitted across the plasma membrane by hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2), which is cleaved into diacylglycerol and inositol-1,4,5-trisphosphate by phosphoinositide-specific phospholipase C (PI-PLC). We present structural, biochemical, and RNA expression data for three distinct PI-PLC isoforms, StPLC1, StPLC2, and StPLC3, which were cloned from a guard cell-enriched tissue preparation of potato (Solanum tuberosum) leaves. All three enzymes contain the catalytic X and Y domains, as well as C2-like domains also present in all PI-PLCs. Analysis of the reaction products obtained from PIP2 hydrolysis unequivocally identified these enzymes as genuine PI-PLC isoforms. Recombinant StPLCs showed an optimal PIP2-hydrolyzing activity at 10 microM Ca2+ and were inhibited by Al3+ in equimolar amounts. In contrast to PI-PLC activity in plant plasma membranes, however, recombinant enzymes could not be activated by Mg2+. All three stplc genes are expressed in various tissues of potato, including leaves, flowers, tubers, and roots, and are affected by drought stress in a gene-specific manner.

• Huwiler A, Fabbro D, Pfeilschifter J
• Selective ceramide binding to protein kinase C-alpha and -delta isoenzymes in renal mesangial cells.
• Biochemistry. 1998; 37: 14556-62
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• Ceramide is an important lipid second messenger produced by sphingolipid metabolism in cells exposed to a limited number of agonists and in turn triggers several cell responses in a protein kinase C (PKC)-dependent manner. Stimulation of mesangial cells with a radioiodinated photoaffinity labeling analogue of ceramide, (N-[3-[[[2-(125I)iodo-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benz yl] oxy]carbonyl]propanoyl]-D-erythro-sphingosine) ([125I]-TID-ceramide), defines PKC-alpha and PKC-delta as direct targets of ceramide. No binding of ceramide to PKC-epsilon and PKC-zeta could be detected. Moreover, TID-ceramide selectively binds to recombinant PKC-alpha and -delta but not to PKC-epsilon and -zeta isoenzymes. In vitro kinase activity assays reveal that only the binding of ceramide to PKC-alpha is accompanied by an increase in kinase activity. In contrast, there is no change in in vitro kinase activity of the other isoforms tested, i.e., PKC-delta, -epsilon, and -zeta, toward any of the conventional substrates tested. However, it is noteworthy that PKC-delta shows a decreased autophosphorylation upon ceramide binding. In vivo, activation of PKC-alpha by ceramide is monitored by a delayed translocation of the isoform from the cytosol to the membrane fraction, detectable after 1 h of stimulation. In contrast, neither PKC-delta, nor -epsilon nor -zeta is redistributed by ceramide. One functional cell response mediated by PKC-alpha in mesangial cells is a negative feedback regulation of ligand-stimulated phosphoinositide hydrolysis. When cells are pretreated with ceramide, ATP-induced inositol trisphosphate formation is time-dependently reduced. A maximal inhibition is observed after 2 h of ceramide exposure. In summary, these results suggest that ceramide selectively interacts with the alpha- and delta-isoforms of PKC in mesangial cells. Whereas PKC-alpha is activated with pronounced inhibition of hormone-stimulated phosphoinositide signaling, PKC-delta displays a decrease in its autophosphorylation, suggesting a negative role of ceramide binding on PKC-delta activity.

• Grobler JA, Hurley JH
• Catalysis by phospholipase C delta1 requires that Ca2+ bind to the catalytic domain, but not the C2 domain.
• Biochemistry. 1998; 37: 5020-8
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• The hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphoinositide-specific phospholipase C (PLC) is absolutely dependent on Ca2+. The PH domain truncated catalytic core of rat phospholipase C delta1 (PLC-delta1) has Ca2+ binding sites in its catalytic and C2 domains, and potential Ca2+ binding sites in two EF-hands. A catalytically inactive PLC-delta1 catalytic core bound with low affinity to PIP2-containing vesicles in the presence of Ca2+. A mutant PLC-delta1 has been engineered which lacks the C2 domain Ca2+ binding site and the surrounding loops known as the jaws. Isothermal calorimetric titration showed four Ca2+ ions bind to the wild-type PLC-delta1 catalytic core in solution but only one binds to the C2 domain jaws deletion mutant. The activity and Ca2+ dependence of wild-type and mutant phospholipase Cs were determined using substrate incorporated in detergent micelles and in large unilamellar vesicles. The activities of wild-type and mutant were identical to each other in both assay systems. Wild-type and the C2 jaws deletion mutant of PLC have Hill coefficients of 1.12-1.16 with respect to [Ca2+]. We conclude that a single Ca2+ bound to the catalytic domain is entirely responsible for the Ca2+ dependence of the basal activity of PLC-delta1.

• Hondal RJ et al.
• Mechanism of phosphatidylinositol-specific phospholipase C: a unified view of the mechanism of catalysis.
• Biochemistry. 1998; 37: 4568-80
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• The mechanism of phosphatidylinositol-specific phospholipase C (PI-PLC) has been suggested to resemble that of ribonuclease A. The goal of this work is to rigorously evaluate the mechanism of PI-PLC from Bacillus thuringiensis by examining the functional and structural roles of His-32 and His-82, along with the two nearby residues Asp-274 and Asp-33 (which form a hydrogen bond with His-32 and His-82, respectively), using site-directed mutagenesis. In all, twelve mutants were constructed, which, except D274E, showed little structural perturbation on the basis of 1D NMR and 2D NOESY analyses. The H32A, H32N, H32Q, H82A, H82N, H82Q, H82D, and D274A mutants showed a 10(4)-10(5)-fold decrease in specific activity toward phosphatidylinositol; the D274N, D33A, and D33N mutants retained 0. 1-1% activity, whereas the D274E mutant retained 13% activity. Steady-state kinetic analysis of mutants using (2R)-1, 2-dipalmitoyloxypropane-3-(thiophospho-1d-myo-inositol) (DPsPI) as a substrate generally agreed well with the specific activity toward phosphatidylinositol. The results suggest a mechanism in which His-32 functions as a general base to abstract the proton from 2-OH and facilitates the attack of the deprotonated 2-oxygen on the phosphorus atom. This general base function is augmented by the carboxylate group of Asp-274 which forms a diad with His-32. The H82A and D33A mutants showed an unusually high activity with substrates featuring low pKa leaving groups, such as DPsPI and p-nitrophenyl inositol phosphate (NPIPs). These results suggest that His-82 functions as the general acid with assistance from Asp-33, facilitating the departure of the leaving group by protonation of the glycerol O3 oxygen. The Bronsted coefficients obtained for the WT and the D33N mutant indicate a high degree of proton transfer to the leaving group and further underscore the "helper" function of Asp-33. The complete mechanism also includes activation of the phosphate group toward nucleophilic attack by a hydrogen bond between Arg-69 and a nonbridging oxygen atom. The overall mechanism can be described as "complex" general acid-general base since three elements are required for efficient catalysis.

• Yagisawa H et al.
• Replacements of single basic amino acids in the pleckstrin homology domain of phospholipase C-delta1 alter the ligand binding, phospholipase activity, and interaction with the plasma membrane.
• J Biol Chem. 1998; 273: 417-24
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• The pleckstrin homology (PH) domain of phosphatidylinositol-specific phospholipase C-delta1 (PLC-delta1) binds to both D-myo-inositol 1,4, 5-trisphosphate (Ins(1,4,5)P3) and phosphatidylinositol 4, 5-bisphosphate (PtdIns(4,5)P2) with high affinities. We have previously identified a region rich in basic amino acids within the PH domain critical for ligand binding (Yagisawa, H., Hirata, M., Kanematsu, T., Watanabe, Y., Ozaki, S., Sakuma, K., Tanaka, H., Yabuta, N., Kamata, H., Hirata, H., and Nojima, H. (1994) J. Biol. Chem. 269, 20179-20188; Hirata, M., Kanematsu, T., Sakuma, K., Koga, T., Watanabe, Y., Ozaki, S., and Yagisawa, H. (1994) Biochem. Biophys. Res. Commun. 205, 1563-1571). To investigate the role of these basic residues, we have performed site-directed mutagenesis replacing each of the basic amino acid in the N-terminal 60 residues of PLC-delta1 (Lys24, Lys30, Lys32, Arg37, Arg38, Arg40, Lys43, Lys49, Arg56, Lys57, and Arg60) with a neutral or an acidic amino acid. The effects of these mutations on the PH domain ligand binding properties and their consequence for substrate hydrolysis and membrane interactions of PLC-delta1 were analyzed using several assay systems. Analysis of [3H]-Ins(1,4,5)P3 binding, measurement of the binding affinities, and measurements of phospholipase activity using PtdIns(4,5)P2-containing phospholipid vesicles, demonstrated that residues Lys30, Lys32, Arg37, Arg38, Arg40, and Lys57 were required for these PLC-delta1 functions; in comparison, other mutations resulted in a moderate reduction. A subset of selected mutations was further analyzed for the enzyme activity toward substrate present in cellular membranes of permeabilized cells and for interaction with the plasma membrane after microinjection. These experiments demonstrated that mutations affecting ligand binding and PtdIns(4,5)P2 hydrolysis in phospholipid vesicles also resulted in reduction in the hydrolysis of cellular polyphosphoinositides and loss of membrane attachment. All residues (with the exception of the K43E substitution) found to be critical for the analyzed PLC-delta1 functions are present at the surface of the PH domain shown to contain the Ins(1,4,5)P3 binding pocket.

• Naylor CE et al.
• Structure of the key toxin in gas gangrene.
• Nat Struct Biol. 1998; 5: 738-46
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• Clostridium perfringens alpha-toxin is the key virulence determinant in gas gangrene and has also been implicated in the pathogenesis of sudden death syndrome in young animals. The toxin is a 370-residue, zinc metalloenzyme that has phospholipase C activity, and can bind to membranes in the presence of calcium. The crystal structure of the enzyme reveals a two-domain protein. The N-terminal domain shows an anticipated structural similarity to Bacillus cereus phosphatidylcholine-specific phospholipase C (PC-PLC). The C-terminal domain shows a strong structural analogy to eukaryotic calcium-binding C2 domains. We believe this is the first example of such a domain in prokaryotes. This type of domain has been found to act as a phospholipid and/or calcium-binding domain in intracellular second messenger proteins and, interestingly, these pathways are perturbed in cells treated with alpha-toxin. Finally, a possible mechanism for alpha-toxin attack on membrane-packed phospholipid is described, which rationalizes its toxicity when compared to other, non-haemolytic, but homologous phospholipases C.

• Katan M
• Families of phosphoinositide-specific phospholipase C: structure and function.
• Biochim Biophys Acta. 1998; 1436: 5-17
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• A large number of extracellular signals stimulate hydrolysis of phosphatidylinositol 4,5-bisphosphate by phosphoinositide-specific phospholipase C (PI-PLC). PI-PLC isozymes have been found in a broad spectrum of organisms and although they have common catalytic properties, their regulation involves different signalling pathways. A number of recent studies provided an insight into domain organisation of PI-PLC isozymes and contributed towards better understanding of the structural basis for catalysis, cellular localisation and molecular changes that could underlie the process of their activation.

• Bae YS, Cantley LG, Chen CS, Kim SR, Kwon KS, Rhee SG
• Activation of phospholipase C-gamma by phosphatidylinositol 3,4,5-trisphosphate.
• J Biol Chem. 1998; 273: 4465-9
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• Signal transduction across cell membranes often involves the activation of both phosphatidylinositol (PI)-specific phospholipase C (PLC) and phosphoinositide 3-kinase (PI 3-kinase). Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a substrate for both enzymes, is converted to phosphatidylinositol 3,4, 5-trisphosphate (PI(3,4,5)P3) by the action of PI 3-kinase. Here, we show that PI(3,4,5)P3 activates purified PLC-gamma isozymes by interacting with their Src homology 2 domains. Furthermore, the expression of an activated catalytic subunit of PI 3-kinase in COS-7 cells resulted in an increase in inositol phosphate formation, whereas platelet-derived growth factor-induced PLC activation in NIH 3T3 cells was markedly inhibited by the specific PI 3-kinase inhibitor LY294002. These results suggest that receptors coupled to PI 3-kinase may activate PLC-gamma isozymes indirectly, in the absence of PLC-gamma tyrosine phosphorylation, through the generation of PI(3,4,5)P3.

• Perisic O, Fong S, Lynch DE, Bycroft M, Williams RL
• Crystal structure of a calcium-phospholipid binding domain from cytosolic phospholipase A2.
• J Biol Chem. 1998; 273: 1596-604
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• Cytosolic phospholipase A2 (cPLA2) is a calcium-sensitive 85-kDa enzyme that hydrolyzes arachidonic acid-containing membrane phospholipids to initiate the biosynthesis of eicosanoids and platelet-activating factor, potent inflammatory mediators. The calcium-dependent activation of the enzyme is mediated by an N-terminal C2 domain, which is responsible for calcium-dependent translocation of the enzyme to membranes and that enables the intact enzyme to hydrolyze membrane-resident substrates. The 2.4-A x-ray crystal structure of this C2 domain was solved by multiple isomorphous replacement and reveals a beta-sandwich with the same topology as the C2 domain from phosphoinositide-specific phospholipase C delta 1. Two clusters of exposed hydrophobic residues surround two adjacent calcium binding sites. This region, along with an adjoining strip of basic residues, appear to constitute the membrane binding motif. The structure provides a striking insight into the relative importance of hydrophobic and electrostatic components of membrane binding for cPLA2. Although hydrophobic interactions predominate for cPLA2, for other C2 domains such as in "conventional" protein kinase C and synaptotagmins, electrostatic forces prevail.

• Neri LM, Borgatti P, Capitani S, Martelli AM
• Nuclear diacylglycerol produced by phosphoinositide-specific phospholipase C is responsible for nuclear translocation of protein kinase C-alpha.
• J Biol Chem. 1998; 273: 29738-44
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• It is well established that an independent inositide cycle is present within the nucleus, where it is involved in the control of cell proliferation and differentiation. Previous results have shown that when Swiss 3T3 cells are treated with insulin-like growth factor-I (IGF-I) a rapid and sustained increase in mass of diacylglycerol (DAG) occurs within the nuclei, accompanied by a decrease in the levels of both phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. However, it is unclear whether or not other lipids could contribute to this prolonged rise in DAG levels. We now report that the IGF-I-dependent increase in nuclear DAG production can be inhibited by the specific phosphatidylinositol phospholipase C inhibitor 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine or by neomycin sulfate but not by the purported phosphatidylcholine-phospholipase C specific inhibitor D609 or by inhibitors of phospholipase D-mediated DAG generation. Treatment of cells with 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine or neomycin sulfate inhibited translocation of protein kinase C-alpha to the nucleus. Moreover, exposure of cells to 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine, but not to D609, dramatically reduced the number of cells entering S-phase upon stimulation with IGF-I. These results suggest that the only phospholipase responsible for generation of nuclear DAG after IGF-I stimulation of 3T3 cells is PI-PLC. When this activity is inhibited, neither DAG rise is seen nor PKC-alpha translocation to the nucleus occurs. Furthermore, this PI-PLC activity appears to be essential for the G0/G1 to S-phase transition.

• Kavran JM et al.
• Specificity and promiscuity in phosphoinositide binding by pleckstrin homology domains.
• J Biol Chem. 1998; 273: 30497-508
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• Pleckstrin homology (PH) domains are small protein modules involved in recruitment of signaling molecules to cellular membranes, in some cases by binding specific phosphoinositides. We describe use of a convenient "dot-blot" approach to screen 10 different PH domains for those that recognize particular phosphoinositides. Each PH domain bound phosphoinositides in the assay, but only two (from phospholipase C-delta1 and Grp1) showed clear specificity for a single species. Using soluble inositol phosphates, we show that the Grp1 PH domain (originally cloned on the basis of its phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) binding) binds specifically to D-myo-inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) (the PtdIns(3,4,5)P3 headgroup) with KD = 27.3 nM, but binds D-myo-inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) or D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) over 80-fold more weakly. We show that this specificity allows localization of the Grp1 PH domain to the plasma membrane of mammalian cells only when phosphatidylinositol 3-kinase (PI 3-K) is activated. The presence of three adjacent equatorial phosphate groups was critical for inositol phosphate binding by the Grp1 PH domain. By contrast, another PH domain capable of PI 3-K-dependent membrane recruitment (encoded by EST684797) does not distinguish Ins(1,3,4)P3 from Ins(1,3,4,5)P3 (binding both with very high affinity), despite selecting strongly against Ins(1,4,5)P3. The remaining PH domains tested appear significantly less specific for particular phosphoinositides. Together with data presented in the literature, our results suggest that many PH domains bind similarly to multiple phosphoinositides (and in some cases phosphatidylserine), and are likely to be regulated in vivo by the most abundant species to which they bind. Thus, using the same simple approach to study several PH domains simultaneously, our studies suggest that highly specific phosphoinositide binding is a characteristic of relatively few cases.

• Heinz DW, Essen LO, Williams RL
• Structural and mechanistic comparison of prokaryotic and eukaryotic phosphoinositide-specific phospholipases C.
• J Mol Biol. 1998; 275: 635-50
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• Phosphoinositide-specific phospholipases C (PI-PLCs) are ubiquitous enzymes that catalyse the hydrolysis of phosphoinositides to inositol phosphates and diacylglycerol (DAG). Whereas the eukaryotic PI-PLCs play a central role in most signal transduction cascades by producing two second messengers, inositol-1,4,5-trisphosphate and DAG, prokaryotic PI-PLCs are of interest because they act as virulence factors in some pathogenic bacteria. Bacterial PI-PLCs consist of a single domain of 30 to 35 kDa, while the much larger eukaryotic enzymes (85 to 150 kDa) are organized in several distinct domains. The catalytic domain of eukaryotic PI-PLCs is assembled from two highly conserved polypeptide stretches, called regions X and Y, that are separated by a divergent linker sequence. There is only marginal sequence similarity between the catalytic domain of eukaryotic and prokaryotic PI-PLCs. Recently the crystal structures of a bacterial and a eukaryotic PI-PLC have been determined, both in complexes with substrate analogues thus enabling a comparison of these enzymes in structural and mechanistic terms. Eukaryotic and prokaryotic PI-PLCs contain a distorted (beta alpha)8-barrel as a structural motif with a surprisingly large structural similarity for the first half of the (beta alpha)8-barrel and a much weaker similarity for the second half. The higher degree of structure conservation in the first half of the barrel correlates with the presence of all catalytic residues, in particular two catalytic histidine residues, in this portion of the enzyme. The second half contributes mainly to the features of the substrate binding pocket that result in the distinct substrate preferences exhibited by the prokaryotic and eukaryotic enzymes. A striking difference between the enzymes is the utilization of a catalytic calcium ion that electrostatically stabilizes the transition state in eukaryotic enzymes, whereas this role is filled by an analogously positioned arginine in bacterial PI-PLCs. The catalytic domains of all PI-PLCs may share not only a common fold but also a similar catalytic mechanism utilizing general base/acid catalysis. The conservation of the topology and parts of the active site suggests a divergent evolution from a common ancestral protein.

• van Dijk MC, van Blitterswijk WJ
• Lipid metabolism in fibroblast growth factor-stimulated L6 myoblasts: a receptor mutation (Y766F) abrogates phospholipase D and diacylglycerol kinase activities.
• Biochim Biophys Acta. 1998; 1391: 273-9
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• Phosphatidylcholine (PC) hydrolysis induced by basic fibroblast growth factor (bFGF) was studied in rat L6 myoblasts expressing the wild-type FGF receptor-1 (FGFR-1) or a mutant (Y766F) that is incapable of activating phospholipase C-gamma (PLCgamma). Stimulation of FGFR-1 activated phospholipase D (PLD) rapidly and transiently, but did not induce PC-specific PLC activity. Downregulation of protein kinase C blocked bFGF-induced PLD activation but not phosphatidic acid formation by diacylglycerol (DG) kinase. Only phosphoinositide (PI)-derived DG, not PC-derived DG, appeared to be a substrate for DG kinase. Stimulation of FGFR-1(Y766F) did not activate PLD or DG kinase, both of which apparently require initial PLCgamma activation. The Y766F mutation reduced mitogen-activated protein kinase activation but not cell proliferation. We conclude that both PI turnover and PC hydrolysis are dispensable for bFGF-induced mitogenesis.

• Hendrickson HS, Hendrickson EK
• A thiophosphate analog of dimyristoylphosphatidyl-inositol-4-phosphate is a substrate for mammalian phosphoinositide-specific phospholipase C.
• Bioorg Med Chem Lett. 1998; 8: 1057-60
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• 1,2-Dimyristoyloxypropane-3-thiophosphate(rac-1-myo-inositol-4- phosphate), a thiophosphate analog of dimyristoyl phosphatidylinositol-4-phosphate was synthesized as a substrate for mammalian phosphoinositide-specific phospholipase C. Its activity with delta(1-132)-PI-PLC-delta 1 (a deletion mutant with the N-terminal pleckstrin homology domain removed) was studied in sonicated dispersions, with and without added Triton X-100. It had an initial activity of about 30 mumol min-1 mg-1, which rapidly decreased due to substrate depletion in the vesicle or micelle. The slower rate of hydrolysis appeared limited by enzyme hopping or exchange of substrate between vesicles or micelles, which was more rapid in the presence of detergent.

• Erneux C, Govaerts C, Communi D, Pesesse X
• The diversity and possible functions of the inositol polyphosphate 5-phosphatases.
• Biochim Biophys Acta. 1998; 1436: 185-99
• Display abstract

• Distinct forms of inositol and phosphatidylinositol polyphosphate 5-phosphatases selectively remove the phosphate from the 5-position of the inositol ring from both soluble and lipid substrates, i.e., inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), inositol 1,3,4, 5-tetrakisphosphate (Ins(1,3,4,5)P4), phosphatidylinositol 4, 5-bisphosphate (PtdIns(4,5)P2) or phosphatidylinositol 3,4, 5-trisphosphate (PtdIns(3,4,5)P3). In mammalian cells, this family contains a series of distinct genes and splice variants. All inositol polyphosphate 5-phosphatases share a 5-phosphatase domain and various protein modules probably responsible for specific cell localisation or recruitment (SH2 domain, proline-rich sequences, prenylation sites, etc.). Type I Ins(1,4,5)P3 5-phosphatase also uses Ins(1,3,4,5)P4 but not the phosphoinositides as substrates. This enzyme is targeted to specific membranes by means of a prenylation site. Type II 5-phosphatases can use both PtdIns(4,5)P2 and PtdIns(3,4,5)P3 as substrates. Five mammalian enzymes and multiple splice variants are known: INPP5P or inositol polyphosphate 5-phosphatase II, OCRL (a Golgi protein implicated in the Lowe oculocerebrorenal syndrome), synaptojanin (a protein involved in the recycling of synaptic vesicles), SHIP 1 and SHIP 2 (or SH2-containing inositol 5-phosphatases). As discussed in this review, the substrate specificity, regulatory mechanisms, subcellular localisation and tissue specificity indicate that the different 5-phosphatase isoforms may play specific roles. As known in the dephosphorylation of tyrosine containing substrates by the tyrosine protein phosphatases or in the metabolism of cyclic nucleotides by the cyclic nucleotide phosphodiesterases, inositol polyphosphate 5-phosphatases directly participate in the control of second messengers in response to both activation or inhibitory cell signalling.

• Falasca M, Logan SK, Lehto VP, Baccante G, Lemmon MA, Schlessinger J
• Activation of phospholipase C gamma by PI 3-kinase-induced PH domain-mediated membrane targeting.
• EMBO J. 1998; 17: 414-22
• Display abstract

• Signaling via growth factor receptors frequently results in the concomitant activation of phospholipase C gamma (PLC gamma) and phosphatidylinositol (PI) 3-kinase. While it is well established that tyrosine phosphorylation of PLC gamma is necessary for its activation, we show here that PLC gamma is regulated additionally by the lipid products of PI 3-kinase. We demonstrate that the pleckstrin homology (PH) domain of PLC gamma binds to phosphatidylinositol 3,4,5-trisphosphate [PdtIns(3,4,5)P3], and is targeted to the membrane in response to growth factor stimulation, while a mutated version of this PH domain that does not bind PdtIns(3,4,5)P3 is not membrane targeted. Consistent with these observations, activation of PI 3-kinase causes PLC gamma PH domain-mediated membrane targeting and PLC gamma activation. By contrast, either the inhibition of PI 3-kinase by overexpression of a dominant-negative mutant or the prevention of PLC gamma membrane targeting by overexpression of the PLC gamma PH domain prevents growth factor-induced PLC gamma activation. These experiments reveal a novel mechanism for cross-talk and mutual regulation of activity between two enzymes that participate in the control of phosphoinositide metabolism.

• Tan CA, Roberts MF
• Engineering of the nonspecific phospholipase C from Bacillus cereus: replacement of glutamic acid-4 by alanine results in loss of interfacial catalysis and enhanced phosphomonoesterase activity.
• Biochemistry. 1998; 37: 4275-9
• Display abstract

• The nonspecific phospholipase C from Bacillus cereus is a zinc metalloenzyme that catalyzes the hydrolysis of phospholipids to yield diacylglycerol and a phosphate monoester. Glu-4 has been proposed as a potential candidate for the general base in the hydrolysis reaction and was shown to interact with the substrate headgroup. Site-specific mutagenesis studies suggest that Glu-4 is important for substrate binding but not for catalysis. This residue is also critical for the enzyme's preference for a phosphodiester substrate. PA, both monomeric and micellar, is shown to be a poor substrate and inhibitor of wild-type PLC. When Glu-4 was mutated to an alanine, a significant increase in PA hydrolysis and a decrease in PC hydrolysis were observed. Unlike the wild type, kinetic studies suggest that the Glu-4-->Ala mutant does not exhibit interfacial activation and processive catalysis. Glu-4 is part of a highly flexible loop flanking the entrance to the active site, suggesting that this loop might constitute an interfacial binding recognition site. This is the first evidence for the presence of an interfacial binding site distinct from the active site in the nonspecific PLC.

• Carnall N, Webb H, Carrington M
• Mutagenesis study of the glycosylphosphatidylinositol phospholipase C of Trypanosoma brucei.
• Mol Biochem Parasitol. 1997; 90: 423-32
• Display abstract

• The glycosylphosphatidylinositol phospholipase C (GPI-PLC) from Trypanosoma brucei is particularly effective in hydrolysing the GPI-anchors of some proteins. The enzyme is inhibited by Zn2+ and p-chloromercurylphenylsulphonic acid, both of which can act as sulphydryl reagents, suggesting that a cysteine residue may be important in catalysis. Single cysteine to serine mutants have been produced for all eight cysteines in GPI-PLC; all the mutants were fully active in vitro and were still susceptible to p-chloromercurylphenylsulphonic acid inhibition. In contrast, a single histidine 34 to glutamine mutation totally inactivated GPI-PLC. The histidine was chosen after a sequence alignment with the Bacillus cereus phosphatidylinositol phospholipase C (PI-PLC) suggested a conservation of active site residues, including histidine 34 which is central to the proposed reaction mechanism (Heinz D.W., Ryan M., Bullock T.L., Griffith O.H. EMBO J 1995;14:3855-3863). The results suggest that the GPI-PLC and bacterial PI-PLCs have conserved active sites and that the inhibition of GPI-PLC by sulphydryl reagents can occur through more than one residue.

• James SR, Downes CP
• Structural and mechanistic features of phospholipases C: effectors of inositol phospholipid-mediated signal transduction.
• Cell Signal. 1997; 9: 329-36
• Display abstract

• The production of the intracellular second messengers inositol (1,4,5)-trisphosphate (InsP3) and sn 1,2-diacylglycerol (DG) in response to a wide variety of extracellular primary messengers is achieved by an extended family of inositol phospholipid phosphodiesterases termed phospholipases C (PLC, E.C. 3.1.4.11). This family has been the subject of extensive research and it is clear that the different isoenzymes exhibit some common characteristics (e.g., interactions with substrates) and other distinctive features (e.g., modes of regulation). The recent description of the X-ray crystal structure of a mammalian PLC has served to clarify much about the behaviour of the PLCs, emphasising the "modular" structure of these enzymes. The main focus of this review will concern the specific adaptations of PLC molecules which make them efficient lipid-metabolising enzymes. We also describe what is known about how these enzymes interact with their lipid substrates, which will serve as a basis for considering how PLCs may be activated.

• da Graca Thrige D, Buur JR, Jorgensen FS
• Substrate binding and catalytic mechanism in phospholipase C from Bacillus cereus: a molecular mechanics and molecular dynamics study.
• Biopolymers. 1997; 42: 319-36
• Display abstract

• For the first time a consistent catalytic mechanism of phospholipase C from Bacillus cereus is reported based on molecular mechanics calculations. We have identified the position of the nucleophilic water molecule, which is directly involved in the hydrolysis of the natural substrate phosphatidylcholine, in phospholipase C. This catalytically essential water molecule, after being activated by an acidic residue (Asp55), performs the nucleophilic attack on the phosphorus atom in the substrate, leading to a trigonal bipyramidal pentacoordinated intermediate (and structurally similar transition state). The subsequent collapse of the intermediate, regeneration of the enzyme, and release of the products has to involve a not yet identified second water molecule. The catalytic mechanism reported here is based on a series of molecular mechanics calculations. First, the x-ray structure of phospholipase C from B cereus including a docked substrate molecule was subjected to a stepwise molecular mechanics energy minimization. Second, the location of the nucleophilic water molecule in the active site of the fully relaxed enzyme-substrate complex was determined by evaluation of nonbonded interaction energies between the complex and a water molecule. The nucleophilic water molecule is positioned at a distance (3.8 A) from the phosphorus atom in the substrate, which is in good agreement with experimentally observed distances. Finally, the stability of the complex between phospholipase C, the substrate, and the nucleophilic water molecule was verified during a 100 ps molecular dynamics simulation. During the simulation the substrate undergoes a conformational change, but retains its localization in the active site. The contacts between the enzyme, the substrate, and the nucleophilic water molecule display some fluctuations, but remain within reasonable limits, thereby confirming the stability of the enzyme-substrate-water complex. The protocol developed for energy minimization of phospholipase C containing three zinc ions located closely together at the bottom of the active site cleft is reported in detail. In order to handle the strong electrostatic interactions in the active site realistically during energy minimization, delocalization of the charges from the three zinc ions was considered. Therefore, quantum mechanics calculations on the zinc ions and the zinc-coordinating residues were carried out prior to the molecular mechanics calculations, and two different sets of partial atomic charges (MNDO-Mulliken and AMI-ESP) were applied. After careful assignment of partial atomic charges, a complete energy minimization of the protein was carried out by a stepwise procedure without explicit solvent molecules. Energy minimization with either set of charges yielded structures, which were very similar both to the x-ray structure and to each other, although using AMI-ESP partial atomic charges and a dielectric constant of 4, yielded the best protein structure.

• Nebigil CG
• Suppression of phospholipase C beta, gamma, and delta families alters cell growth and phosphatidylinositol 4,5-bisphosphate levels.
• Biochemistry. 1997; 36: 15949-58
• Display abstract

• Phosphatidylinositol-specific phospholipase C (PLC) activity reflects a summation of the activities of three families, beta, gamma, and delta, each of which is regulated differently. In order to understand the contribution of each family to cell proliferation signaling, expression of each family was suppressed by use of an inducible expression vector for antisense PLC sequences in a single cell line, FTO-2B rat hepatocytes. Activation of second messengers of PLC [diacylglycerol (DAG) and inositol 1,4,5-tris(phosphate) (IP3)] was dramatically reduced, providing a strategy for probing the consequences of PLC deficiency on cell function. Importantly, while one PLC family was suppressed, the other PLCs actively responded to specific stimuli, suggesting parallel and independent signaling pathways for each PLC family in FTO-2B cells. Selective suppression of each PLC family altered cell growth markedly and differentially. The rank order for suppression of cell growth by loss of a PLC family was gamma > delta > beta. Exploration of down-stream growth regulators revealed that loss of beta and gamma, but not delta, families was associated with markedly reduced basal ras and protein kinase C activity. Moreover, suppression of each of the three PLC families caused remarkably reduced basal and stimulated MAP kinase activities. Interestingly, cellular levels of PIP2 were increased and dramatically correlated with growth inhibition rate in the clones with suppressed PLC activity, suggesting that PIP2 itself can serve as a second messenger of cell growth regulation.

• Hondal RJ et al.
• Phosphatidylinositol-specific phospholipase C: kinetic and stereochemical evidence for an interaction between arginine-69 and the phosphate group of phosphatidylinositol.
• Biochemistry. 1997; 36: 6633-42
• Display abstract

• A new substrate analogue, (2R)-1,2-dipalmitoyloxypropanethiophospho-1-D-myo-inositol (DPsPI), has been used in a new, continuous assay for phosphatidylinositol-specific phospholipase C (PI-PLC). DPsPI is superior to other substrate analogs that have been used for assaying PI-PLC since it is synthesized as a pure diastereomer and maintains both acyl chains of the natural substrate, dipalmitoylphosphatidylinositol (DPPI). The assay that has been developed using this new analogue has allowed us to elucidate detailed kinetic data so far lacking in the field. In addition, several mutants of PI-PLC were constructed and assayed. The results show that Arg-69 is essential for catalysis, since mutations at this position led to a 10(3)- 10(4)-fold decrease in activity with respect that of to the wild-type (WT) enzyme. An alanine mutant of Asp-67, a residue also found at the active site, displays activity similar to that of WT. We have also used nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy to analyze the structural integrity and conformational stability of the mutants. The results show that the overall global conformation of the enzyme is not perturbed by the mutants. The 15N-1H HSQC NMR spectrum of WT PI-PLC is also reported at 600 MHz. The stereoselectivity of the reaction toward the stereoisomers of another analogue, 1,2-dipalmitoyl-sn-glycero-3-thiophospho-1-myo-inositol (DPPsI), was used to probe whether Arg-69 interacts with the phosphate moiety of the substrate. We have calculated that the WT enzyme shows a stereoselectivity ratio of 160000:1 in favor of the Rp isomer versus the Sp isomer. The R69K mutant displayed a significant 10(4)-fold relaxation of stereoselectivity. Our data support the role of Arg-69 in stabilizing the negative charge on the pentacoordinate phosphate in the transition state during catalysis.

• Morris JC, Mensa-Wilmot K
• Role of 2,6-dideoxy-2,6-diaminoglucose in activation of a eukaryotic phospholipase C by aminoglycoside antibiotics.
• J Biol Chem. 1997; 272: 29554-9
• Display abstract

• Recent emergence of microbial resistance to aminoglycoside antibiotics, and the documented cytotoxicity associated with their use, calls for sustained efforts at understanding the effects of the compounds on eukaryotic cells. Using a glycosyl phosphatidylinositol (GPI)-phospholipase C (GPI-PLC) from the protozoan parasite Trypanosoma brucei, we demonstrate that a eukaryotic PLC can be activated 6-fold by aminoglycosides. Neomycin B protected GPI-PLC from a reduction in activity at pH 6.5, and increased the turnover number (kcat) of the enzyme. In structure-activity studies with the neomycin group, 2-deoxy-streptamine was mildly stimulatory; the concentration required to activate GPI-PLC 2-fold (SC200) was 310 microM. Neamine was 150-fold more active (SC200 = 2 microM) than 2-deoxy-streptamine, indicating that a 2,6-dideoxy-2, 6-diaminoglucose substituent at the 4-position of 2-deoxystreptamine plays an important role in activation of GPI-PLC. Ribostamycin and neomycin B also had SC200's of 2 microM, implying that the ribose group in ribostamycin is not involved in activation of GPI-PLC. These conclusions were affirmed in studies with Bacillus thuringiensis phosphatidylinositol-specific phospholipase C. A 2, 6-dideoxy-2,6-diaminoglucose substitution at the 4-OH of 2-deoxystreptamine activates the enzyme 17-fold, while a second 2, 6-dideoxy-2,6-diaminoglucose moiety on the ribose ring of ribostamycin provides an additional 3.5-fold stimulation. Possible implications of these observations for the effects of aminoglycosides on eukaryote cells are discussed.

• Tall E et al.
• Phosphoinositide binding specificity among phospholipase C isozymes as determined by photo-cross-linking to novel substrate and product analogs.
• Biochemistry. 1997; 36: 7239-48
• Display abstract

• We tested for the presence of high-affinity phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and PI(3,4,5)P3 binding sites in four phospholipase C (PLC) isozymes (delta1, beta1, beta2, and beta3), by probing these proteins with analogs of inositol phosphates, D-Ins(1,4,5)P3, D-Ins(1,3,4,5)P4, and InsP6, and polyphosphoinositides PI(4,5)P2 and PI(3,4,5)P3, which contain a photoactivatable benzoyldihydrocinnamide moiety. Only PLC-delta1 was specifically radiolabeled. More than 90% of the label was found in tryptic and chymotryptic fragments which reacted with antisera against the pleckstrin homology (PH) domain, whereas less than 5% was recovered in fragments that encompassed the catalytic core. In separate experiments, the isolated delta1-PH domain was also specifically labeled. Equilibrium binding of D-Ins(1,4,5)P3 to PLC-delta1 indicated the presence of a single, high-affinity binding site; binding of D-Ins(1,4,5)P3 to PLC-beta1, -beta2, or -beta3 was not detected. The catalytic activity of PLC-delta1 was inhibited by the product D-Ins(1,4,5)P3, whereas no inhibition of PLC-beta1, -beta2, or -beta3 activity was observed. These results demonstrate that the PH domain is the sole high-affinity PI(4,5)P2 binding site of PLC-delta1 and that a similar site is not present in PLC-beta1, -beta2, or -beta3. The data are consistent with the idea that the PH domain of PLC-delta1, but not the beta isozymes, directs the catalytic core to membranes enriched in PI(4,5)P2 and is subject to product inhibition.

• Liu T, Ryan M, Dahlquist FW, Griffith OH
• Determination of pKa values of the histidine side chains of phosphatidylinositol-specific phospholipase C from Bacillus cereus by NMR spectroscopy and site-directed mutagenesis.
• Protein Sci. 1997; 6: 1937-44
• Display abstract

• Two active site histidine residues have been implicated in the catalysis of phosphatidylinositol-specific phospholipase C (PI-PLC). In this report, we present the first study of the pKa values of histidines of a PI-PLC. All six histidines of Bacillus cereus PI-PLC were studied by 2D NMR spectroscopy and site-directed mutagenesis. The protein was selectively labeled with 13C epsilon 1-histidine. A series of 1H-13C HSQC NMR spectra were acquired over a pH range of 4.0-9.0. Five of the six histidines have been individually substituted with alanine to aid the resonance assignments in the NMR spectra. Overall, the remaining histidines in the mutants show little chemical shift changes in the 1H-13C HSQC spectra, indicating that the alanine substitution has no effect on the tertiary structure of the protein. H32A and H82A mutants are inactive enzymes, while H92A and H61A are fully active, and H81A retains about 15% of the wild-type activity. The active site histidines, His32 and His82, display pKa values of 7.6 and 6.9, respectively. His92 and His227 exhibit pKa values of 5.4 and 6.9. His61 and His81 do not titrate over the pH range studied. These values are consistent with the crystal structure data, which shows that His92 and His227 are on the surface of the protein, whereas His61 and His81 are buried. The pKa value of 6.9 corroborates the hypothesis of His82 acting as a general acid in the catalysis. His32 is essential to enzyme activity, but its putative role as the general base is in question due to its relatively high pKa.

• Gassler CS, Ryan M, Liu T, Griffith OH, Heinz DW
• Probing the roles of active site residues in phosphatidylinositol-specific phospholipase C from Bacillus cereus by site-directed mutagenesis.
• Biochemistry. 1997; 36: 12802-13
• Display abstract

• The role of amino acid residues located in the active site pocket of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus[Heinz, D. W., Ryan, M., Bullock, T., & Griffith, O. H. (1995) EMBO J. 14, 3855-3863] was investigated by site-directed mutagenesis, kinetics, and crystal structure analysis. Twelve residues involved in catalysis and substrate binding (His32, Arg69, His82, Gly83, Lys115, Glu117, Arg163, Trp178, Asp180, Asp198, Tyr200, and Asp274) were individually replaced by 1-3 other amino acids, resulting in a total number of 21 mutants. Replacements in the mutants H32A, H32L, R69A, R69E, R69K, H82A, H82L, E117K, R163I, D198A, D198E, D198S, Y200S, and D274S caused essentially complete inactivation of the enzyme. The remaining mutants (G83S, K115E, R163K, W178Y, D180S, Y200F, and D274N) exhibited reduced activities up to 57% when compared with wild-type PI-PLC. Crystal structures determined at a resolution ranging from 2.0 to 2.7 A for six mutants (H32A, H32L, R163K, D198E, D274N, and D274S) showed that significant changes were confined to the site of the respective mutation without perturbation of the rest of the structure. Only in mutant D198E do the side chains of two neighboring arginine residues move across the inositol binding pocket toward the newly introduced glutamic acid. An analysis of these structure-function relationships provides new insight into the catalytic mechanism, and suggests a molecular explanation of some of the substrate stereospecificity and inhibitor binding data available for this enzyme.

• Wu Y, Zhou C, Roberts MF
• Stereocontrolled syntheses of water-soluble inhibitors of phosphatidylinositol-specific phospholipase C: inhibition enhanced by an interface.
• Biochemistry. 1997; 36: 356-63
• Display abstract

• Three inositol 1,2-(cyclic)-phosphate analogs, inositol cyclic phosphonates with different stereochemistry at the C-2 position of the inositol ring, have been synthesized as water-soluble inhibitors of phosphatidylinositol-specific phospholipase C (PI-PLC). Their inhibition of both phosphotransferase and cyclic phosphodiesterase activities has been studied in the absence and presence of an interface. Key results include the following. (i) Only the analog with the same stereochemistry at the C-2 position of the inositol ring as the natural substrate, myo-inositol 1,2-(cyclic)-phosphate (cIP), exhibits effective inhibition of PI-PLC. (ii) The inhibition of the PI-PLC cyclic phosphodiesterase activity by this cIP analog is enhanced by the presence of an interface (Triton X-100 or diC7PC micelles). This is the first observation of detergent enhancing the effectiveness of a water-soluble inhibitor competing with a water-soluble substrate. (iii) For the cyclic phosphodiesterase activity measured in the presence of 8 mM of the best (e.g., most activating) interface, diC7PC, myo-inositol 1,2-(cyclic)-2-methylenephosphonate (cICH2P) was shown to be a competitive inhibitor with a Ki of 12.3 mM. (iv) The IC50 obtained for the same compound inhibiting the PI-PLC hydrolysis of PI dispersed in DiC7PC micelles was consistent with a Ki approximately 10 mM for the phosphotransferase activity. The similarity of Ki for both PI and cIP processing by PI-PLC suggests both reactions occur at the same site on the enzyme.

• Bromann PA, Boetticher EE, Lomasney JW
• A single amino acid substitution in the pleckstrin homology domain of phospholipase C delta1 enhances the rate of substrate hydrolysis.
• J Biol Chem. 1997; 272: 16240-6
• Display abstract

• The pleckstrin homology (PH) domain has been postulated to serve as an anchor for enzymes that operate at a lipid/water interface. To understand further the relationship between the PH domain and enzyme activity, a phospholipase C (PLC) delta1/PH domain enhancement-of-activity mutant was generated. A lysine residue was substituted for glutamic acid in the PH domain of PLC delta1 at position 54 (E54K). Purified native and mutant enzymes were characterized using a phosphatidylinositol 4,5-bisphosphate (PI(4, 5)P2)/dodecyl maltoside mixed micelle assay and kinetics measured according to the dual phospholipid model of Dennis and co-workers (Hendrickson, H. S., and Dennis, E. A. (1984) J. Biol. Chem. 259, 5734-5739; Carmen, G. M., Deems, R. A., and Dennis, E. A. (1995) J. Biol. Chem. 270, 18711-18714). Our results show that both PLC delta1 and E54K bind phosphatidylinositol bisphosphate cooperatively (Hill coefficients, n = 2.2 +/- 0.2 and 2.0 +/- 0.1, respectively). However, E54K shows a dramatically increased rate of (PI(4, 5)P2)-stimulated PI(4,5)P2 hydrolysis (interfacial Vmax for PLC delta1 = 4.9 +/- 0.3 micromol/min/mg and for E54K = 31 +/- 3 micromol/min/mg) as well as PI hydrolysis (Vmax for PLC delta1 = 27 +/- 3.4 nmol/min/mg and for E54K = 95 +/- 12 nmol/min/mg). In the absence of PI(4,5)P2 both native and mutant enzyme hydrolyze PI at similar rates. E54K also has a higher affinity for micellar substrate (equilibrium dissociation constant, Ks = 85 +/- 36 microM for E54K and 210 +/- 48 microM for PLC delta1). Centrifugation binding assays using large unilamelar phospholipid vesicles confirm that E54K binds PI(4,5)P2 with higher affinity than native enzyme. E54K is more active even though the interfacial Michaelis constant (Km) for E54K (0.034 +/- 0.01 mol fraction PI(4,5)P2) is higher than the Km for native enzyme (0.012 +/- 0.002 mol fraction PI(4,5)P2). D-Inositol trisphosphate is less potent at inhibiting E54K PI(4,5)P2 hydrolysis compared with native enzyme. These results demonstrate that a single amino acid substitution in the PH domain of PLC delta1 can dramatically enhance enzyme activity. Additionally, the marked increase in Vmax for E54K argues for a direct role of PH domains in regulating catalysis by allosteric modulation of enzyme structure.

• Kozawa O, Blume-Jensen P, Heldin CH, Ronnstrand L
• Involvement of phosphatidylinositol 3'-kinase in stem-cell-factor-induced phospholipase D activation and arachidonic acid release.
• Eur J Biochem. 1997; 248: 149-55
• Display abstract

• We have shown previously that the stem cell factor (SCF) receptor undergoes phosphorylation on serine residues following ligand stimulation, and that this phopshorylation is dependent mainly on the activity of protein kinase C (PKC). In the present study, we have further investigated the molecular mechanisms behind SCF-stimulated activation of PKC, and found that SCF does not activate phosphatidylinositol-specific phospholipase C. In contrast, phospholipase D (PLD) is activated in response to SCF in a dose-dependent manner. Activation of PLD was not inhibited by calphostin C, an inhibitor of PKC. On the other hand, inhibitors of phosphatidylinositol PtdIns 3'-kinase (PtdIns 3'-kinase), i.e. wortmannin and LY294002, inhibited SCF-induced PLD activation. Moreover, a mutant SCF receptor in which Tyr721, which is responsible for activation of PtdIns 3'-kinase, is mutated to a phenylalanine residue was unable to mediate activation of PLD. Thus, PtdIns 3'-kinase appears to be essential for SCF-induced PLD activation. Furthermore, we demonstrate that phosphatidic acid (PtdH), generated through the action of PLD in response to SCF, is metabolized to diacylglycerol by dephosphorylation. Diacylglycerol can then activate PKC, and, moreover, after deacylation by a diacylglycerol lipase, yield arachidonic acid, an important second messenger in cell signaling.

• Rapuano BE, Bockman RS
• Protein kinases A and C positively regulate G protein-dependent activation of phosphatidylinositol-specific phospholipase C by tumor necrosis factor-alpha in MC3T3-E1 osteoblasts.
• J Cell Biochem. 1997; 65: 198-208
• Display abstract

• The role(s) of protein kinases in the regulation of G protein-dependent activation of phosphatidylinositol-specific phospholipase C by tumor necrosis factor-alpha was investigated in the osteoblast cell line MC3T3-E1. We have previously reported the stimulatory effects of tumor necrosis factor-alpha and A1F4-, an activator of G proteins, on this phospholipase pathway documented by a decrease in mass of PI and release of diacylglycerol. In this study, we further explored the mechanism(s) by which the tumor necrosis factor or A1F4(-)-promoted breakdown of phosphatidylinositol and the polyphosphoinositides by phospholipase C is regulated. Tumor necrosis factor-alpha was found to elicit a 4-5-fold increase in the formation of [3H]inositol-1,4-phosphate and [3H]inositol-1,4,5-phosphate; and a 36% increase in [3H]inositol-1-phosphate within 5 min in prelabeled cells. [3H]inositol-4-phosphate, a metabolite of [3H]inositol-1,4-phosphate and [3H]inositol-1,4,5-phosphate, was found to be the predominant phosphoinositol product of tumor necrosis factor-alpha and A1F4(-)-activated phospholipase C hydrolysis after 30 min. In addition, the preincubation of cells with pertussis toxin decreased the tumor necrosis factor-induced release of inositol phosphates by 53%. Inhibitors of protein kinase C, including Et-18-OMe and H-7, dramatically decreased the formation of [3H]inositol phosphates stimulated by either tumor necrosis factor-alpha or A1F4- by 90-100% but did not affect basal formation. The activation of cAMP-dependent protein kinase, or protein kinase A, by the treatment of cells with forskolin or 8-BrcAMP augmented basal, tumor necrosis factor-alpha and A1F4(-)-induced [3H]inositol phosphate formation. Therefore, we report that protein kinases can regulate tumor necrosis factor-alpha-initiated signalling at the cell surface in osteoblasts through effects on the coupling between receptor, G-protein and phosphatidylinositol-specific phospholipase C.

• Zhou C, Garigapati V, Roberts MF
• Short-chain phosphatidylinositol conformation and its relevance to phosphatidylinositol-specific phospholipase C.
• Biochemistry. 1997; 36: 15925-31
• Display abstract

• The solution conformation of chiral diheptanoylphosphatidylinositol (D- and L-inositol isomers) has been characterized by NMR spectroscopy. A positive NOE between the inositol C2 proton and an sn-3 glycerol CH2 proton has been observed in the D- but not in the L-inositol isomer of diheptanoylphosphatidylinositol (PI). Computer modeling using QUANTA constrained by this NOE and ring coupling constants suggests that the inositol ring is nearly parallel to the chain packing direction, leaving the phosphate ester accessible to attack by phosphatidylinositol-specific phospholipase C enzymes. In this model, the hydroxyl groups in the 2- and 6-positions of inositol form hydrogen bonds with the pro-R and ester oxygens, respectively. Chemical shifts and 13C spin-lattice relaxation times were also used to assess conformation and lipid dynamics in monomer and micelle states. The 13C T1's of inositol C2 and C6 in monomeric phosphatidylinositol were markedly less than for other inositol ring carbons. These results are consistent with the hydrogen bonds to the phosphate constraining the motions of C2 and C6. Diheptanoylphosphatidyl-2-O-methylinositol is a good inhibitor of PI-specific phospholipase C because it blocks the initial phosphotransferase step in PI hydrolysis. Introduction of the methyl group on the C-2 hydroxyl group lowers the CMC of the derivative compared to diheptanoylphosphatidylinositol. However, an NOE between an sn-3 glycerol proton and the inositol C2 proton constrains the orientation of the inositol ring with respect to the glycerol backbone in a conformation similar to diheptanoylphosphatidylinositol. Modeling of the 2-O-methylinositol derivative suggests that the methyl group blocks one side of the phosphate, consistent with the observation that nonspecific phospholipase C enzymes which are able to hydrolyze PI, albeit poorly, are unable to hydrolyze diheptanoylphosphatidyl-2-O-methylinositol.

• Sun B, Murray NR, Fields AP
• A role for nuclear phosphatidylinositol-specific phospholipase C in the G2/M phase transition.
• J Biol Chem. 1997; 272: 26313-7
• Display abstract

• Protein kinase C (PKC) is activated at the nucleus during the G2 phase of cell cycle, where it is required for mitosis. However, the mechanisms controlling cell cycle-dependent activation of nuclear PKC are not known. We now report that nuclear levels of the major physiologic PKC activator diacylglycerol (DAG) fluctuate during cell cycle. Specifically, nuclear DAG levels in G2/M phase cells are 2. 5-3-fold higher than in G1 phase cells. In synchronized cells, nuclear DAG levels rise to a peak coincident with the G2/M phase transition and return to basal levels in G1 phase cells. This increase in DAG level is sufficient to stimulate betaII PKC-mediated phosphorylation of its mitotic nuclear envelope substrate lamin B in vitro. Isolated nuclei from G2 phase cells contain an active phospholipase activity capable of generating DAG in vitro. Nuclear phospholipase activity is inhibited by the selective phosphatidylinositol-specific phospholipase C (PI-PLC) inhibitor 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine and neomycin sulfate, but not by the phosphatidylcholine-PLC selective inhibitor D609 or inhibitors of phospholipase D-mediated DAG generation. Treatment of synchronized cells with 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine leads to decreased nuclear PI-PLC activity and cell cycle blockade in the G2 phase, suggesting a role for nuclear PI-PLC in the G2/M phase transition. Our data are consistent with the hypothesis that nuclear PI-PLC generates DAG to activate nuclear betaII PKC, whose activity is required for mitosis.

• Wu Y, Perisic O, Williams RL, Katan M, Roberts MF
• Phosphoinositide-specific phospholipase C delta1 activity toward micellar substrates, inositol 1,2-cyclic phosphate, and other water-soluble substrates: a sequential mechanism and allosteric activation.
• Biochemistry. 1997; 36: 11223-33
• Display abstract

• The kinetics of full-length and PH domain truncated cloned PI-PLC delta1 from rat toward soluble substrates [inositol 1, 2-(cyclic)-phosphate (cIP) and glycerophosphoinositol phosphates (GPIPx)] as well as PI in detergent micelles provide the following insights into the mechanism of this enzyme. (i) That cIP is a substrate for the enzyme implies a two-step mechanism for PI hydrolysis [intramolecular phosphotransferase reaction to form cIP followed by cyclic phosphodiesterase activity to form inositol-1-phosphate (I-1-P)]. The dependence of enzyme activity on cIP is sigmoidal, suggesting a transition between less active and more active forms of the enzyme that is affected by substrate. (ii) Interfaces increase the kcat for cIP (but do not affect the cooperativity), and this allosteric activation requires an intact PH domain. (iii) Phosphorylation of the soluble inositol phosphodiesters GPI, GPIP, and GPIP2 enhances PI-PLC delta1 activity by dramatically increasing kcat and decreasing Km. For these phosphodiesters, the substrate saturation curve is no longer sigmoidal but hyperbolic, indicating the phosphorylated substrate can shift the enzyme to the activated form. (iv) Given the kinetic parameters for cIP hydrolysis and the constant ratio of cIP/I-1-P generated during PI hydrolysis, the cIP produced in situ is either released (and not readily rebound since its concentration is well below Km) or attacked by a water molecule for the generation of the acyclic product.

• Irvine R
• Phospholipid signalling. Taking stock of PI-PLC.
• Nature. 1996; 380: 581-3

• James SR, Smith S, Paterson A, Harden TK, Downes CP
• Time-dependent inhibition of phospholipase C beta-catalysed phosphoinositide hydrolysis: a comparison of different assays.
• Biochem J. 1996; 314: 917-21
• Display abstract

• The properties of three different beta-isoforms of phospholipase C (PLC) were analysed using substrate lipids dispersed in phospholipid vesicles, phospholipid-detergent mixed micelles and phospholipid monolayers spread at an air-water interface. Phosphatidylinositol 4,5-bisphosphate hydrolysis went virtually to completion in monolayers, but inositol trisphosphate production was curtailed prematurely in vesicular and micellar assays. Assays were linear for less than 2 min with vesicles; the linear portion could be significantly extended in micelles by increasing the ratio of micelles to enzyme molecules. However, onset of a second lower rate of substrate hydrolysis always occurred when < or = 10% of PtdIns(4,5)P(2) had been utilized. This was not due to enzyme inactivation in the micellar interface, determined by addition of fresh substrate or fresh enzyme after the slow phase of activity had started, nor was it due to overt product inhibition of PLC or apparent entrapment of PLC at the micelle surface. These results are similar to those seen in assays using bacterial PLC and we suggest that the biphasic kinetics may be due to product-dependent changes in the presentation of substrate lipic to PLC in lamellar assays, leading to reduced activity.

• Venkatakrishnan G, Exton JH
• Identification of determinants in the alpha-subunit of Gq required for phospholipase C activation.
• J Biol Chem. 1996; 271: 5066-72
• Display abstract

• A series of chimeras between a constitutively active mutant of the alpha-subunit of Gq and the alpha-subunit of Gs was constructed to identify the domains in alphaq specifically involved in interaction with its effector phosphoinositide phospholipase C (PLC). Transient expression of the chimeric proteins and measurement of the production of inositol phosphates and cAMP in HEK-293 cells revealed that the Ile217-Lys276 sequence of alphaq contained the PLC interaction sites, whereas the residues for activation of adenylyl cyclase were in the Ile235-Leu294 sequence of alphas. Alanine scanning mutagenesis of the Ile217-Lys276 region of alphaq further identified two clusters of amino acids (Asp243,Asn244,Glu245 and Arg256,Thr257) that were specifically required for interaction with PLC. Comparison of the sequences of alphaq, alphas, and alphat showed that the PLC-interacting residues identified in alphaq are different from the corresponding residues in alphas and alphat that are involved in effector activation. Alignment of the sequences of alphaq and alphat, based on the crystal structure of alphat (Noel, J. P., Hamm, H. E., and Sigler, P. D. (1993) Nature 366, 654-663), indicated that the PLC-activating residues of alphaq are located in alpha-helix 3 and its linker to beta-sheet 4, which are adjacent to a switch region whose conformation changes with activation. It is proposed that the selectivity of alphaq for PLC involves relatively few amino acids, but that the effector may interact with other nonselective sequences in the alpha-subunit.

• Huber A, Sander P, Gobert A, Bahner M, Hermann R, Paulsen R
• The transient receptor potential protein (Trp), a putative store-operated Ca2+ channel essential for phosphoinositide-mediated photoreception, forms a signaling complex with NorpA, InaC and InaD.
• EMBO J. 1996; 15: 7036-45
• Display abstract

• The transient receptor potential protein (Trp) is a putative capacitative Ca2+ entry channel present in fly photoreceptors, which use the inositol 1,4,5-trisphosphate (InsP3) signaling pathway for phototransduction. By immunoprecipitation studies, we find that Trp is associated into a multiprotein complex with the norpA-encoded phospholipase C, an eye-specific protein kinase C (InaC) and with the InaD protein (InaD). InaD is a putative substrate of InaC and contains two PDZ repeats, putative protein-protein interaction domains. These proteins are present in the photoreceptor membrane at about equimolar ratios. The Trp homolog analyzed here is isolated together with NorpA, InaC and InaD from blowfly (Calliphora) photoreceptors. Compared to Drosophila Trp, the Calliphora Trp homolog displays 77% amino acid identity. The highest sequence conservation is found in the region that contains the putative transmembrane domains S1-S6 (91% amino acid identity). As investigated by immunogold labeling with specific antibodies directed against Trp and InaD, the Trp signaling complex is located in the microvillar membranes of the photoreceptor cells. The spatial distribution of the signaling complex argues against a direct conformational coupling of Trp to an InsP3 receptor supposed to be present in the membrane of internal photoreceptor Ca2+ stores. It is suggested that the organization of signal transducing proteins into a multiprotein complex provides the structural basis for an efficient and fast activation and regulation of Ca2+ entry through the Trp channel.

• Breshnahan BA, Kelefiotis D, Stratidakis I, Lianos EA
• PGF2alpha-induced signaling events in glomerular mesangial cells.
• Proc Soc Exp Biol Med. 1996; 212: 165-73
• Display abstract

• Of the various arachidonate cyclooxygenation eicosanoids synthesized in the normal and injured renal glomerular capillary, prostaglandin F2alpha (PGF2alpha) is the most abundant and potent in eliciting signaling events and biologic responses including contraction and proliferation of glomerular capillary pericytes known as mesangial cells. The regulation of PGF2alpha-induced signaling in these cells is unknown. The present studies assessed two key signaling events in response to PGF2alpha in mesangial cells; activation of phospholipase C (PLC) and protein kinase C (PKC). Mechanisms regulating PLC activation were also explored. Incubation of cultured growth arrested rat mesangial cells with PGF2alpha (1 microM) resulted in activation of a phosphatidyl inositol-specific phospholipase C (PI-PLC) assessed as increased generation of polyphosphates in myo-[3H]-inositol-labeled cells and as increased diacylglycerol (DAG) mass levels measured by a radioenzymatic assay. Generation of both inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate occurred, the former constituting 70% of total inositol trisphosphates. Enhanced generation of inositol 1,4-bisphosphate (IP2) also occurred and was greater than that of inositol 1,4,5-trisphosphate (IP3), indicating that PI-PLC utilized the phosphatidyl inositol monophosphate (PIP) to a greater extent than the phosphatidyl inositol bisphosphate (PIP2) substrate. Generation of DAG in response to PGF2alpha occurred in a biphasic pattern characterized by an early transient rise that peaked concomitantly with IP3 at 15 sec, and a late sustained increase at 2, 5, and 15 min that was not associated with an increase in IP3. PGF2alpha also activated PKC assessed as translocation of enzyme activity from cytosolic to membrane fractions. Inhibition of PKC using H-7 enhanced PGF2alpha-induced generation of IP3 at 15 sec but attenuated generation of DAG at 15 min. A more selective PKC inhibitor, Calphostin C, dose-dependently increased basal IP3 generation and also attenuated generation of DAG in response to PGF2alpha. This indicates that PKC negatively modulates PGF2alpha-induced PI-PLC activation, and that the late sustained DAG generation in response to PGF2alpha is regulated by a PKC-dependent phospholipase other than PLC. The mechanisms of PI-PLC stimulation in response to PGF2alpha were further explored using inhibitors of protein tyrosine phosphorylation and of guanine nucleotide-binding (G) protein activation. Inhibition of protein tyrosine phosphorylation using genistein had no effect on IP3 or DAG generation. ADP ribosylation of Gi using pertussis toxin (PTx) had no effect on IP3 generation in response to PGF2alpha. The inhibitor of receptor-coupled PI-PLC activation aminosteroid compound U-73122 that blocks G(PLC) was also ineffective. The observations indicate that PGF2alpha stimulates a PI-PLC which is under negative feedback regulatory control by PKC, and a phospholipase other than PLC which is under positive regulatory control by PKC. PGF2alpha-induced PI-PLC activation is independent of protein tyrosine phosphorylation and of PTx-sensitive G proteins.

• Heinz DW, Ryan M, Smith MP, Weaver LH, Keana JF, Griffith OH
• Crystal structure of phosphatidylinositol-specific phospholipase C from Bacillus cereus in complex with glucosaminyl(alpha 1-->6)-D-myo-inositol, an essential fragment of GPI anchors.
• Biochemistry. 1996; 35: 9496-504
• Display abstract

• Numerous proteins on the external surface of the plasma membrane are anchored by glycosylated derivatives of phosphatidylinositol (GPI), rather than by hydrophobic amino acids embedded in the phospholipid bilayer. These GPI anchors are cleaved by phosphatidylinositol-specific phospholipases C (PI-PLCs) to release a water-soluble protein with an exposed glycosylinositol moiety and diacylglycerol, which remains in the membrane. We have previously determined the crystal structure of Bacillus cereus PI-PLC, the enzyme which is widely used to release GPI-anchored proteins from membranes, as free enzyme and also in complex with myo-inositol [Heinz, D.W., Ryan, M. Bullock, T.L., & Griffith, O. H. (1995) EMBO J. 14, 3855-3863]. Here we report the refined 2.2 A crystal structure of this enzyme complexed with a segment of the core of all GPI anchors, glucosaminyl(alpha 1-->6)-D-myo-inositol [GlcN-(alpha 1-->6)Ins ]. The myo-inositol moiety of GlcN(alpha 1-->6)Ins is well-defined and occupies essentially the same position in the active site as does free myo-inositol, which provides convincing evidence that the enzyme utilizes the same catalytic mechanism for cleavage of PI and GPI anchors. The myo-inositol moiety makes several specific hydrogen bonding interactions with active site residues. In contrast, the glucosamine moiety lies exposed to solvent at the entrance of the active site with minimal specific protein contacts. The glucosamine moiety is also less well-defined, suggesting enhanced conformational flexibility. On the basis of the positioning of GlcN(alpha 1-->6)Ins in the active site, it is predicted that the remainder of the GPI-glycan makes little or no specific interactions with B. cereus PI-PLC. This explains why B. cereus PI-PLC can cleave GPI anchors having variable glycan structures.

• Grobler JA, Hurley JH
• Expression, characterization, and crystallization of the catalytic core of rat phosphatidylinositide-specific phospholipase C delta 1.
• Protein Sci. 1996; 5: 680-6
• Display abstract

• Phosphatidylinositide-specific phospholipase Cs (PI-PLCs) catalyze the calcium-dependent hydrolysis of phosphatidylinositides in response to diverse stimuli in higher eukaryotes. Mammalian PI-PLCs contain divergent regulatory regions, but all share three conserved regions: an N-terminal pleckstrin homology (PH) domain, X, and Y. We report the high-level expression and characterization of a recombinant "catalytic core" of rat PI-PLC delta 1 that contains the catalytically essential X and Y regions, but not the PH domain. The expressed protein, PI-PLC delta delta 1-134, is catalytically active versus phosphatidylinositol 4,5-bisphosphate in deoxycholate micelles with a K(m) of 182 microM and a Vmax of 27 mumol/min/mg. PI-PLC delta delta 1-134 is monomeric and monodisperse as judged by dynamic light scattering. Far-UV CD indicates a structure with approximately 35% alpha-helix. A reversible change in the near-UV CD spectrum is observed on addition of calcium, suggesting that calcium can bind PI-PLC delta delta 1-134 in the absence of phospholipid. Triclinic crystals of PI-PLC delta delta 1-134 have been obtained that diffract beyond 2.4 A resolution under cryogenic conditions. Based on Vm = 2.72 Da/A3 and on the self-rotation function, there are two PI-PLC delta delta 1-134 molecules per asymmetric unit that are related to each other by a noncrystallographic axis of approximate twofold symmetry parallel to a.

• Milting H, Heilmeyer LM Jr, Thieleczek R
• Cloning of a phospholipase C-delta 1 of rabbit skeletal muscle.
• J Muscle Res Cell Motil. 1996; 17: 79-84
• Display abstract

• The phospholipase C isoform responsible for the increase in the total myoplasmic inositol 1,4,5-trisphosphate concentration during tetanic contraction of isolated skeletal muscle and its mechanism of activation is not known. We have cloned and sequenced a phospholipase C cDNA of rabbit skeletal muscle coding for a protein of 745 amino acids with a molecular mass of 84,440 kDa. The deduced amino acid sequence exhibits the phospholipase C-specific domains X and Y which according to current knowledge very likely represent the catalytic centre of the enzyme. An overall sequence homology of 88% to the phospholipase C-delta 1 of rat brain suggests that the encoded protein represents a phospholipase C-delta 1 isoform of rabbit skeletal muscle. Northern blot analysis shows, that this phospholipase C-delta is dominantly expressed in skeletal muscle, less strongly in smooth muscle (uterus) and lung and weakly in heart, kidney and brain. In the N-terminal part of the primary structure a consensus sequence for a canonical EF-hand Ca2+ binding domain can be identified together with a short positively charged motif which recently has been suggested to be essential for the binding of phosphatidylinositol 4,5-bisphosphate. If these two domains which are unique for phospholipase C-delta are sufficient in establishing a mechanism for the activation of the enzyme, inositol 1,4,5-trisphosphate formation in skeletal muscle could be the consequence of an increase in myoplasmic Ca2+.

• Roberts MF
• Phospholipases: structural and functional motifs for working at an interface.
• FASEB J. 1996; 10: 1159-72
• Display abstract

• Phospholipases form a ubiquitous class of enzymes optimized to catalyze the hydrolysis of phospholipids. Because their products are often second messengers, they are highly regulated by the cell. For a given ester bond, there are separate secreted as well as cytoplasmic phospholipases with different substrate specificities and modes of regulation. As it becomes available, structural information provides a view of interfacial catalysis for several of these phospholipases on a molecular level. Recent structural advances include solution structures of a pancreatic phospholipase A2 in the absence and presence of a micellar interface, crystal structures of a bacterial phosphatidylinositol-phospholipase C whose active site is reminiscent of ribonuclease, and a Ca2+ lipid binding domain with high homology to regions in several cytoplasmic phospholipases that can model the way those proteins interact with the membrane surface. Phospholipases also have a wide and complex array of regulatory mechanisms involving cytoplasmic proteins, notably G-proteins, as well as different effector lipids (e.g., phosphatidylinositol-4,5-biphosphate, or PIP2) or Ca2+. Deconvolution of these interactions is necessary to understand their roles in different signal transduction pathways.-Roberts, M. F. Phospholipases: structural and functional motifs for working at an interface.

• Wang X et al.
• Variant GPI structure in relation to membrane-associated functions of a murine folate receptor.
• Biochemistry. 1996; 35: 16305-12
• Display abstract

• Two variant sublines of murine L1210 leukemia cells (L1210A and L1210JF) overexpress the cell surface folate receptor (FR). The membrane bound FR in L1210A cells exhibited significantly (up to 17-fold) greater relative affinities for (6S)-N5-methyltetrahydrofolate, (6S)-N5-formyltetrahydrofolate and methotrexate compared to the FR in L1210JF cells. Furthermore, receptor-mediated transport of [3H]-(6S)-N5-methyltetrahydrofolate was much more efficient in L1210A cells compared to L1210JF cells. When solubilized with Triton X-100, the ligand binding characteristics of FR from both sublines resembled those of the receptor associated with L1210 JF cell membranes. N-terminal amino acid sequence analysis as well as RT-PCR analysis of the entire coding region revealed a single species of FR in both cells, identical to murine FR-alpha. The FR in L1210JF cells was sensitive to phosphatidylinositol specific phospholipase C (PI-PLC) indicating the presence of a glycosyl-phosphatidylinositol (GPI) membrane anchor while the FR in L1210A cells was resistant to PI-PLC; however, the FR in L1210A cells was released from plasma membranes by nitrous acid, as expected for GPI and its PI-PLC resistant structural variants. Treatment of L1210A cell membranes with mild base rendered the protein PI-PLC sensitive as expected for GPI anchors acylated in the inositol ring and also decreased the affinities of the membrane associated FR for reduced folates. When the cDNA for murine FR-alpha was expressed in parental L1210 cells the protein was PI-PLC resistant but was sensitive to PI-PLC when the cDNA was expressed in human 293 fibroblasts. In L1210JF, L1210A, and parental L1210 cells, several cell surface proteins, including FR, incorporated [3H]ethanolamine, a component of the GPI membrane anchor; however, the labeled proteins were released by PI-PLC only in L1210JF cells. The above results preclude any peculiarity of the FR polypeptide in either L1210 subline as the basis for the observed differences in PI-PLC sensitivity and membrane-associated functions of FR. Partial deglycosylation of membrane associated FR from either cell with N-glycanase did not influence its ligand binding characteristics. The results of this study lead to the hypothesis that variant GPI structures may modulate the function of a protein by influencing its conformation/topography in the membrane. Such effects may be identified by their disappearance/reduction upon detergent solubilization or mild base treatment of the membrane.

• Cunningham E, Tan SK, Swigart P, Hsuan J, Bankaitis V, Cockcroft S
• The yeast and mammalian isoforms of phosphatidylinositol transfer protein can all restore phospholipase C-mediated inositol lipid signaling in cytosol-depleted RBL-2H3 and HL-60 cells.
• Proc Natl Acad Sci U S A. 1996; 93: 6589-93
• Display abstract

• The mammalian phosphatidylinositol transfer proteins (PITP) and the yeast Saccharomyces cerevisiae PITP (SEC14p) that show no sequence homology both catalyze exchange of phosphatidylinositol (PI) between membranes compartments in vitro. In HL-60 cells where the cytosolic proteins are depleted by permeabilization, exogenously added PITPalpha is required to restore G protein-mediated phospholipase Cbeta (PLCbeta) signaling. Recently, a second mammalian PITPbeta form has been described that shows 77% identity to rat PITPalpha. We have examined the ability of the two mammalian PITPs and SEC14p to restore PLC-mediated signaling in cytosol-depleted HL-60 and RBL-2H3 cells. Both PITPalpha and PITPbeta isoforms as well as SEC14p restore G protein-mediated PLCbeta signaling with a similar potency. In RBL-2H3 cells, crosslinking of the IgE receptor by antigen stimulates inositol lipid hydrolysis by tyrosine phosphorylation of PLCgamma1. Permeabilization of RBL cells leads to loss of PLCgamma1 as well as PITP into the extracellular medium and this coincides with loss of antigen-stimulated lipid hydrolysis. Both PLCgamma1 and PITP were required to restore inositol lipid signaling. We conclude that (i) because the PI binding/transfer activities of PITP/SEC14p is the common feature shared by all three transfer proteins, it must be the relevant activity that determines their abilities to restore inositol lipid-mediated signaling and (ii) PITP is a general requirement for inositol lipid hydrolysis regardless of how and which isoform of PLC is activated by the appropriate agonist.

• Grobler JA, Essen LO, Williams RL, Hurley JH
• C2 domain conformational changes in phospholipase C-delta 1.
• Nat Struct Biol. 1996; 3: 788-95
• Display abstract

• The structure of the PH-domain truncated core of rat phosphoinositide-specific phospholipase C-delta 1 has been determined at 2.4 A resolution and compared to the structure previously determined in a different crystal form. The stereochemical relationship between the EF, catalytic, and C2 domains is essentially identical. The Ca2+ analogue Sm3+ binds at two sites between the jaws of the C2 domain. Sm3+ binding ejects three lysine residues which bridge the gap between the jaws and occupy the Ca2+ site in the apoenzyme, triggering a conformational change in the jaws. The distal sections of the C2 jaws move apart, opening the mouth by 9 A and creating a gap large enough to bind a phospholipid headgroup.

• Lomasney JW et al.
• Phosphatidylinositol 4,5-bisphosphate binding to the pleckstrin homology domain of phospholipase C-delta1 enhances enzyme activity.
• J Biol Chem. 1996; 271: 25316-26
• Display abstract

• The pleckstrin homology (PH) domain is a newly recognized protein module believed to play an important role in signal transduction. While the tertiary structures of several PH domains have been determined, some co-complexed with ligands, the function of this domain remains elusive. In this report, the PH domain located in the N terminus of human phospholipase C-delta1 (PLCdelta1) was found to regulate enzyme activity. The hydrolysis of phosphatidylinositol (PI) was stimulated by phosphatidylinositol 4,5-bisphosphate (PIP2) in a dose-dependent manner with an EC50 = 1 microM (0.3 mol%), up to 9-fold higher when 5 microM (1.5 mol%) of PIP2 was incorporated into the PI/phosphatidylserine (PS)/phosphatidylcholine (PC) vesicles (30 microM of PI with a molar ratio of PI:PS:PC = 1:5:5). Stimulation was specific for PIP2, since other anionic phospholipids including phosphatidylinositol 4-phosphate had no stimulatory effect. PIP2-mediated stimulation was, however, inhibited by inositol 1,4, 5-triphosphate (IP3) in a dose-dependent manner, suggesting a modulatory role for this inositol. When a nested set of PH domain deletions up to 70 amino acids from the N terminus of PLCdelta1 were constructed, the deletion mutant enzymes all catalyzed the hydrolysis of the micelle forms of PI and PIP2 with specific activities comparable with those of the wild type enzyme. However, the stimulatory effect of PIP2 was greatly diminished when more than 20 amino acid residues were deleted from the N terminus. To identify the specific residues involved in PIP2-mediated enzyme activation, amino acids with functional side chains between residues 20 and 40 were individually changed to glycine. While all these mutations had little effect on the ability of the enzyme to catalyze the hydrolysis of PI or PIP2 micelles, the catalytic activity of mutants K24G, K30G, K32G, R38G, or W36G was markedly unresponsive to PIP2. Analysis of PIP2-stimulated PI hydrolysis by a dual substrate binding model of catalysis revealed that the micellar dissociation constant (Ks) of PLCdelta1 for the PI/PS/PC vesicles was reduced from 558 microM to 53 microM, and the interfacial Michaelis constant (Km) was reduced from 0.21 to 0.06 by PIP2. The maximum rate of PI hydrolysis (Vmax) was not affected by PIP2. These results demonstrate that a major function of the PH domain of PLCdelta1 is to modulate enzyme activity. Further, our results identify PIP2 as a functional ligand for a PH domain and suggest a general mechanism for the regulation of other proteins by PIP2.

• Horstman DA, DeStefano K, Carpenter G
• Enhanced phospholipase C-gamma1 activity produced by association of independently expressed X and Y domain polypeptides.
• Proc Natl Acad Sci U S A. 1996; 93: 7518-21
• Display abstract

• The X and Y domains of phospholipase C (PLC)-gamma1, which are conserved in all mammalian phosphoinositide-specific PLC isoforms and are proposed to interact to form the catalytic site, have been expressed as individual hexahistidine-tagged fusion proteins in the baculovirus system. Following coinfection of insect cells with recombinant viruses, association of X and Y polypeptides was demonstrated in coprecipitation assays. When enzyme activity was examined, neither domain possessed catalytic activity when expressed alone; however, coexpression of the X and Y polypeptides produced a functional enzyme. This reconstituted phospholipase activity remained completely dependent on the presence of free Ca2+. The specific activity of the X:Y complex was significantly greater (20- to 100-fold) than that of holoPLC-gamma1 and was only moderately influenced by varying the concentration of substrate. The enzyme activities of holoPLC-gamma1 and the X:Y complex exhibited distinct pH optima. For holoPLC-gamma1 maximal activity was detected at pH 5.0, while activity of the X:Y complex was maximal at pH 7.2.

• Exton JH
• Regulation of phosphoinositide phospholipases by hormones, neurotransmitters, and other agonists linked to G proteins.
• Annu Rev Pharmacol Toxicol. 1996; 36: 481-509
• Display abstract

• The actions of many hormones, neurotransmitters, and growth factors are mediated by the hydrolysis of phosphatidylinositol 4,5-bisphosphate catalyzed by specific isozymes of phospholipase C. This hydrolysis releases inositol 1,4,5-trisphosphate, which mobilizes Ca2+ ions from components of the endoplasmic reticulum, and 1,2-diacylglycerol, which activates isozymes of protein kinase C. The hormones and neurotransmitters activate beta-isozymes of phospholipase C through receptors that have seven transmembrane segments and couple to G proteins of the Gq and Gi/o families. Activation of phospholipase C by the Gq family involves their alpha-subunits, whereas activation by the Gi/o family involves their beta gamma-subunits. The growth factors activate gamma-isozymes of phospholipase C through receptors that become autophosphorylated due to their stimulated tyrosine kinase activity and provide binding sites for the Src homology domains of the isozymes. The molecular mechanisms by which agonists activate phopholipase isozymes are described in detail.

• Vizitiu D, Kriste AG, Campbell AS, Thatcher GR
• Inhibition of phosphatidylinositol-specific phospholipase C: studies on synthetic substrates, inhibitors and a synthetic enzyme.
• J Mol Recognit. 1996; 9: 197-209
• Display abstract

• Enzyme inhibition studies on phosphatidylinositol-specific phospholipase C (PI-PLC) from B. Cereus were performed in order to gain an understanding of the mechanism of the PI-PLC family of enzymes and to aid inhibitor design. Inhibition studies on two synthetic cyclic phosphonate analogues (1,2) of inositol cyclic-1:2-monophosphate (cIP), glycerol-2-phosphate and vanadate were performed using natural phosphatidylinositol (PI) substrate in Triton X100 co-micelles and an NMR assay. Further inhibition studies on PI-PLC from B. Cereus were performed using a chromogenic, synthetic PI analogue (DPG-PI), an HPLC assay and Aerosol-OT (AOT), phytic acid and vanadate as inhibitors. For purposes of comparison, a model PI-PLC enzyme system was developed employing a synthetic Cu(II)-metallomicelle and a further synthetic PI analogue (IPP-PI). The studies employing natural PI substrate in Triton X100 co-micelles and synthetic DPG-PI in the absence of surfactant indicate three classes of PI-PLC inhibitors: (1) active-site directed inhibitors (e.g. 1,2); (2) water-soluble polyanions (e.g. tetravanadate, phytic acid); (3) surfactant anions (e.g. AOT). Three modes of molecular recognition are indicated to be important: (1) active site molecular recognition; (2) recognition at an anion-recognition site which may be the active site, and; (3) interfacial (or hydrophobic) recognition which may be exploited to increase affinity for the anion-recognition site in anionic surfactants such as AOT. The most potent inhibition of PI-PLC was observed by tetravanadate and AOT. The metallomicelle model system was observed to mimic PI-PLC in reproducing transesterification of the PI analogue substrate to yield cIP as product and in showing inhibition by phytic acid and AOT.

• Waldo GL, Paterson A, Boyer JL, Nicholas RA, Harden TK
• Molecular cloning, expression and regulatory activity of G alpha 11- and beta gamma-subunit-stimulated phospholipase C-beta from avian erythrocytes.
• Biochem J. 1996; 316: 559-68
• Display abstract

• A turkey erythrocyte phospholipase C (PLC) has been instrumental in delineating the role of G-proteins in receptor-regulated inositol lipid signalling. This isoenzyme is uniquely regulated both by alpha-subunits of the Gq family and by G-protein beta gamma-subunits. A 4819 bp cDNA encoding this PLC has been cloned from a turkey erythrocyte cDNA library. The open reading frame of this cDNA encodes a 1211-amino-acid protein (calculated molecular mass 139050 Da) that contains amino acid sequences of 16 peptides sequenced from the turkey erythrocyte PLC. The predicted sequence of the turkey PLC shows considerable similarity with the sequences of previously cloned members of the PLC-beta family, with the highest identity (71%) shared with PLC-beta 2 and lesser identities observed with PLC-beta 1 (49%), PLC-beta 3 (46%) and PLC-beta 4 (37%). The largest differences in sequence between the turkey PLC-beta and other PLC-beta isoenzymes occur in the C-terminal domain and in the region between the X- and Y-domains. The turkey isoenzyme and PLC-beta 2, which differ in their regulation by G-protein alpha-subunits, are only 44% similar across the approx. 400 amino acid residues of the C-terminal domain that has been implicated in alpha q activation of these proteins. Recombinant turkey PLC-beta was purified to homogeneity following expression from a recombinant baculovirus in Sf9 insect cells. The immunoreactivity and mobility on SDS/PAGE of the recombinant enzyme were the same as observed with native turkey erythrocyte PLC-beta. Moreover, the catalytic activities of the recombinant enzyme were indistinguishable from those of native turkey erythrocyte PLC-beta in assays carried out in the presence of cholate and Ca2+, or in assays of activity after reconstitution with G alpha 11 or G-protein beta gamma-subunits. The turkey PLC-beta was more sensitive to activation by G alpha 11 than was PLC-beta 2, and was more sensitive to activation by beta gamma-subunits than either PLC-beta 2 or PLC-beta 1.

• Ryan M, Smith MP, Vinod TK, Lau WL, Keana JF, Griffith OH
• Synthesis, structure-activity relationships, and the effect of polyethylene glycol on inhibitors of phosphatidylinositol-specific phospholipase C from Bacillus cereus.
• J Med Chem. 1996; 39: 4366-76
• Display abstract

• Substrate analog inhibitors of Bacillus cereus phosphatidylinositol-specific phospholipase C (PI-PLC) were synthesized and screened for their suitability to map the active site region of the enzyme by protein crystallography. Analogs of the natural substrate phosphatidylinositol (PI) were designed to examine the importance of the lipid portion and the inositol phosphate head group for binding to the enzyme. The synthetic compounds contained pentyl, hexyl, or hexanoyl and octyl lipid chains at the sn-1 and sn-2 positions of the glycerol backbone and phosphonoinositol, phosphonic acid, methyl phosphonate, phosphatidic acid, or methyl phosphate at the sn-3 position. The most hydrophobic compound, dioctyl methyl phosphate 14, was also the best inhibitor with an IC50 of 12 microM. In a series of dihexyl lipids, compounds with phosphonoinositol head groups inhibited more strongly than those that do not contain inositol but are otherwise identical. Compound 29, a short-chain lipid with a phosphonoinositol head group, was found to be a competitive inhibitor and the most potent in this series with an IC50 of 18 microM (Ki = 14 microM). Analogs with dihexyl chains were better inhibitors than those with dihexanoyl chains, presumably because the ether-linked lipids are more hydrophobic than the ester-linked lipids. No appreciable difference in inhibition was found between a phosphonoinositol lipid and the corresponding difluorophosphonoinositol lipid. Inositols and inositol derivatives that do not contain lipid moieties show IC50s about 3 orders of magnitude above those of the short-chain lipids. In this group, glucosaminyl(alpha 1-->6)-D-myo-inositol inhibited more strongly than myo-inositol, which in turn is a better inhibitor than inositol phosphate. The addition of polyethylene glycol (PEG-600) resulted in a marked decrease in inhibition by the short-chain lipids, but had little effect on the water-soluble head group analogs. This is accounted for in terms of solubilization of the amphipathic inhibitors by PEG. Since PEG is required in the crystallization, these data indicate that the best strategy for obtaining enzyme inhibitor complexes is to start by cocrystallizing PI-PLC with the head group analogs. The next step is to synthetically add the shortest possible hydrophobic moieties to the analogs and cocrystallize these with the enzyme. This strategy may be applicable to other lipolytic enzymes.

• Ellis MV, U S, Katan M
• Mutations within a highly conserved sequence present in the X region of phosphoinositide-specific phospholipase C-delta 1.
• Biochem J. 1995; 307: 69-75
• Display abstract

• Phosphoinositide-specific phospholipase C (PI-PLC) enzymes have considerable structural similarity within limited regions (X and Y) implicated in catalysis. The role of residues contained within a highly conserved sequence present in the X region was investigated by site-directed mutagenesis of PLC-delta 1 isoenzyme. Seven residues (Ser-308, Ser-309, Ser-310, His-311, Thr-313, Tyr-314, and Gln-319) were individually replaced by alanine or glutamine (His-311). Replacement of two residues, His-311 and Tyr-314, resulted in a dramatic reduction of enzyme activity. The kcat of hydrolysis of phosphatidylinositol 4,5-bisphosphate by H311A and Y314A mutants was reduced 1000- and 10-fold respectively, with little effect on Km. Further analysis of H311A and Y314A mutants, using limited proteolysis and circular dichroism, had shown that no major structural alterations had occurred. Since site-directed mutagenesis demonstrated the importance of histidine residues, their role in enzyme function was also analysed by chemical modification with diethyl pyrocarbonate. This modification of histidine residues resulted in the reduction of enzyme activity and also indicated that more than one residue could be important.

• Koblan KS, Schaber MD, Edwards G, Gibbs JB, Pompliano DL
• src-homology 2 (SH2) domain ligation as an allosteric regulator: modulation of phosphoinositide-specific phospholipase C gamma 1 structure and activity.
• Biochem J. 1995; 305: 745-51
• Display abstract

• Phosphoinositide-specific phospholipase C gamma 1 (PI-PLC gamma 1) catalyses the hydrolysis of PtdIns(4,5)P2 to generate the second messengers diacylglycerol and Ins(1,4,5)P3. PI-PLC gamma 1, an src-homology 2/3 (SH2/SH3)-domain-containing enzyme, is activated in response to growth-factor-induced tyrosine phosphorylation, and, in vivo, is translocated from the cytosol to the particulate cell fraction. Here we report the bacterial expression of rat brain PI-PLC gamma 1 under the control of the T7 promoter. Production of the active enzyme in amounts suitable for structure-function analysis depended on coupling the translation of PLC gamma 1 to the expression of the phage-phi 10 coat protein. Purification of the enzyme was facilitated by the presence of a three-amino-acid C-terminal antibody epitope tag (Glu-Glu-Phe) engineered into the cloned PLC gamma 1. Examination of the specific activity, pH-rate profile, [Ca2+]-dependence and substrate specificity of bacterially expressed PLC gamma indicated that it had kinetic properties similar to those of PLC gamma isolated from bovine brain. The substrate specificity was dependent on [Ca2+]: at low [Ca2+] (1-10 microM) PtdIns(4,5)P2 was a better substrate than PtdIns. Addition of phosphotyrosine-containing peptides (12-mers) with the cognate sequence of the high-affinity binding site for PLC gamma 1 on the activated epidermal-growth-factor (EGF) receptor (Tyr-992) increased enzyme activity (up to 85%) in vitro. Cognate non-phosphorylated peptides had no effect on activity. When c.d. spectroscopy was used to monitor the effect of added phosphotyrosine-containing peptide on the structure of recombinant PLC gamma 1, significant spectral shifts, indicative of a conformational change, were observed upon complexation with the EGF-receptor phosphotyrosine-containing 12-residue peptide (Tyr*-992). How SH2 domains from PLC gamma 1 can mediate structural rearrangements and modulate enzymic activity on their ligation by growth-factor receptors is discussed.

• Garcia P et al.
• The pleckstrin homology domain of phospholipase C-delta 1 binds with high affinity to phosphatidylinositol 4,5-bisphosphate in bilayer membranes.
• Biochemistry. 1995; 34: 16228-34
• Display abstract

• The pleckstrin homology (PH) domain of phospholipase C-delta 1 (PLC-delta 1) binds to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in phospholipid membranes with an affinity (Ka approximately 10(6) M-1) and specificity comparable to those of the native enzyme. PLC-delta 1 and its PH domain also bind inositol 1,4,5-trisphosphate, the polar head group of PI(4,5)P2, with comparable affinity and approximately 1:1 stoichiometry. A peptide corresponding to amino acids 30-43 of the PLC-delta 1 PH domain contains several basic residues predicted to bind PI(4,5)P2, but binds weakly and with little specificity for PI(4,5)P2; hence the tertiary structure of the isolated PH domain is required for high affinity PI(4,5)P2 binding. Our PI-(4,5)P2 binding results support the hypothesis that the intact PH domain, serving as a specific tether, directs PLC-delta 1 to membranes enriched in PI(4,5)P2 and permits the active site, located elsewhere in the protein, to hydrolyze multiple substrate molecules before this enzyme dissociates from the membrane surface.

• Nakashima S et al.
• Deletion and site-directed mutagenesis of EF-hand domain of phospholipase C-delta 1: effects on its activity.
• Biochem Biophys Res Commun. 1995; 211: 365-9
• Display abstract

• In order to elucidate a role of a putative EF-hand motif (144-172) in phospholipase C-delta 1 (PLC-delta 1), deletion and point mutation of the enzyme were performed and the mutated cDNAs were expressed in CHO cells and E. coli AD202 strain. Deletion of amino acid residues of 141-236 or 173-236 resulted in abolition of PLC activity. However, the decreased PLC activity to 15-20% by deletion of the EF-hand motif (144-172) was still Ca(2+)-dependent. Furthermore, mutants, in which conserved Asp153, Asp157, Glu164 or all these acidic amino acids in the EF-hand motif were replaced with alanine residues, showed nearly the same PLC activity and Ca(2+)-dependency as those of wild-type. These results suggest that the region containing the EF-hand motif may not play a role in regulation of Ca(2+)-sensitivity of PLC-delta 1, but is important for its activity.

• Paterson HF et al.
• Phospholipase C delta 1 requires a pleckstrin homology domain for interaction with the plasma membrane.
• Biochem J. 1995; 312: 661-6
• Display abstract

• The structural requirements of phospholipase C delta 1 for interaction with the plasma membrane were analysed by immunofluorescence after microinjection into living cells. Microinjection of deletion mutants revealed that the region required for membrane attachment and binding of inositol 1,4,5-trisphosphate in vitro corresponded to the pleckstrin homology domain, a structural module described in more than 90 proteins.

• Cheng HF et al.
• Cloning and identification of amino acid residues of human phospholipase C delta 1 essential for catalysis.
• J Biol Chem. 1995; 270: 5495-505
• Display abstract

• In vitro single point mutagenesis, inositol phospholipid hydrolysis, and substrate protection experiments were used to identify catalytic residues of human phosphatidylinositide-specific phospholipase C delta 1 (PLC delta 1) isolated from a human aorta cDNA library. Invariant amino acid residues containing a functional side chain in the highly conserved X region were changed by in vitro mutagenesis. Most of the mutant enzymes were still able to hydrolyze inositol phospholipid with activity ranging from 10 to 100% of levels in the wild type enzyme. Exceptions were mutants with the conversion of Arg338 to Leu (R338L), Glu341 to Gly (E341G), or His356 to Leu (H356L), which made the enzyme severely defective in hydrolyzing inositol phospholipid. Phospholipid vesicle binding experiments showed that these three cleavage-defective mutant forms of PLC delta 1 could specifically bind to phosphatidylinositol 4,5-bisphosphate (PIP2) with an affinity similar to that of wild type enzyme. Western blotting analysis of trypsin-treated enzyme-PIP2 complexes revealed that a 67-kDa major protein fragment survived trypsin digestion if the wild type enzyme, E341G, or H356L mutant PLC delta 1 was preincubated with 7.5 microM PIP2, whereas if it was preincubated with 80 microM PIP2, the size of major protein surviving was comparable to that of intact enzyme. However, mutant enzyme R338L was not protected from trypsin degradation by PIP2 binding. These observations suggest that PLC delta 1 can recognize PIP2 through a high affinity and a low affinity binding site and that residues Glu341 and His356 are not involved in either high affinity or low affinity PIP2 binding but rather are essential for the Ca(2+)-dependent cleavage activity of PLC.

• McDonald LJ, Mamrack MD
• Phosphoinositide hydrolysis by phospholipase C modulated by multivalent cations La(3+), Al(3+), neomycin, polyamines, and melittin.
• J Lipid Mediat Cell Signal. 1995; 11: 81-91
• Display abstract

• Second messenger production from phosphoinositide hydrolysis is regulated by different pathways, such as G-proteins or tyrosine phosphorylation of phosphoinositide phospholipase C (PI-PLC). Another means of altering the activity of PI-PLC is through cation interaction with the phosphoinositide substrate. A variety of organic and inorganic multi-valent cations were examined for their effects on the activity of purified PI-PLC delta. Surprisingly, the cations produced both stimulation and inhibition of PI-PLC catalyzed phosphoinositide hydrolysis, depending on the substrate and the ion to phosphoinositide stoichiometry. These data support the hypothesis that ionic complexes with phosphoinositides may alter their hydrolysis by PI-PLC.

• Tompkins TA, Moscarello MA
• The mechanism of stimulation of brain phospholipase C-alpha by myelin basic protein involves specific interactions.
• Biochim Biophys Acta. 1994; 1206: 208-14
• Display abstract

• The modulation of a brain phosphoinositide-specific phospholipase C-alpha activity was studied using a variety of compounds of different charge. Detergents such as sodium deoxycholate and cetyltrimethylammonium bromide stimulated the phospholipase C activity when used alone but when used together the effects were not additive. Spermine was an effective inhibitor of the enzyme activity while the cationic peptide, Melittin, had no effect. The inositol phosphates produced by hydrolysis with phosphoinositide-specific phospholipase C were inhibitory while diacylglycerol and inositol did not affect the phospholipase activity. Myelin basic protein, which was previously shown to stimulate phospholipase C activity by 2.5-fold, did not interact with the anionic inositol phosphatases to any significant extent. Thus we concluded that the mechanism of stimulation was not due to relief of product inhibition. Crosslinking studies with the photoactivatable reagent, N-hydroxysuccinimidyl-4-azidosalicylic acid, showed that peptide 24-33 of myelin basic protein, which stimulated the activity almost as much as the native protein, interacted specifically with the phospholipase C. Thus the mechanism by which myelin basic protein stimulated the enzyme appeared to be through specific protein-protein interaction.

• Yagisawa H et al.
• Expression and characterization of an inositol 1,4,5-trisphosphate binding domain of phosphatidylinositol-specific phospholipase C-delta 1.
• J Biol Chem. 1994; 269: 20179-88
• Display abstract

• It was previously found that the 85-kDa protein purified from rat brain using an inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-immobilized matrix was the delta 1 isoform of phosphatidylinositol-specific phospholipase C (PLC). We expressed rat PLC-delta 1 in Escherichia coli as a fusion protein with glutathione S-transferase, and found that the bacterial lysate shows a significant amount of Ins(1,4,5)P3 binding. The lysate was applied to Ins(1,4,5)P3-immobilized column chromatography and the eluate with 2 M NaCl solution containing only a 100-kDa protein showed high Ins(1,4,5)P3 binding. The lysate was also purified to near homogeneity using a glutathione-Sepharose 4B affinity system. Bacterially-expressed enzyme thus purified showed essentially the same inositol phosphate binding characteristics as the brain-derived enzyme. PLC-delta 1 consists of the amino-terminal nonconserved region and two well-conserved regions among isozymes, designated as X and Y, which are thought to constitute a catalytic core of the enzyme. Using a combination of deletion mutants and proteolytic products of the enzyme, we were able to locate an Ins(1,4,5)P3 binding domain in the molecule. Deletion of 223 residues from the amino terminus completely abolished the binding activity, while deletion of X region only partially inhibited the binding and deletion of Y region did not affect the binding. A 76-kDa proteolytic product of the expressed PLC-delta 1 which lacked 60 amino acids at the amino terminus showed a minimal Ins(1,4,5)P3 binding activity. A peptide consists of 14 amino acids corresponding to residues 30-43 of PLC-delta 1, which contains 6 basic amino acids, binds to an Ins(1,4,5)P3-immobilized matrix. Moreover, Ins(1,4,5)P3 binding was blocked by phospholipid vesicles containing phosphatidylinositol 4,5-bisphosphate. These results, taken together, indicate that the amino-terminal domain of PLC-delta 1 is important for the binding of both Ins(1,4,5)P3 and phosphatidylinositol 4,5-bisphosphate.

• Cifuentes ME, Delaney T, Rebecchi MJ
• D-myo-inositol 1,4,5-trisphosphate inhibits binding of phospholipase C-delta 1 to bilayer membranes.
• J Biol Chem. 1994; 269: 1945-8
• Display abstract

• The binding of phosphoinositide-specific phospholipase C-delta 1 (PLC-delta 1) to bilayer membranes composed of phosphatidylcholine (PC) and phosphatidylinositol 4,5-bisphosphate (PIP2) was measured in the presence or absence of inositol phosphates. Binding was inhibited by the natural D-isomer of myo-inositol 1,4,5-trisphosphate (D-InsP3), but not by the L-isomer. The concentration of D-InsP3 required to decrease binding by 50% was 5.4 +/- 0.5 microM. 1-(alpha-Glycerophosphoryl)-D-myo-inositol 4,5-bisphosphate and D-myo-inositol 2,4,5-trisphosphate were nearly as effective as D-Ins(1,4,5)P3. D-myo-inositol monophosphate with phosphate esterified at either positions 1 or 2 of the myo-inositol ring, had no significant effect on binding. D-myo-inositol 1,4-bisphosphate weakly inhibited the binding, whereas the 4,5-isomer was nearly as potent as D-InsP3. Neither ATP nor inorganic phosphate significantly affected binding. As expected, D-Ins(1,4,5)P3 but not L-Ins(1,4,5)P3 decreased the initial rate of PIP2 hydrolysis in bilayer vesicles. The concentration required to decrease hydrolysis by 50% was 12.4 +/- 0.5 microM. A catalytic fragment of PLC-delta 1 that lacks a domain necessary for high affinity PIP2 binding was prepared as previously described (Cifuentes, M. E., Honkanen, L., and Rebecchi, M. J. (1993) J. Biol. Chem. 268, 11586-11593). In contrast to the native enzyme, the rate of PIP2 hydrolysis, catalyzed by the fragment, was not affected by D-Ins(1,4,5)P3. These data suggest that high affinity binding of the enzyme to PIP2 and processive catalysis, involve specific recognition of the 4- and 5-position phosphates of the inositol ring. Our results are consistent with feedback inhibition by the polar head group product, D-Ins(1,4,5)P3, at a step that precedes catalysis, namely interfacial recognition.

• Sundell S, Hansen S, Hough E
• A proposal for the catalytic mechanism in phospholipase C based on interaction energy and distance geometry calculations.
• Protein Eng. 1994; 7: 571-7
• Display abstract

• The non-specific phospholipase C from Bacillus cereus preferentially hydrolyses phosphatidylcholine but is also active against phosphatidylserine, phosphatidylethanolamine and at a much lower level, sphingomyelin. A minimal substrate model containing all required structural and configurational elements of a high affinity substrate was docked into the active site. The enzyme-substrate attachment points were from molecular interaction energy calculations using the program GRID and from a previous phosphate inhibitor complex structure. Available conformational space for the substrate was sampled by distance geometry calculations using the program DGEOM. This investigation clearly identifies the attacking nucleophile, a catalytically favourable orientation of the phosphate group in its tetra-, as well as its penta-, coordinated state and a crucial stabilizing environment for the alkoxide intermediate. Based on this information a complete catalytic cycle is proposed.

• Bruzik KS, Tsai MD
• Toward the mechanism of phosphoinositide-specific phospholipases C.
• Bioorg Med Chem. 1994; 2: 49-72

• Homma Y
• [Structure and function of inositol phospholipid-specific phospholipase C]
• Seikagaku. 1994; 66: 1091-110

• Pokorski M, Strosznajder R
• PO2-dependence of phospholipase C in the cat carotid body.
• Adv Exp Med Biol. 1993; 337: 191-5

• Flick JS, Thorner J
• Genetic and biochemical characterization of a phosphatidylinositol-specific phospholipase C in Saccharomyces cerevisiae.
• Mol Cell Biol. 1993; 13: 5861-76
• Display abstract

• Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphatidylinositol-specific phospholipase C (PI-PLC) generates two second messengers, inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. The polymerase chain reaction was used to isolate a Saccharomyces cerevisiae gene (PLC1) that encodes a protein of 869 amino acids (designated Plc1p) that bears greatest resemblance to the delta isoforms of mammalian PI-PLC in terms of overall sequence similarity and domain arrangement. Plc1p contains the conserved X and Y domains found in all higher eukaryotic PI-PLCs (51 and 29% identity, respectively, to the corresponding domains of rat delta 1 PI-PLC) and also contains a presumptive Ca(2+)-binding site (an E-F hand motif). Plc1p, modified by in-frame insertion of a His6 tract and a c-myc epitope near its amino terminus, was overexpressed from the GAL1 promoter, partially purified by nickel chelate affinity chromatography, and shown to be an active PLC enzyme in vitro with properties similar to those of its mammalian counterparts. Plc1p activity was strictly Ca2+ dependent: at a high Ca2+ concentration (0.1 mM), the enzyme hydrolyzed PIP2 at a faster rate than phosphatidylinositol, and at a low Ca2+ concentration (0.5 microM), it hydrolyzed PIP2 exclusively. Cells carrying either of two different deletion-insertion mutations (plc1 delta 1::HIS3 and plc1 delta 2::LEU2) were viable but displayed several distinctive phenotypes, including temperature-sensitive growth (inviable above 35 degrees C), osmotic sensitivity, and defects in the utilization of galactose, raffinose, and glycerol at permissive temperatures (23 to 30 degrees C). The findings reported here suggest that hydrolysis of PIP2 in S. cerevisiae is required for a number of nutritional and stress-related responses.

• Ellis MV, Carne A, Katan M
• Structural requirements of phosphatidylinositol-specific phospholipase C delta 1 for enzyme activity.
• Eur J Biochem. 1993; 213: 339-47
• Display abstract

• Phosphatidylinositol-specific phospholipase C delta 1 isozyme of phosphoinositol-specific phospholipase C has been used for studies of structural requirements for the catalytic function. The enzyme was expressed in a bacterial system and purified to homogeneity. Using a combination of deletion mutant analysis and limited proteolysis, it was found that the large proportion of the molecule participated in formation of a catalytic domain (residues 139-756); it included regions of high and low conservation with other phospholipase-C molecules. These studies also showed that the residues spanning regions of conservation, designated as X and Y, were exposed and highly susceptible to proteolysis by trypsin. Two of the fragments resulting from the cleavage (30 kDa and 40 kDa) interacted and, under non-denaturing conditions, formed a protein of 70 kDa.

• Bolt MJ, Bissonnette BM, Wali RK, Hartmann SC, Brasitus TA, Sitrin MD
• Characterization of phosphoinositide-specific phospholipase C in rat colonocyte membranes.
• Biochem J. 1993; 292: 271-6
• Display abstract

• The phosphoinositide signal transduction pathway mediates important processes in intestinal physiology, yet the key enzyme, phosphoinositide-specific phospholipase C (PI-PLC), is not well-characterized in the colon. PI-PLC activity was examined in rat colonic membranes using exogenous [3H]phosphatidylinositol 4,5-bisphosphate (PIP2) as substrate, and beta-glycerophosphate to suppress degradation of substrate or product. The activity of membrane PI-PLC increased 6-fold with the addition of alamethicin, and a further 2-3-fold enhancement was observed with 10 microM guanosine 5'-[gamma-thio]triphosphate (GTP[S]), suggesting the involvement of G-protein(s). The effect of GTP[S] appeared to be specific, as up to 100 microM adenosine 5'-[gamma-thio]-triphosphate failed to stimulate PI-PLC activity, and guanosine 5'-[beta-thio]diphosphate inhibited activity. The response of membrane PI-PLC to Ca2+ was biphasic, while > 0.5 mM Mg2+ was inhibitory with or without GTP[S]. Comparable total PI-PLC activities and responses to GTP[S] and Ca2+ were observed in purified brush-border and basolateral membranes. Western immunoblots probed with monoclonal antibodies to PLC isoenzymes PLC-beta 1, -gamma 1 and -delta 1 demonstrated that these antipodal plasma membranes contain predominantly the PLC-delta 1 isoform, with small amounts of PLC-gamma 1 present but no detectable PLC-beta 1. PLC-gamma 1 was the major isoform detected in cytosol.

• Cifuentes ME, Honkanen L, Rebecchi MJ
• Proteolytic fragments of phosphoinositide-specific phospholipase C-delta 1. Catalytic and membrane binding properties.
• J Biol Chem. 1993; 268: 11586-93
• Display abstract

• Active proteolytic fragments of phosphoinositide-specific phospholipase C-delta 1 (PLC-delta 1) were generated by trypsin digestion of the native protein. Brief proteolysis produced a 77-kDa fragment that contained the highly conserved X and Y regions but lacked the amino-terminal domain (amino acids 1-60). Prolonged digestion of PLC-delta 1 produced two fragments, one of 45 kDa that contained the entire X region and another of 32 kDa that consisted of the entire Y region and COOH-terminal domain. The 45- and 32-kDa fragments were isolated as an active heterodimeric complex. The 77-kDa fragment and the complex catalyzed calcium-dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) in detergent/phospholipid mixed micelles. When compared with the native enzyme, both the 77-kDa fragment and the complex exhibited a reduced capacity to processively hydrolyze PIP2; increasing the mole fraction of PIP2 in the mixed micelle surface greatly increased the rate of PIP2 hydrolysis catalyzed by the native enzyme but not the fragments. Both fragments also exhibited a reduced affinity for substrate; the native enzyme bound to bilayer vesicles consisting of phosphatidylcholine and PIP2 with high affinity (Ka approximately 10(6) M-1), whereas the fragments bound weakly (Ka < 10(4) M-1). These results demonstrate that the X, Y, and COOH-terminal regions form a calcium-dependent catalytic core that is resistant to proteolysis. The amino-terminal domain appears to be essential for high affinity binding to PIP2 but not catalysis. These observations are consistent with the idea that the amino-terminal domain forms part of a PIP2 binding site, which anchors PLC-delta 1 to the membrane surface during processive hydrolysis of its substrate.

• Leonis MA, Silbert DF
• Characterization of a second hamster lung fibroblast mutant with defects in phosphatidylinositol-specific phospholipase C.
• J Biol Chem. 1993; 268: 9416-24
• Display abstract

• We report here further characterization of the Chinese hamster lung fibroblast mutant D1-9b displaying elevated agonist-induced phosphatidylinositol (PI) turnover responses relative to CCL39, the parental cell line (Rath, H. M., Doyle, G. A. R., and Silbert, D. F. (1989) J. Biol. Chem. 264, 13387-13390). These differences in PI turnover responses are further enhanced by long term (24 h) pretreatment of cells with the phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). Short term pretreatment (15 min) with PMA attenuates the agonist-induced PI turnover response of both wild type and mutant D1-9b cell lines to that of unstimulated controls, suggesting that PMA-induced desensitization responses in D1-9b are intact. Both cytosolic and membrane/particulate fractions prepared from mutant D1-9b have decreased phospholipase C (PLC) activities relative to wild type when assayed with either of four different PI/phosphatidylinositol 4,5-bisphosphate-mixed micelle/vesicle substrate combinations. Thermolability studies, Mono Q anion-exchange chromatography, and Western blot studies identified the source of cytosolic PLC deficiency in D1-9b as being due to the absence of PLC delta 1, one of at least two PLC isozymes previously shown to be in wild type CCL39 cytosol (Rath, H. M., Fee, J. A., Rhee, S. G., and Silbert, D. F. (1990) J. Biol. Chem. 265, 3080-3087). We also report here the presence of two peaks of PLC activities (i.e. peaks mA and mB) following Mono Q chromatography of wild type CCL39 membrane/particulate extracts; membrane/particulate extracts prepared from mutant D1-9b are missing the first of these two peaks of activities (peak mA). Immunoblot analysis confirms the presence of PLC beta 1, but not PLC gamma 1 or PLC delta 1, in peak mB. Comparisons are made between D1-9b and the previously characterized mutant D1-6b, which displays diminished agonist-induced PI turnover responses yet in vitro biochemical deficiencies similar to those of mutant D1-9b. Somatic cell hybridization experiments suggest that the defects found in mutant D1-9b are codominant relative to wild type phenotype and that mutant D1-9b belongs to the same genetic complementation group as mutant D1-6b. Possible explanations to explain these different agonist-induced responses in light of the two mutants similar in vitro biochemical deficiencies are addressed.

• Hansen S, Hansen LK, Hough E
• Crystal structures of phosphate, iodide and iodate-inhibited phospholipase C from Bacillus cereus and structural investigations of the binding of reaction products and a substrate analogue.
• J Mol Biol. 1992; 225: 543-9
• Display abstract

• The crystal structure of the complex formed between phospholipase C (PLC) from Bacillus cereus and inorganic phosphate (Pi), which is an inhibitor, has been determined and refined to 2.1 A resolution. The final R-factor is 19.7%. We have also studied the binding of two other inhibitors, iodide and iodate, to PLC. X-ray data for these two complexes were collected to 2.8 A resolution during the search for heavy-atom derivatives. A series of screening experiments where PLC crystals have been treated with several reaction products and a substrate analogue were carried out to clarify the question of substrate binding. The results have so far been ambiguous but are discussed briefly. Phosphate and iodate are both found to bind to the three metal ions in the protein molecule, suggesting that these ions are involved directly in the catalytic process and thereby identifying the active site. PLC also binds nine iodide ions, eight of which are on the surface of the molecule and of lower occupancy. The ninth blocks the entrance to the active site cleft and is of higher occupancy. Altogether, these results suggest that the substrate, a phospholipid, is associated directly with the metal ions during catalysis.

• Byberg JR, Jorgensen FS, Hansen S, Hough E
• Substrate-enzyme interactions and catalytic mechanism in phospholipase C: a molecular modeling study using the GRID program.
• Proteins. 1992; 12: 331-8
• Display abstract

• Based on the high-resolution X-ray crystallographic structure of phospholipase C from Bacillus cereus, the orientation of the phosphatidylcholine substrate in the active site of the enzyme is proposed. The proposal is based on extensive calculations using the GRID program and molecular mechanics geometry relaxations. The substrate model has been constructed by successively placing phosphate, choline and diacylglycerol moieties in the positions indicated from GRID calculations. On the basis of the resulting orientation of a complete phosphatidylcholine molecule, we propose a mechanism for the hydrolysis of the substrate.

• Rhee SG, Choi KD
• Regulation of inositol phospholipid-specific phospholipase C isozymes.
• J Biol Chem. 1992; 267: 12393-6

• Leigh AJ, Volwerk JJ, Griffith OH, Keana JF
• Substrate stereospecificity of phosphatidylinositol-specific phospholipase C from Bacillus cereus examined using the resolved enantiomers of synthetic myo-inositol 1-(4-nitrophenyl phosphate).
• Biochemistry. 1992; 31: 8978-83
• Display abstract

• The substrate stereospecificity of phosphatidylinositol-specific phospholipase C from Bacillus cereus is examined using the resolved optical isomers of synthetic myo-inositol 1-(4-nitrophenyl phosphate), a chromogenic substrate for the phospholipase. The synthetic route employs mild acid-labile protecting groups and separation of the substituted myo-inositol enantiomers as the (-)-camphanyl ester diastereomers. Measurements of the initial rates of cleavage of the D and L enantiomers of the nitrophenyl substrate by phosphatidylinositol-specific phospholipase C from B. cereus show that this enzyme is essentially stereospecific for the D enantiomer. Under identical conditions, the rate of cleavage of the L isomer is less than 0.2% of that observed for the D isomer. The same is observed for the highly homologous enzyme from Bacillus thuringiensis. There is no measurable inhibition by the L enantiomer of the B. cereus enzyme acting on the D enantiomer, even when the molar ratio of L:D is 5, indicating that binding of the L enantiomer to the phospholipase is negligible. Thus, the enzyme active site is exquisitely sensitive to the stereochemistry of the myo-inositol group of the substrate.

• Shashidhar MS, Volwerk JJ, Griffith OH, Keana JF
• A chromogenic substrate for phosphatidylinositol-specific phospholipase C: 4-nitrophenyl myo-inositol-1-phosphate.
• Chem Phys Lipids. 1991; 60: 101-10
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• A chromogenic water-soluble substrate for phosphatidylinositol-specific phospholipase C was synthesized starting from myo-inositol employing isopropylidene and 4-methoxytetrahydropyranyl protecting groups. In this analogue of phosphatidylinositol, 4-nitrophenol replaces the diacylglycerol moiety, resulting in synthetic, racemic 4-nitrophenyl myo-inositol-1-phosphate. Using this synthetic substrate a rapid, convenient and sensitive spectrophotometric assay for the phosphatidylinositol-specific phospholipase C from Bacillus cereus was developed. Initial rates of the cleavage of the nitrophenol substrate were linear with time and the amount of enzyme used. At pH 7.0, specific activities for the B. cereus enzyme were 77 and 150 mumol substrate cleaved min-1 (mg protein)-1 at substrate concentrations of 1 and 2 mM, respectively. Under these conditions, less than 50 ng quantities of enzyme were easily detected. The chromogenic substrate was stable during long term storage (6 months) as a solid at -20 degrees C.

• Shashidhar MS, Volwerk JJ, Keana JF, Griffith OH
• A fluorescent substrate for the continuous assay of phosphatidylinositol-specific phospholipase C: synthesis and application of 2-naphthyl myo-inositol-1-phosphate.
• Anal Biochem. 1991; 198: 10-4
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• A fluorescent water-soluble substrate for phosphatidylinositol-specific phospholipase C was synthesized. The diacylglycerol moiety of the natural substrate, phosphatidylinositol, was replaced by the fluorescent moiety, 2-naphthol, resulting in the synthetic substrate, racemic 2-naphthyl myo-inositol-1-phosphate. The synthetic substrate provided a continuous fluorometric assay for the phosphatidylinositol-specific phospholipase C from Bacillus cereus. Initial rates of the cleavage of the 2-naphthyl substrate by the phospholipase measured by fluorometry were linear with time and the amount of enzyme added. The specific enzyme activity at pH 8.5 and 25 degrees C was about 0.04 mumol/min mg protein at an initial substrate concentration of 0.8 mM. 31P NMR experiments suggest that, as with phosphatidylinositol itself, cleavage of the fluorescent substrate proceeds in two steps via a myo-inositol-1,2-cyclic phosphate intermediate, and that only the D-isomer is a substrate for the B. cereus phospholipase. The synthetic substrate was stable during long-term storage as a solid in the dark at -20 degrees C. It was also stable for several weeks when stored in the dark frozen in aqueous solution near neutral pH.

• Volwerk JJ, Shashidhar MS, Kuppe A, Griffith OH
• Phosphatidylinositol-specific phospholipase C from Bacillus cereus combines intrinsic phosphotransferase and cyclic phosphodiesterase activities: a 31P NMR study.
• Biochemistry. 1990; 29: 8056-62
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• The inositol phosphate products formed during the cleavage of phosphatidylinositol by phosphatidylinositol-specific phospholipase C from Bacillus cereus were analyzed by 31P NMR. 31P NMR spectroscopy can distinguish between the inositol phosphate species and phosphatidylinositol. Chemical shift values (with reference to phosphoric acid) observed are 0.41, 3.62, 4.45, and 16.30 ppm for phosphatidylinositol, myo-inositol 1-monophosphate, myo-inositol 2-monophosphate, and myo-inositol 1,2-cyclic monophosphate, respectively. It is shown that under a variety of experimental conditions this phospholipase C cleaves phosphatidylinositol via an intramolecular phosphotransfer reaction producing diacylglycerol and D-myo-inositol 1,2-cyclic monophosphate. We also report the new and unexpected observation that the phosphatidylinositol-specific phospholipase C from B. cereus is able to hydrolyze the inositol cyclic phosphate to form D-myo-inositol 1-monophosphate. The enzyme, therefore, possesses phosphotransferase and cyclic phosphodiesterase activities. The second reaction requires thousandfold higher enzyme concentrations to be observed by 31P NMR. This reaction was shown to be regiospecific in that only the 1-phosphate was produced and stereospecific in that only D-myo-inositol 1,2-cyclic monophosphate was hydrolyzed. Inhibition with a monoclonal antibody specific for the B. cereus phospholipase C showed that the cyclic phosphodiesterase activity is intrinsic to the bacterial enzyme. We propose a two-step mechanism for the phosphatidyl-inositol-specific phospholipase C from B. cereus involving sequential phosphotransferase and cyclic phosphodiesterase activities. This mechanism bears a resemblance to the well-known two-step mechanism of pancreatic ribonuclease, RNase A.

• Bairoch A, Cox JA
• EF-hand motifs in inositol phospholipid-specific phospholipase C.
• FEBS Lett. 1990; 269: 454-6

• Crooke ST, Bennett CF
• Mammalian phosphoinositide-specific phospholipase C isoenzymes.
• Cell Calcium. 1989; 10: 309-23
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• Procaryotic and eucaryotic cells have evolved multiple pathways for communication with their external environment. The inositol 1,4,5-trisphosphate/diacylglycerol second messenger system is an example of such a signal transduction pathway which is present in multicellular eucaryotic organisms. Binding of an agonist to a specific cell surface receptor promotes rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate. The pivotal enzyme for this second messenger system is phosphoinositide-specific phospholipase C which hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate the two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol. Recently, much progress has been made in the purification, characterization and cDNA cloning of multiple PI-PLC isoenzymes. The results of the recent studies on phosphoinositide-specific phospholipase C are reviewed.

• Hough E et al.
• High-resolution (1.5 A) crystal structure of phospholipase C from Bacillus cereus.
• Nature. 1989; 338: 357-60
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• Both the phosphatidylinositol-hydrolysing and the phosphatidylcholine-hydrolysing phospholipases C have been implicated in the generation of second messengers in mammalian cells. The phosphatidylcholine-hydrolysing phospholipase C (PLC) from Bacillus cereus, a monomeric protein containing 245 amino-acid residues, is similar to some of the corresponding mammalian proteins. This, together with the fact that the bacterial enzyme can mimic the action of mammalian PLC in causing, for example, enhanced prostaglandin biosynthesis, suggests that B. cereus PLC can be used as a model for the hitherto poorly characterized mammalian PLCs. We report here the three-dimensional structure of B. cereus PLC at 1.5 A resolution. The enzyme is an all-helix protein belonging to a novel structural class and contains, at least in the crystalline state, three Zn2+ in the active site. We also present preliminary results from a study at 1.9 A resolution of the complex between PLC and inorganic phosphate (Pi) which indicate that the substrate binds directly to the metal ions.

• Bennett CF, Balcarek JM, Varrichio A, Crooke ST
• Molecular cloning and complete amino-acid sequence of form-I phosphoinositide-specific phospholipase C.
• Nature. 1988; 334: 268-70
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• We report the molecular cloning and sequence of a phosphoinositide-specific phospholipase C (PI-PLC), an enzyme that is of particular interest because of its central role in cell signal transduction. The signals in question are those delivered by hormones to their cell-surface receptors that activate PI-PLC by means of a guanine nucleotide binding protein. Activation of the enzyme leads to the hydrolysis of phosphatidylinositol 4,5-bisphosphate to two second messengers, 1,2-diacylglycerol and inositol 1,4,5-trisphosphate, the second of which ultimately mobilizes internal pools of calcium. There are at least five PI-PLC isoenzymes, whose differences in structure and function are unknown. We have focused on isoenzyme I, which we have recently purified and characterized from guinea pig uterus. We have now determined the sequence of a full length complementary DNA of this isoenzyme from the rat. Although the sequence has little similarity with the only other sequenced PI-PLC isoenzyme, it has a surprising degree of similarity to thioredoxins, protein co-factors in thiol-dependent redox reactions.

• Homma Y, Imaki J, Nakanishi O, Takenawa T
• Isolation and characterization of two different forms of inositol phospholipid-specific phospholipase C from rat brain.
• J Biol Chem. 1988; 263: 6592-8
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• Two different forms of inositol phospholipid-specific phospholipase C (PLC) have been purified 2810- and 4010-fold, respectively, from a crude extract of rat brain. The purification procedures consisted of chromatography of both enzymes on Affi-Gel blue and cellulose phosphate, followed by three sequential high performance liquid chromatography steps, which were different for the two enzymes. The resultant preparations each contained homogeneous enzyme with a Mr of 85,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One of these enzymes (PLC-II) was found to hydrolyze phosphatidyl-inositol 4,5-bisphosphate (PIP2) at a rate of 15.3 mumol/min/mg of protein and also phosphatidylinositol 4-monophosphate and phosphatidylinositol (PI) at slower rates. For hydrolysis of PI, this enzyme was activated by an acidic pH and a high concentration of Ca2+ and showed a Vmax value of 19.2 mumol/min/mg of protein. The other enzyme (PLC-III) catalyzed hydrolysis of PIP2 preferentially at a Vmax rate of 12.9 mumol/min/mg of protein and catalyzed that of phosphatidylinositol 4-monophosphate slightly. The rate of PIP2 hydrolysis by this enzyme exceeded that of PI under all conditions tested. Neither of these enzymes had any activity on phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, or phosphatidic acid. These two enzymes showed not only biochemical but also structural differences. Western blotting showed that antibodies directed against PLC-II did not react with PLC-III. Furthermore, the two enzymes gave different peptide maps after digestion with alpha-chymotrypsin or Staphylococcus aureus V8 protease. These results suggest that these two forms of PLC belong to different families of PLC.

• Nakanishi O, Homma Y, Kawasaki H, Emori Y, Suzuki K, Takenawa T
• Purification of two distinct types of phosphoinositide-specific phospholipase C from rat liver. Enzymological and structural studies.
• Biochem J. 1988; 256: 453-9
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• Two kinds of phosphoinositide-specific phospholipase C (PLC) were purified from rat liver by acid precipitation and several steps of column chromatography. About 50% of the activity could be precipitated when the pH of the liver homogenate was lowered to pH 4.7. The redissolved precipitate yielded two peaks, PLC I and PLC II, in an Affi-gel Blue column, and each was further purified to homogeneity by three sequential h.p.l.c. steps, which were different for the two enzymes. The purified PLC I and PLC II had estimated Mr values of 140,000 and 71,000 respectively on SDS/polyacrylamide-gel electrophoresis. Both enzymes hydrolysed phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) in a Ca2+- and pH-dependent manner. PLC I was most active at 10 microM- and 0.1 mM-Ca2+ for hydrolysis of PI and PIP2 respectively, whereas PLC II showed the highest activity at 5 mM- and 10 microM-Ca2+ for that of PI and PIP2 respectively. The optimal pH of the two enzymes also differed with substrates or Ca2+ concentration, in the range pH 5.0-6.0. Hydrolysis of phosphoinositides by these enzymes was completely inhibited by Hg2+ and was affected by other bivalent cations. From data obtained by peptide mapping and partial amino acid sequencing, it was clarified that PLC I and PLC II had distinct structures. Moreover, partial amino acid sequences of three proteolytic fragments of PLC I completely coincided with those of PLC-148 [Stahl, Ferenz, Kelleher, Kriz & Knopf (1988) Nature (London) 332, 269-272].

• Connolly TM, Wilson DB, Bross TE, Majerus PW
• Isolation and characterization of the inositol cyclic phosphate products of phosphoinositide cleavage by phospholipase C. Metabolism in cell-free extracts.
• J Biol Chem. 1986; 261: 122-6
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• The phosphoinositides are metabolized by phospholipase C in response to hormone or agonist stimulation in many cell types to produce diglyceride and water-soluble inositol phosphates. We have recently shown that the phospholipase C reaction products include cyclic phosphate esters of inositol. One of these, inositol 1, 2-cyclic 4,5-trisphosphate, is active in promoting Ca2+ mobilization in platelets and in inducing changes in conductance in Limulus photoreceptors similar to those produced by light (Wilson, D. B., Connolly, T. M., Bross, T. E., Majerus, P. W., Sherman, W. R., Tyler, A., Rubin, L. J., and Brown, J. E. (1985) J. Biol. Chem. 260, 13496-13501. In the current study, we have examined the metabolism of the inositol phosphates. We find that both cyclic and non-cyclic inositol trisphosphates are metabolized by inositol 1,4,5-trisphosphate 5-phosphomonoesterase, to inositol 1,2-cyclic bisphosphate and inositol 1,4-bisphosphate, respectively. However, the apparent Km of the enzyme for the cyclic substrate is approximately 10-fold higher than for the non-cyclic substrate. These inositol bisphosphates are more slowly degraded to inositol 1,2-cyclic phosphate and inositol 1-phosphate, respectively. Inositol 1,2-cyclic phosphate is then hydrolyzed to inositol 1-phosphate, which in turn is degraded to inositol and inorganic phosphate by inositol 1-phosphate phosphatase. The human platelet inositol 1,2-cyclic phosphate hydrolase enzyme and a similar rat kidney hydrolase do not utilize the cyclic polyphosphate esters of inositol as substrates. These results suggest that the inositol cyclic phosphates and the non-cyclic inositol phosphates are metabolized separately by phosphatases to cyclic and non-cyclic inositol monophosphates. The cyclic monophosphate is then converted to inositol 1-phosphate by a cyclic hydrolase. We suggest that the enzymes that metabolize the inositol phosphates may serve to regulate cellular responses to these compounds.

• Hardman KD, Ainsworth CF
• Myo-inositol binding site of concanavalin A.
• Nat New Biol. 1972; 237: 54-5

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