Secondary literature sources for the DAGKa domain

For each of the hand selected primary papers associated with the DAGKa domain, 100 neighbouring papers are retrieved from PubMed. If one of these neighbouring papers is referenced by more than two primary papers, it is shown in the table below.

Gene structure and expression of an unusual protein kinase from Plasmodium falciparum homologous at its carboxyl terminus with the EF hand calcium-binding proteins.

Zhao Y, Kappes B, Franklin RM
J Biol Chem. 1993; 268: 4347-54

An unusual protein kinase gene, termed PfCPK, was isolated from Plasmodium falciparum. The gene, which contains five exons and four introns, encodes a product with a predicted length of 524 amino acids. The amino-terminal segment of the predicted protein contains all of the conserved sequences characteristic of a protein kinase catalytic domain and has a high homology to several protein serine-threonine kinase subfamilies (30-41% amino acid identities). These subfamilies include calcium/calmodulin-dependent protein kinases, calcium-dependent protein kinase, ribosomal S6 protein kinase, cyclic nucleotide-dependent protein kinases, protein kinase C, and the yeast SNF1 subfamily. All of these protein kinases are relatively close in the phylogeny tree and within the kinase catalytic domains have about 35% amino acid identities to each other, suggesting that PfCPK is also in this region of the phylogeny tree. An unusual feature of PfCPK is that its carboxyl-terminal segment displays homology to the EF hand calcium-binding proteins, for example 34% amino acid identity to chicken fast skeletal muscle troponin C and 35% amino acid identity to human calmodulin. Like troponin Cs and calmodulins, PfCPK also contains four EF hand calcium-binding motifs. Furthermore, the four introns in the PfCPK gene are all located in the carboxyl-terminal putative EF hand calcium-binding region (EF hand calcium-binding proteins from higher eukaryotes generally contain multiple introns). This combination of a protein kinase and an EF hand calcium-binding protein in a single polypeptide implies that PfCPK may be directly activated by calcium. Constructs containing the full-length PfCPK cDNA have been expressed in Escherichia coli at a high level to generate a 60-kDa recombinant protein. Compared with similar fractions from control cells, the fraction containing PfCPK recombinant protein exhibited an elevated protein kinase activity which was Ca(2+)-dependent.

EF-hand motifs of alpha, beta and gamma isoforms of diacylglycerol kinase bind calcium with different affinities and conformational changes.

Yamada K, Sakane F, Matsushima N, Kanoh H
Biochem J. 1997; 321: 59-64

The three diacylglycerol kinase isoenzymes (DGK alpha, DGK beta and DGK gamma) cloned so far contain in common a tandem repeat of EF-hand motifs. However, the Ca2+ dependences of the DGK activities are known to be variable between isoenzymes, and the Ca(2+)-binding activities of these motifs have not been tested except for those present in DGK alpha. We therefore attempted to define the intrinsic properties of EF-hands occurring in the DGK isoenzymes. For this purpose we bacterially expressed and purified the EF-hand motifs (termed DKE forms) of the three DGKs. Equilibrium dialysis with the purified DKE forms showed that all of the expressed proteins could bind approx. 2 mol of Ca2+ per mol. However, the apparent dissociation constant (Kd) for calcium binding to alpha-DKE (9.9 microM) was an order of magnitude greater than those estimated for beta-DKE (0.89 microM) and gamma-DKE (0.40 microM). Experiments with 2-p-toluidinyl-naphthalene 6-sulphonate, a probe for hydrophobic regions of proteins, showed that the binding of Ca2+ to beta-DKE resulted in the exposure of hydrophobic amino acids, whereas hydrophobic regions of alpha-DKE and gamma-DKE were masked by the addition of Ca2+. Taken together, these results indicate that DGK alpha, DGK beta and DGK gamma possess EF-hand structures with intrinsic properties different from each other with respect to affinities for Ca2+ and Ca(2+)-induced conformational changes.

Immunoquantitation of 80 kDa diacylglycerol kinase in pig and human lymphocytes and several other cells.

Yamada K, Sakane F, Kanoh H
FEBS Lett. 1989; 244: 402-6

80 kDa diacylglycerol kinase (DGK) was immunoquantitated in cell homogenates and subcellular fractions. It was extremely abundant in the cytosol of various lymphocytes and comprised, in the highest case, more than 0.2% of the total soluble protein in T cell-enriched pig splenocytes. The lymphocyte membrane contained less than 10% of the total cellular DGK protein. The content of 80 kDa DGK in the human T cell leukemic cell line, Jurkat (360 ng/mg homogenate protein), was similar to those in pig and human peripheral blood lymphocytes. In contrast, the enzyme level was very low in the human promyeloblastic cell line, HL-60 (less than 10 ng/mg homogenate protein), and was undetectable in human polymorphonuclear leukocytes. These findings indicate that the content of 80 kDa DGK is markedly variable depending on the type of cells, even though all these cells are known to accumulate phosphatidate rapidly upon cell stimulation.

Sphingosine activates cellular diacylglycerol kinase in intact Jurkat cells, a human T-cell line.

Yamada K, Sakane F, Imai S, Takemura H
Biochim Biophys Acta. 1993; 1169: 217-24

Sphingosine is known to regulate a variety of cellular functions through protein kinase C-dependent or independent pathways. In an attempt to investigate differential functions of diacylglycerol kinase (DGK) isozymes, we tested the effect of sphingosine on DGK operating in intact Jurkat cells, a human T-cell line. We found that phosphatidic acid (PA) synthesized from endogenous diacylglycerol (DG) and exogenously added short-chain DGs like dioctanoylglycerol were markedly enhanced by approx. 20 microM sphingosine. Further studies such as the use of protein kinase C down-regulated cells, mass measurements of cellular DGs, analysis of molecular species of PA and the effect of exogenous DG on the conversion of endogenous DG to PA suggested that sphingosine directly activated cellular DGK having a broad specificity toward DG molecular species.

The different effects of sphingosine on diacylglycerol kinase isozymes in Jurkat cells, a human T-cell line.

Yamada K, Sakane F
Biochim Biophys Acta. 1993; 1169: 211-6

We studied the effect of sphingosine on the activities of soluble and membrane-bound isozymes from Jurkat cells using combinations of different substrates (arachidonoyl- and didecanoyl DGs) and assay methods (octylglucoside mixed micellar and deoxycholate suspension assays). The results suggested the presence of at least four DGK isoforms, which could be distinguished from each other with respect to intracellular localization, specificity to DG molecular species, responsiveness to sphingosine, and reactivity to anti-80 kDa DGK antibody. We confirmed the presence of arachidonoyl DG-specific DGK in membranes, though this isozyme was not activated by sphingosine. We detected in the cytosol at least two species of sphingosine-activatable and non-activatable DGK isoforms, the major species being the 80 kDa DGK. We postulate that both or either of the two soluble DGKs may be the target of the sphingosine action.

Occurrence of immunoreactive 80 kDa and non-immunoreactive diacylglycerol kinases in different pig tissues.

Yamada K, Kanoh H
Biochem J. 1988; 255: 601-8

We surveyed diacylglycerol kinase in different pig tissues by using rabbit antibody immunospecific to the brain 80 kDa enzyme [Kanoh, Iwata, Ono & Suzuki (1986) J. Biol. Chem. 261, 5597-5602]. Among the other tissues examined, the immunoreactive 80 kDa enzyme was found only in the thymus and, to a much lesser extent, in the spleen, although this enzyme species was widely distributed in a variety of brain regions. Other tissues such as platelets, kidney, heart and liver contained little, if any, immunoreactive enzymes. Gel filtration of cytosolic enzymes from several tissues revealed the presence of three major activity peaks, apparently corresponding to 280, 120 and 80 kDa. Thymus and spleen contained the immunoreactive 80 kDa species together with non-immunoreactive 280 kDa enzyme. In the case of platelets, the kinase consisted almost exclusively of non-immunoreactive 120 kDa species with some 280 kDa enzyme. In an attempt to characterize the different kinase forms, the thymus enzyme was chosen for further studies because of its high activity. No immunoreactive proteins were detected in Western-blot analysis when the 280 kDa enzyme was solvent-extracted, proteinase-treated or preincubated in the presence of Ca2+. In comparison with the 80 kDa species, the 280 kDa enzyme was much more heat-stable and less dependent on deoxycholate in the assay mixture. Although the purification of different forms of the kinase is required to confirm the presence of isoenzymes, the results show that there exist several immunologically distinct diacylglycerol kinase species.

Regulation of neurite outgrowth in N1E-115 cells through PDZ-mediated recruitment of diacylglycerol kinase zeta.

Yakubchyk Y, Abramovici H, Maillet JC, Daher E, Obagi C, Parks RJ, Topham MK, Gee SH
Mol Cell Biol. 2005; 25: 7289-302

Syntrophins are scaffold proteins that regulate the subcellular localization of diacylglycerol kinase zeta (DGK-zeta), an enzyme that phosphorylates the lipid second-messenger diacylglycerol to yield phosphatidic acid. DGK-zeta and syntrophins are abundantly expressed in neurons of the developing and adult brain, but their function is unclear. Here, we show that they are present in cell bodies, neurites, and growth cones of cultured cortical neurons and differentiated N1E-115 neuroblastoma cells. Overexpression of DGK-zeta in N1E-115 cells induced neurite formation in the presence of serum, which normally prevents neurite outgrowth. This effect was independent of DGK-zeta kinase activity but dependent on a functional C-terminal PDZ-binding motif, which specifically interacts with syntrophin PDZ domains. DGK-zeta mutants with a blocked C terminus acted as dominant-negative inhibitors of outgrowth from serum-deprived N1E-115 cells and cortical neurons. Several lines of evidence suggest DGK-zeta promotes neurite outgrowth through association with the GTPase Rac1. DGK-zeta colocalized with Rac1 in neuronal processes and DGK-zeta-induced outgrowth was inhibited by dominant-negative Rac1. Moreover, DGK-zeta directly interacts with Rac1 through a binding site located within its C1 domains. Together with syntrophin, these proteins form a tertiary complex in N1E-115 cells. A DGK-zeta mutant that mimics phosphorylation of the MARCKS domain was unable to bind an activated Rac1 mutant (Rac1(V12)) and phorbol myristate acetate-induced protein kinase C activation inhibited the interaction of DGK-zeta with Rac1(V12), suggesting protein kinase C-mediated phosphorylation of the MARCKS domain negatively regulates DGK-zeta binding to active Rac1. Collectively, these findings suggest DGK-zeta, syntrophin, and Rac1 form a regulated signaling complex that controls polarized outgrowth in neuronal cells.

Purification and characterization of cytosolic diacylglycerol kinases of human platelets.

Yada Y, Ozeki T, Kanoh H, Nozawa Y
J Biol Chem. 1990; 265: 19237-43

Three isozymes of diacylglycerol kinase (DGK), DGK-I, DGK-II, and DGK-III, were purified from the cytosol of human platelets by successive chromatography on DEAE-cellulose, Ultrogel AcA34, heparin-Sepharose, ATP-agarose, Mono Q, phenyl-Superose, HCA-hydroxyapatite, Wakopak G40, and TSK-3000SW columns. Two DGK species (DGK-I and DGK-III) were purified to apparent homogeneity, and upon SDS-polyacrylamide gel electrophoresis, they showed a single band of apparent molecular mass of 152 kDa (DGK-I) or 58 kDa (DGK-III). The peptide mapping analysis showed that DGK-I and DGK-III are structurally different. DGK-II was only partially purified, and its apparent Mr was estimated to be 75,000 by gel filtration. The specific enzyme activities of the three isozymes were increased 1,480-fold (DGK-I), 690-fold (DGK-II) and 2,100-fold (DGK-III) over original platelet cytosol. The activities of DGK-II and DGK-III were markedly enhanced by the presence of deoxycholate or phosphatidylserine, whereas DGK-I activity was not much affected by the anionic compounds. All of the three activities were strongly suppressed by phosphatidylcholine. Triton X-100 and octyl glucoside were strongly inhibitory to all of the enzymes, although to different extents. The DGK inhibitor, R59022, inhibited DGK-II and to a lesser extent DGK-III, but little affected DGK-I activity. DGK-I was much more heat-stable than DGK-II and DGK-III. The Km values for ATP were 150 microM for DGK-I, 245 microM for DGK-II, and 450 microM for DGK-III. The apparent Km values for suspended diolein were not much different among the DGKs and were in the range of 50-80 microM. These observations indicate that human platelet cytosol contains DGK isozymes with different enzymological properties. Furthermore, the three DGKs isolated from human platelets were found not to cross-react with the antibody raised against porcine brain 80-kDa DGK, thus indicating that human platelets contain novel species of DGK.

Production of a novel calmodulin-binding DGK by alternative splicing.

Wood NT
Trends Plant Sci. 2001; 6: 50-50

Isolation and molecular cloning of human sorcin a calcium-binding protein in vincristine-resistant HOB1 lymphoma cells.

Wang SL, Tam MF, Ho YS, Pai SH, Kao MC
Biochim Biophys Acta. 1995; 1260: 285-93

A vincristine-resistant lymphoma cell line (HOB1/VCR1.0) that is resistant to 1.0 microM of vincristine has been established from a human immunoblastic B lymphoma cell line, HOB1. HOB1/VCR1.0 cells demonstrated the typical multidrug resistant phenotypes. Using two-dimensional gel electrophoresis, we discovered one protein with a molecular mass of 22 kDa and pI 5.7 that was overexpressed in HOB1/VCR1.0 cells. This protein was purified to the degree of apparent homogeneity by preparative isoelectric focusing and sodium dodecylsulfate-polyacrylamide gel electrophoresis. The identification of this protein with sorcin was revealed by comparing the internal amino acid sequence of three Lys-C digested peptides from the purified protein with the sequence previously determined for hamster sorcin. The complete primary structure of the human sorcin was deduced from nucleotide sequence analysis of its cDNA clones. It is composed of 198 amino acid residues with a calculated molecular weight of 21,676, and its sequence is highly similar to that of hamster sorcin (95%). Direct-binding assay with calcium showed that human sorcin is a calcium-binding protein with four 'E-F hand' structures typical of calcium-binding sites. Like the sorcin of hamster, two of the calcium-binding sites of human sorcin contain putative recognition sites for cAMP-dependent protein kinase. Southern and Northern blot analyses showed that the human sorcin gene was greatly amplified and overexpressed in resistant HOB1/VCR1.0 cells but not detected in the parental HOB1 cells. The overproduction of this protein in resistant cells implies that sorcin plays a role in expression of the resistant phenotype.

Cloning and expression of human mitochondrial deoxyguanosine kinase cDNA.

Wang L, Hellman U, Eriksson S
FEBS Lett. 1996; 390: 39-43

Mammalian mitochondrial deoxyguanosine kinase (dGK) is responsible for phosphorylation of purine deoxyribonucleosides in the mitochondrial matrix. Using a RT-PCR-generated probe, based on amino acid sequence information from proteolytic fragments of purified bovine dGK, we have cloned a cDNA from a human brain cDNA library that encodes a 30 kDa protein. The deduced amino acid sequence of this protein included the sequence of all six peptides isolated and sequenced from purified dGK. Expression and purification of recombinant protein from induced Escherichia coli extracts revealed that it catalyses efficient phosphorylation of dGuo, arabinosyl guanine, dAdo, 2-chloro-2'-deoxyadenosine and dIno similar to purified dGK. Northern blot analysis demonstrated one dominant positive mRNA of 1.35 kb and it was found in several tissues at similar levels. The coding sequence of dGK showed 46% identity to the coding sequence of cytosolic deoxycytidine kinase, and conserved sequence motifs among the known deoxynucleoside kinase were identified.

Arachidonoyl-diacylglycerol kinase from bovine testis. Purification and properties.

Walsh JP, Suen R, Lemaitre RN, Glomset JA
J Biol Chem. 1994; 269: 21155-64

Previous work in our laboratory demonstrated the existence of a membrane-bound diacylglycerol kinase highly selective for diacylglycerols containing arachidonate as the sn-2 fatty acyl moiety (MacDonald, M. L., Mack K. F., Richardson, C. N., and Glomset, J. A. (1988) J. Biol. Chem. 263, 1575-1583). We now report the purification of arachidonoyl-diacylglycerol kinase 34,400-fold to apparent homogeneity from bovine testis. High concentrations of both salt and detergent were required to extract the enzyme from membranes and stabilize its activity, suggesting that in vivo the enzyme is part of a complex with other membrane or cytoskeletal proteins. Arachidonoyl-diacylglycerol kinase had an apparent M(r) of 58,000 both on SDS-polyacrylamide gels and by size exclusion chromatography. The enzyme appeared to be an integral membrane protein. In a mixed micellar assay, arachidonoyl-diacylglycerol kinase followed surface dilution kinetics with respect to diacylglycerol. The purified enzyme retained the arachidonate selectivity observed previously in membranes. Kinetic analyses indicated a Km for sn-1-stearoyl-2-arachidonoylglycerol of 2.4 mol %, as compared to 43 mol % for sn-1-palmitoyl-2-oleoylglycerol. Calcium, an activator of some other diacylglycerol kinases, had no apparent effect on the arachidonate-specific enzyme. Guanosine triphosphate could effectively substitute for ATP as the phosphoryl donor and Mg2+ could be replaced by Mn2+ or Ca2+. Phosphatidylserine and, to a lesser extent, phosphatidylinositol inhibited the purified enzyme. Phosphatidylcholine and phosphatidylethanolamine had only small effects.

Diacylglycerol kinase theta is translocated and phosphoinositide 3-kinase-dependently activated by noradrenaline but not angiotensin II in intact small arteries.

Walker AJ, Draeger A, Houssa B, van Blitterswijk WJ, Ohanian V, Ohanian J
Biochem J. 2001; 353: 129-137

Diacylglycerol (DG) kinase (DGK) phosphorylates the lipid second messenger DG to phosphatidic acid. We reported previously that noradrenaline (NA), but not angiotensin II (AII), increases membrane-associated DGK activity in rat small arteries [Ohanian and Heagerty (1994) Biochem. J. 300, 51-56]. Here, we have identified this DGK activity as DGKtheta, present in both smooth muscle and endothelial cells of these small vessels. Subcellular fractionation of artery homogenates revealed that DGKtheta was present in nuclear, plasma membrane (and/or Golgi) and cytosolic fractions. Upon NA stimulation, DGKtheta translocated towards the membrane and cytosol (155 and 153% increases relative to the control, respectively) at 30 s, followed by a return to near-basal levels at 5 min; AII was without effect. Translocation to the membrane was to both Triton-soluble and -insoluble fractions. NA, but not AII, transiently increased DGKtheta activity in immunoprecipitates (126% at 60 s). Membrane translocation and DGKtheta activation were regulated differently: NA-induced DGKtheta activation, but not translocation, was dependent on transient activation of phosphoinositide 3-kinase (PI 3-K). In addition, DGK activity co-immunoprecipitated with protein kinase B, a downstream effector of PI 3-K, and was increased greatly by NA stimulation. The rapid and agonist-specific activation of DGKtheta suggests that this pathway may have a physiological role in vascular smooth-muscle responses.

PPARgamma agonists ameliorate endothelial cell activation via inhibition of diacylglycerol-protein kinase C signaling pathway: role of diacylglycerol kinase.

Verrier E, Wang L, Wadham C, Albanese N, Hahn C, Gamble JR, Chatterjee VK, Vadas MA, Xia P
Circ Res. 2004; 94: 1515-22

Subject- Peroxisome proliferator-activated receptor (PPAR)-gamma agonists are emerging as potential protectors against inflammatory cardiovascular diseases including atherosclerosis and diabetic complications. However, their molecular mechanism of action within vasculature remains unclear. We report here that PPARgamma agonists, thiazolidinedione class drugs (TZDs), or 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) were capable of activating diacylglycerol (DAG) kinase (DGK), resulting in attenuation of DAG levels and inhibition of protein kinase C (PKC) activation. The PPARgamma agonist-induced DGK was completely blocked by a dominant-negative mutant of PPARgamma, indicating an essential receptor-dependent action. Importantly, the suppression of DAG-PKC signaling pathway was functional linkage to the anti-inflammatory properties of PPARgamma agonists in endothelial cells (EC), characterized by the inhibition of proinflammatory adhesion molecule expression and adherence of monocytes to the activated EC induced by high glucose. These findings thus demonstrate a novel molecular action of PPARgamma agonists to suppress the DAG-PKC signaling pathway via upregulation of an endogenous attenuator, DGK.

Tissue- and cell-specific expression of Ins(1,4,5)P3 3-kinase isoenzymes.

Vanweyenberg V, Communi D, D'Santos CS, Erneux C
Biochem J. 1995; 306: 429-35

The phosphorylation of Ins(1,4,5)P3 (InsP3) to Ins(1,3,4,5)P4 (InsP4) is catalysed by InsP3 3-kinase. Molecular-biological data have shown the presence of two human isoenzymes of InsP3 3-kinase, namely InsP3 3-kinases A and B. We have isolated from a rat thymus cDNA library a 2235 bp cDNA (clone B15) encoding rat InsP3 3-kinase B. Northern-blot analysis of mRNA isolated from rat tissues (thymus, testis, brain, spleen, liver, kidney, heart, lung and intestine) revealed that a rat InsP3 3-kinase B probe hybridized to a 6 kb mRNA in lung, thymus, testis, brain and heart. In contrast, Northern-blot analysis of the same tissues probed under stringent conditions with a rat InsP3 3-kinase A probe hybridized to a 2 kb mRNA only in brain and a 1.8-2.0 kb mRNA species in testis. Northern-blot analysis of three human cell lines (HL-60, SH-SY5Y and HTB-138) probed with a human InsP3 3-kinase B probe showed the presence of a 6 kb mRNA in all cell lines, except in the human neuroblastoma cell line (SH-SY5Y), where two mRNA species of 5.7 and 6 kb were detected. Using the same blot, no hybridization signal could be seen with a human InsP3 3-kinase A probe. Altogether, our data are consistent with the notion that the two InsP3 3-kinase isoenzymes, A and B, are specifically expressed in different tissues and cells.

Diacylglycerol kinases in signal transduction.

van Blitterswijk WJ, Houssa B
Chem Phys Lipids. 1999; 98: 95-108

Diacylglycerol kinase (DGK) phosphorylates the second messenger diacylglycerol (DAG) to phosphatidic acid. A family of nine mammalian isotypes have been identified. Their primary structure shows a diverse array of conserved domains, such as a catalytic domain, zinc fingers, pleckstrin homology domains and EF-hand structures, known to interact with other proteins, lipids or Ca2+, in signal transduction processes. DGK is believed to act in the phosphoinositide cycle in which DAG is enriched with arachidonoyl moieties, but the majority of DGK isotypes do not show specificity for this DAG species in vitro. This could imply that DGKs may also have other functions in the cell. DGK activity is not only found in membranes, but also in the nucleus and at the cytoskeleton. Agonist-induced translocations of DGK to or from these subcellular sites are known to occur. Some isotypes are contained in signaling complexes in specific association with members of the Rho family of small GTP binding proteins, suggesting that they are involved in Rho-mediated processes such as cytoskeletal reorganization.

Properties and functions of diacylglycerol kinases.

van Blitterswijk WJ, Houssa B
Cell Signal. 2000; 12: 595-605

Diacylglycerol kinases (DGKs) phosphorylate the second-messenger diacylglycerol (DAG) to phosphatidic acid (PA). The family of DGKs is well conserved among most species. Nine mammalian isotypes have been identified, and are classified into five subgroups based on their primary structure. DGKs contain a conserved catalytic domain and an array of other conserved motifs that are likely to play a role in lipid-protein and protein-protein interactions in various signalling pathways dependent on DAG and/or PA production. DGK is therefore believed to be activated at the (plasma) membrane where DAG is generated. Some isotypes are found associated with and/or regulated by small GTPases of the Rho family, presumably acting in cytoskeletal rearrangements. Others are (also) found in the nucleus, in association with other regulatory enzymes of the phosphoinositide cycle, and have an effect on cell cycle progression. Most DGK isotypes show high expression in the brain, often in distinct brain regions, suggesting that each individual isotype has a unique function.

Translocation of diacylglycerol kinase theta from cytosol to plasma membrane in response to activation of G protein-coupled receptors and protein kinase C.

van Baal J, de Widt J, Divecha N, van Blitterswijk WJ
J Biol Chem. 2005; 280: 9870-8

Diacylglycerol kinase (DGK) phosphorylates the second messenger diacylglycerol (DAG) to phosphatidic acid. We previously identified DGK as one of nine mammalian DGK isoforms and reported on its regulation by interaction with RhoA and by translocation to the plasma membrane in response to noradrenaline. Here, we have investigated how the localization of DGK, fused to green fluorescent protein, is controlled upon activation of G protein-coupled receptors in A431 cells. Extracellular ATP, bradykinin, or thrombin induced DGK translocation from the cytoplasm to the plasma membrane within 2-6 min. This translocation, independent of DGK activity, was preceded by protein kinase C (PKC) translocation and was blocked by PKC inhibitors. Conversely, activation of PKC by 12-O-tetradecanoylphorbol-13-acetate induced DGK translocation. Membrane-permeable DAG (dioctanoylglycerol) also induced DGK translocation but in a PKC (staurosporin)-independent fashion. Mutations in the cysteine-rich domains of DGK abrogated its hormone- and DAG-induced translocation, suggesting that these domains are essential for DAG binding and DGK recruitment to the membrane. We show that DGK interacts selectively with and is phosphorylated by PKCepsilon and -eta and that peptide agonist-induced selective activation of PKCepsilon directly leads to DGK translocation. Our data are consistent with the concept that hormone-induced PKC activation regulates the intracellular localization of DGK, which may be important in the negative regulation of PKCepsilon and/or PKCeta activity.

Molecular cloning and characterization of protein kinase D: a target for diacylglycerol and phorbol esters with a distinctive catalytic domain.

Valverde AM, Sinnett-Smith J, Van Lint J, Rozengurt E
Proc Natl Acad Sci U S A. 1994; 91: 8572-6

A serine/threonine protein kinase that binds phorbol esters and diacylglycerol (named protein kinase D, PKD) has been identified. PKD contains membrane localization signals and a cysteine-rich repeat sequence homologous to that seen in the regulatory domain of protein kinase C (PKC). A bacterially expressed N-terminal domain of PKD exhibited high-affinity phorbol ester binding activity (Kd = 35 nM). The diacylglycerol analog 1-oleoyl-2-acetylglycerol inhibited phorbol ester binding in a dose-dependent manner. The catalytic domain of PKD contains all characteristic sequence motifs of serine protein kinases but shows only a low degree of sequence similarity to PKCs. The highest identity is with the catalytic domain of myosin light-chain kinase from Dictyostelium (41%). The bacterially expressed catalytic domain of PKD efficiently phosphorylated the exogenous peptide substrate syntide 2 in serine but did not catalyze significant phosphorylation of a variety of other substrates used by PKCs and other major second messenger regulated kinases. PKD may be an unusual component in the transduction of diacylglycerol and phorbol ester signals.

Nck-2, a novel Src homology2/3-containing adaptor protein that interacts with the LIM-only protein PINCH and components of growth factor receptor kinase-signaling pathways.

Tu Y, Li F, Wu C
Mol Biol Cell. 1998; 9: 3367-82

Many of the protein-protein interactions that are essential for eukaryotic intracellular signal transduction are mediated by protein binding modules including SH2, SH3, and LIM domains. Nck is a SH3- and SH2-containing adaptor protein implicated in coordinating various signaling pathways, including those of growth factor receptors and cell adhesion receptors. We report here the identification, cloning, and characterization of a widely expressed, Nck-related adaptor protein termed Nck-2. Nck-2 comprises primarily three N-terminal SH3 domains and one C-terminal SH2 domain. We show that Nck-2 interacts with PINCH, a LIM-only protein implicated in integrin-linked kinase signaling. The PINCH-Nck-2 interaction is mediated by the fourth LIM domain of PINCH and the third SH3 domain of Nck-2. Furthermore, we show that Nck-2 is capable of recognizing several key components of growth factor receptor kinase-signaling pathways including EGF receptors, PDGF receptor-beta, and IRS-1. The association of Nck-2 with EGF receptors was regulated by EGF stimulation and involved largely the SH2 domain of Nck-2, although the SH3 domains of Nck-2 also contributed to the complex formation. The association of Nck-2 with PDGF receptor-beta was dependent on PDGF activation and was mediated solely by the SH2 domain of Nck-2. Additionally, we have detected a stable association between Nck-2 and IRS-1 that was mediated primarily via the second and third SH3 domain of Nck-2. Thus, Nck-2 associates with PINCH and components of different growth factor receptor-signaling pathways via distinct mechanisms. Finally, we provide evidence indicating that a fraction of the Nck-2 and/or Nck-1 proteins are associated with the cytoskeleton. These results identify a novel Nck-related SH2- and SH3-domain-containing protein and suggest that it may function as an adaptor protein connecting the growth factor receptor-signaling pathways with the integrin-signaling pathways.

Gene expression, cellular localization, and enzymatic activity of diacylglycerol kinase isozymes in rat ovary and placenta.

Toya M, Hozumi Y, Ito T, Takeda M, Sakane F, Kanoh H, Saito H, Hiroi M, Kurachi H, Kondo H, Goto K
Cell Tissue Res. 2005; 320: 525-33

Female reproductive organs show remarkable cyclic changes in morphology and function in response to a combination of hormones. Evidence has accumulated suggesting that phosphoinositide turnover and the consequent diacylglycerol (DG) protein kinase C (PKC) pathway are intimately involved in these mechanisms. The present study has been performed to investigate the gene expression, cellular localization, and enzymatic activity of the DG kinase (DGK) isozymes that control the DG-PKC pathway. Gene expression for DGKalpha, -epsilon, -zeta, and -iota was detected in the ovary and placenta. Intense expression signals for DGKzeta and -alpha were observed in the theca cells and moderate signals in the interstitium and corpora lutea of the ovary. On the other hand, signals for DGKepsilon were seen more intensely in granulosa cells. In the placenta, signals for DGKalpha and -iota were observed in the junctional zone, whereas those for DGKzeta were detected in the labyrinthine zone. At higher magnification, the signals for DGKalpha were mainly discerned in giant cytotrophoblasts, and those for DGKiota were found in small cytotrophoblasts of the junctional zone. DGKzeta signals were observed in all cellular components of the labyrinthine zone, including mesenchyme, trabecular trophoblasts, and cytotrophoblasts. DGKepsilon signals were detected in the junctional zone on day 13 and 15 of pregnancy and were diffusely distributed both in the labyrinthine and junctional zones at later stages. The present study reveals distinct patterns of mRNA localization for DGK isozymes in the rat ovary and placenta, suggesting that each isozyme plays a unique role in distinct cell types in these organs.

Protein kinase C regulates the nuclear localization of diacylglycerol kinase-zeta.

Topham MK, Bunting M, Zimmerman GA, McIntyre TM, Blackshear PJ, Prescott SM
Nature. 1998; 394: 697-700

Diacylglycerol kinases (DGKs) terminate signalling from diacylglycerol by converting it to phosphatidic acid. Diacylglycerol regulates cell growth and differentiation, and its transient accumulation in the nucleus may be particularly important in this regulation. Here we show that a fraction of DGK-zeta is found in the nucleus, where it regulates the amount of nuclear diacylglycerol. Reducing nuclear diacylglycerol levels by conditional expression of DGK-zeta attenuates cell growth. The nuclear-localization signal of DGK-zeta is located in a region that is homologous to the phosphorylation-site domain of the MARCKS protein. This is, to our knowledge, the first evidence that this domain, which is a major target for protein kinase C, can localize a protein to the nucleus. Two isoforms of protein kinase C, but not others, regulate the localization of DGK-zeta. Our results define a cycle in which diacylglycerol activates protein kinase C, which then regulates the metabolism of diacylglycerol by alternating the intracellular location of DGK-zeta. This may be a general mechanism to control mitogenic signals that depend on nuclear diacylglycerol.

Cloning and expression in Escherichia coli of a rat brain cDNA encoding a Ca2+/calmodulin-sensitive inositol 1,4,5-trisphosphate 3-kinase.

Takazawa K, Vandekerckhove J, Dumont JE, Erneux C
Biochem J. 1990; 272: 107-12

Inositol 1,4,5-trisphosphate (InsP3) 3-kinase catalyses the phosphorylation of InsP3 to inositol 1,3,4,5-tetrakisphosphate (InsP4). InsP3 3-kinase activity was stimulated by Ca2+ in the presence of calmodulin (CaM) and the protein was associated with two silver-stained bands which migrated with an apparent Mr of approx. 50,000 on SDS/polyacrylamide gels. Upon limited proteolysis with trypsin, the native InsP3 3-kinase was converted into polypeptides of Mr 44,000 and 36,000. Both tryptic fragments displayed InsP3 3-kinase activity that was Ca2+/CaM-sensitive. A cDNA clone, C5, that encodes the C-terminal part of the InsP3 3-kinase, was isolated by immunoscreening of a rat brain cDNA library. The 5' end of this clone was used in turn to probe the same library, yielding a clone (CP16) containing the entire coding sequence of InsP3 3-kinase. The encoding protein of 459 amino acids (calculated Mr 50,868) has several putative phosphorylation sites for cyclic AMP-dependent protein kinase, protein kinase C and CaM-dependent protein kinase II. When clone C5 was expressed in Escherichia coli, the truncated fusion protein showed Ca2+/CaM-sensitive InsP3 3-kinase activity. Our data demonstrate that the N-terminal part of the protein is not essential for either enzymic or CaM-regulatory properties.

Adenovirus-mediated overexpression of diacylglycerol kinase-zeta inhibits endothelin-1-induced cardiomyocyte hypertrophy.

Takahashi H, Takeishi Y, Seidler T, Arimoto T, Akiyama H, Hozumi Y, Koyama Y, Shishido T, Tsunoda Y, Niizeki T, Nozaki N, Abe J, Hasenfuss G, Goto K, Kubota I
Circulation. 2005; 111: 1510-6

BACKGROUND: Diacylglycerol (DAG) is a lipid second messenger that transiently accumulates in cells stimulated by endothelin-1 (ET-1) and other Galphaq protein-coupled receptor agonists. Diacylglycerol kinase (DGK) is thought to be an enzyme that controls the cellular levels of DAG by converting it to phosphatidic acid; however, the functional role of DGK has not been examined in cardiomyocytes. Because DGK inactivates DAG, a strong activator of protein kinase C (PKC), we hypothesized that DGK inhibited ET-1-induced activation of a DAG-PKC signaling cascade and subsequent cardiomyocyte hypertrophy. METHODS AND RESULTS: Real-time reverse transcription-polymerase chain reaction demonstrated a significant increase of DGK-zeta mRNA by ET-1 in cardiomyocytes. To determine the functional role of DGK-zeta, we overexpressed DGK-zeta in cardiomyocytes using a recombinant adenovirus encoding rat DGK-zeta (Ad-DGKzeta). ET-1-induced translocation of PKC-epsilon was blocked by Ad-DGKzeta (P<0.01). Ad-DGKzeta also inhibited ET-1-induced activation of extracellular signal-regulated kinase (P<0.01). Luciferase reporter assay revealed that ET-1-mediated increase of activator protein-1 (AP1) DNA-binding activity was significantly inhibited by DGK-zeta (P<0.01). In cardiomyocytes transfected with DGK-zeta, ET-1 failed to cause gene induction of atrial natriuretic factor, increases in [3H]-leucine uptake, and increases in cardiomyocyte surface area. CONCLUSIONS: We demonstrated for the first time that DGK-zeta blocked ET-1-induced activation of the PKC-epsilon-ERK-AP1 signaling pathway, atrial natriuretic factor gene induction, and resultant cardiomyocyte hypertrophy. DGK-zeta might act as a negative regulator of hypertrophic program in response to ET-1, possibly by controlling cellular DAG levels.

Diacylglycerol kinase-theta is localized in the speckle domains of the nucleus.

Tabellini G, Bortul R, Santi S, Riccio M, Baldini G, Cappellini A, Billi AM, Berezney R, Ruggeri A, Cocco L, Martelli AM
Exp Cell Res. 2003; 287: 143-54

It is well established that the nucleus is endowed with enzymes that are involved in lipid-dependent signal transduction pathways. Diacylglycerol (DAG) is a fundamental lipid second messenger that is produced in the nucleus. Previous reports have shown that the nucleus contains diacylglycerol kinases (DGKs), i.e., the enzymes that, by converting DAG into phosphatidic acid (PA), terminate DAG-dependent events. Here, we show, by immunofluorescence staining and confocal analysis, that DGK-theta localizes mainly to the nucleus of various cell lines, such as MDA-MB-453, MCF-7, PC12, and HeLa. Nuclear DGK-theta co-localizes with phosphatidylinositol 4,5-bisphosphate (PIP(2)) in domains that correspond to nuclear speckles, as revealed by the use of an antibody to the splicing factor SC-35, a well-established marker for these structures. The spatial distribution of nuclear DGK-theta was dynamic in that it was affected by inhibition of mRNA transcription with alpha-amanitin. Immuno-electron microscopy analysis demonstrated that DGK-theta, PIP(2), and phosphoinositide-specific phospholipase Cbeta1 (PLCbeta1) associated with electron-dense particles within the nucleus that correspond to interchromatin granule clusters. Cell fractionation experiments performed in MDA-MB-453, HeLa, and PC12 cells showed a preferential association of DGK-theta with the nucleus. Western blots demonstrated that DGK-theta was enriched in the nuclear matrix fraction prepared from MDA-MB-453 cells. Immunoprecipitation experiments with an antibody to PLCbeta1 revealed in MDA-MB-453 cells an association between this enzyme and both DGK-theta and phosphatidylinositol phosphate kinase Ialpha (PIPKIalpha). Our findings strengthen the contention that speckles represent a crucial site for the nuclear-based inositol lipid cycle. We may speculate that nuclear speckle-located DGK-theta, on cell stimulation with an agonist, converts to PA the DAG derived from PLCbeta1-dependent PIP(2) hydrolysis.

Nuclear diacylglycerol kinase-theta is activated in response to nerve growth factor stimulation of PC12 cells.

Tabellini G, Billi AM, Fala F, Cappellini A, Evagelisti C, Manzoli L, Cocco L, Martelli AM
Cell Signal. 2004; 16: 1263-71

Previous evidence from independent laboratories has shown that the nucleus contains diacylglycerol kinase (DGK) isoforms, i.e., the enzymes, which yield phosphatidic acid from diacylglycerol, thus terminating protein kinase C-mediated signaling events. A DGK isoform, which resides in the nucleus of PC12 cells, is DGK-theta. Here, we show that nerve growth factor (NGF) treatment of serum-starved PC12 cells results in the stimulation of both a cytoplasmic and a nuclear DGK activity. However, time course analysis shows that cytoplasmic DGK activity peaked earlier than its nuclear counterpart. While nuclear DGK activity was dramatically down-regulated by a monoclonal antibody known for selectively inhibiting DGK-theta, cytoplasmic DGK activity was not. Moreover, nuclear DGK activity was stimulated by phosphatidylserine, an anionic phospholipid that had no effect on cytoplasmic DGK activity. Upon NGF stimulation, the amount and the activity of DGK-theta, which was bound to the insoluble nuclear matrix fraction, substantially increased. Epidermal growth factor up-regulated a nuclear DGK activity insensitive to anti-DGK-theta monoclonal antibody. Overall, our findings identify nuclear DGK-theta as a down-stream target of NGF signaling in PC12 cells.

Molecular cloning and characterization of the human protein kinase D2. A novel member of the protein kinase D family of serine threonine kinases.

Sturany S, Van Lint J, Muller F, Wilda M, Hameister H, Hocker M, Brey A, Gern U, Vandenheede J, Gress T, Adler G, Seufferlein T
J Biol Chem. 2001; 276: 3310-8

We have isolated the full-length cDNA of a novel human serine threonine protein kinase gene. The deduced protein sequence contains two cysteine-rich motifs at the N terminus, a pleckstrin homology domain, and a catalytic domain containing all the characteristic sequence motifs of serine protein kinases. It exhibits the strongest homology to the serine threonine protein kinases PKD/PKC&mgr; and PKCnu, particularly in the duplex zinc finger-like cysteine-rich motif, in the pleckstrin homology domain and in the protein kinase domain. In contrast, it shows only a low degree of sequence similarity to other members of the PKC family. Therefore, the new protein has been termed protein kinase D2 (PKD2). The mRNA of PKD2 is widely expressed in human and murine tissues. It encodes a protein with a molecular mass of 105 kDa in SDS-polyacrylamide gel electrophoresis, which is expressed in various human cell lines, including HL60 cells, which do not express PKC&mgr;. In vivo phorbol ester binding studies demonstrated a concentration-dependent binding of [(3)H]phorbol 12,13-dibutyrate to PKD2. The addition of phorbol 12,13-dibutyrate in the presence of dioleoylphosphatidylserine stimulated the autophosphorylation of PKD2 in a synergistic fashion. Phorbol esters also stimulated autophosphorylation of PKD2 in intact cells. PKD2 activated by phorbol esters efficiently phosphorylated the exogenous substrate histone H1. In addition, we could identify the C-terminal Ser(876) residue as an in vivo phosphorylation site within PKD2. Phosphorylation of Ser(876) of PKD2 correlated with the activation status of the kinase. Finally, gastrin was found to be a physiological activator of PKD2 in human AGS-B cells stably transfected with the CCK(B)/gastrin receptor. Thus, PKD2 is a novel phorbol ester- and growth factor-stimulated protein kinase.

cDNA cloning, chromosome assignment, and genomic structure of a human gene encoding a novel member of the RBM family.

Stover C, Gradl G, Jentsch I, Speicher MR, Wieser R, Schwaeble W
Cytogenet Cell Genet. 2001; 92: 225-30

We have cloned and characterised a novel human gene mapping to chromosome 20q11.2. A partial transcript was initially isolated from a human cDNA library transcribed from RNA of the colon carcinoma cell line T-84. In order to determine the full coding sequence of this novel mRNA, we isolated seven cDNA clones from a human cDNA library transcribed from RNA of the acute monocytic leukemia cell line THP1 by colony hybridization. On Northern blot analysis of four human cell lines, the cDNAs isolated hybridize with an abundantly expressed mRNA species of 3.5 kb. A full-length cDNA transcript of this novel mRNA has an open reading frame of 2,796 bp encoding a protein with a calculated molecular weight of 97 kDa. Two repetitive structural consensus motifs are contained within the deduced protein sequence, namely five distinct RNA binding motifs and two proline rich regions. The derived protein sequence also contains putative transmembrane domains. These structural motifs identify this novel protein as a member of an expanding protein family containing RNA binding motifs (RBM). As seen from recently completed sequence of the genomic area encoding this novel mRNA by the Sanger Centre Human Genome Project, the coding region of this gene, RBM12, is intronless.

Alternative splicing of a novel diacylglycerol kinase in tomato leads to a calmodulin-binding isoform.

Snedden WA, Blumwald E
Plant J. 2000; 24: 317-26

Calmodulin is a regulatory protein activated during Ca2+ signalling. We have isolated a cDNA, designated LeCBDGK (Lycopersicon esculentum calmodulin-binding diacylglycerol kinase) encoding a novel calmodulin-binding protein with sequence similarity to diacylglycerol kinases from animals. Diacylglycerol kinases convert diacylglycerol to phosphatidic acid. We delineated the calmodulin-binding domain to approximately 25 residues near the C-terminus of LeCBDGK. We have also isolated a second diacylglycerol kinase cDNA, designated LeDGK1, identical to LeCBDGK, except that it lacks the calmodulin-binding domain. Both recombinant LeCBDGK and LeDGK1 were catalytically active in vitro. Anti-DGK antiserum detected two immunoreactive proteins associated with microsomal and plasma membrane fractions from cell suspensions. The higher molecular weight immunoreactive protein was also present in soluble extracts and bound to calmodulin-agarose in the presence of calcium, demonstrating that native LeCBDGK is a calmodulin-binding protein. In the presence of calcium, LeCBDGK associated with membrane cell fractions in vitro, but calmodulin antagonists disrupted this association, suggesting a possible role of calcium in the recruitment of LeCBDGK from soluble to membrane cell fractions. Native LeCBDGK and calmodulin co-immunoprecipitated from tomato soluble cell extracts, suggesting their interaction in vivo. The same gene encodes both LeCBDGK and LeDGK1 and the calmodulin-binding domain of LeCBDGK is encoded by a separate exon. Thus, alternative transcript splicing leads to calmodulin-binding and non-binding forms of diacylglycerol kinases in tomato. Possible roles of LeCBDGK and LeDGK1 in calcium and lipid signalling are discussed.

Purification of matrix Gla protein from a marine teleost fish, Argyrosomus regius: calcified cartilage and not bone as the primary site of MGP accumulation in fish.

Simes DC, Williamson MK, Ortiz-Delgado JB, Viegas CS, Price PA, Cancela ML
J Bone Miner Res. 2003; 18: 244-59

Matrix Gla protein (MGP) belongs to the family of vitamin K-dependent, Gla-containing proteins, and in mammals, birds, and Xenopus, its mRNA was previously detected in extracts of bone, cartilage, and soft tissues (mainly heart and kidney), whereas the protein was found to accumulate mainly in bone. However, at that time, it was not evaluated if this accumulation originated from protein synthesized in cartilage or in bone cells because both coexist in skeletal structures of higher vertebrates and Xenopus. Later reports showed that MGP also accumulated in costal calcified cartilage as well as at sites of heart valves and arterial calcification. Interestingly, MGP was also found to accumulate in vertebra of shark, a cartilaginous fish. However, to date, no information is available on sites of MGP expression or accumulation in teleost fishes, the ancestors of terrestrial vertebrates, who have in their skeleton mineralized structures with both bone and calcified cartilage. To analyze MGP structure and function in bony fish, MGP was acid-extracted from the mineralized matrix of either bone tissue (vertebra) or calcified cartilage (branchial arches) from the bony fish, Argyrosomus regius, separated from the mineral phase by dialysis, and purified by Sephacryl S-100 chromatography. No MGP was recovered from bone tissue, whereas a protein peak corresponding to the MGP position in this type of gel filtration was obtained from an extract of branchial arches, rich in calcified cartilage. MGP was identified by N-terminal amino acid sequence analysis, and the resulting protein sequence was used to design specific oligonucleotides suitable to amplify the corresponding DNA by a mixture of reverse transcription-polymerase chain reaction (RT-PCR) and 5'rapid amplification of cDNA (RACE)-PCR. In parallel, ArBGP (bone Gla protein, osteocalcin) was also identified in the same fish, and its complementary DNA cloned by an identical procedure. Tissue distribution/accumulation was analyzed by Northern blot, in situ hybridization, and immunohistochemistry. In mineralized tissues, the MGP gene was predominantly expressed in cartilage from branchial arches, with no expression detected in the different types of bone analyzed, whereas BGP mRNA was located in bone tissue as expected. Accordingly, the MGP protein was found to accumulate, by immunohistochemical analysis, mainly in the extracellular matrix of calcified cartilage. In soft tissues, MGP mRNA was mainly expressed in heart but in situ hybridization, indicated that cells expressing the MGP gene were located in the bulbus arteriosus and aortic wall, rich in smooth muscle and endothelial cells, whereas no expression was detected in the striated muscle myocardial fibers of the ventricle. These results show that in marine teleost fish, as in mammals, the MGP gene is expressed in cartilage, heart, and kidney tissues, but in contrast with results obtained in Xenopus and higher vertebrates, the protein does not accumulate in vertebra of non-osteocytic teleost fish, but only in calcified cartilage. In addition, our results also indicate that the presence of MGP mRNA in heart tissue is due, at least in fish, to the expression of the MGP gene in only two specific cell types, smooth muscle and endothelial cells, whereas no expression was found in the striated muscle fibers of the ventricle. In light of these results and recent information on expression of MGP gene in these same cell types in mammalian aorta, it is likely that the levels of MGP mRNA previously detected in Xenopus, birds, and mammalian heart tissue may be restricted to regions rich in smooth muscle and endothelial cells. Our results also emphasize the need to re-evaluate which cell types are involved in MGP gene expression in other soft tissues and bring further evidence that fish are a valuable model system to study MGP gene expression and regulation.

Molecular cloning and expression of mouse and human cDNAs encoding heparan sulfate D-glucosaminyl 3-O-sulfotransferase.

Shworak NW, Liu J, Fritze LM, Schwartz JJ, Zhang L, Logeart D, Rosenberg RD
J Biol Chem. 1997; 272: 28008-19

The cellular rate of anticoagulant heparan sulfate proteoglycan (HSPGact) generation is determined by the level of a kinetically limiting microsomal activity, HSact conversion activity, which is predominantly composed of the long sought heparan sulfate D-glucosaminyl 3-O-sulfotransferase (3-OST) (Shworak, N. W., Fritze, L. M. S., Liu, J., Butler, L. D., and Rosenberg, R. D. (1996) J. Biol. Chem. 271, 27063-27071; Liu, J., Shworak, N. W., Fritze, L. M. S., Edelberg, J. M., and Rosenberg, R. D. (1996) J. Biol. Chem. 271, 27072-27082). Mouse 3-OST cDNAs were isolated by proteolyzing the purified enzyme with Lys-C, sequencing the resultant peptides as well as the existing amino terminus, employing degenerate polymerase chain reaction primers corresponding to the sequences of the peptides as well as the amino terminus to amplify a fragment from LTA cDNA, and utilizing the resultant probe to obtain full-length enzyme cDNAs from a lambda Zap Express LTA cDNA library. Human 3-OST cDNAs were isolated by searching the expressed sequence tag data bank with the mouse sequence, identifying a partial-length human cDNA and utilizing the clone as a probe to isolate a full-length enzyme cDNA from a lambda TriplEx human brain cDNA library. The expression of wild-type mouse 3-OST as well as protein A-tagged mouse enzyme by transient transfection of COS-7 cells and the expression of both wild-type mouse and human 3-OST by in vitro transcription/translation demonstrate that the two cDNAs directly encode both HSact conversion and 3-OST activities. The mouse 3-OST cDNAs exhibit three different size classes because of a 5'-untranslated region of variable length, which results from the insertion of 0-1629 base pairs (bp) between residues 216 and 217; however, all cDNAs contain the same open reading frame of 933 bp. The length of the 3'-untranslated region ranges from 301 to 430 bp. The nucleic acid sequence of mouse and human 3-OST cDNAs are approximately 85% similar, encoding novel 311- and 307-amino acid proteins of 35,876 and 35,750 daltons, respectively, that are 93% similar. The encoded enzymes are predicted to be intraluminal Golgi residents, presumably interacting via their C-terminal regions with an integral membrane protein. Both 3-OST species exhibit five potential N-glycosylation sites, which account for the apparent discrepancy between the molecular masses of the encoded enzyme (approximately 34 kDa) and the previously purified enzyme (approximately 46 kDa). The two 3-OST species also exhibit approximately 50% similarity with all previously identified forms of the heparan biosynthetic enzyme N-deacetylase/N-sulfotransferase, which suggests that heparan biosynthetic enzymes share a common sulfotransferase domain.

Cloning, characterization, and expression of a novel Zn2+-binding FYVE finger-containing phosphoinositide kinase in insulin-sensitive cells.

Shisheva A, Sbrissa D, Ikonomov O
Mol Cell Biol. 1999; 19: 623-34

Signaling by phosphorylated species of phosphatidylinositol (PI) appears to regulate diverse responses in eukaryotic cells. A differential display screen for fat- and muscle-specific transcripts led to identification and cloning of the full-length cDNA of a novel mammalian 2,052-amino-acid protein (p235) from a mouse adipocyte cDNA library. Analysis of the deduced amino acid sequence revealed that p235 contains an N-terminal zinc-binding FYVE finger, a chaperonin-like region in the middle of the molecule, and a consensus for phosphoinositide 5-kinases at the C terminus. p235 mRNA appears as a 9-kb transcript, enriched in insulin-sensitive cells and tissues, likely transcribed from a single-copy gene in at least two close-in-size splice variants. Specific antibodies against mouse p235 were raised, and both the endogenously and heterologously expressed proteins were biochemically detected in 3T3-L1 adipocytes and transfected COS cells, respectively. Immunofluorescence microscopy analysis of endogenous p235 localization in 3T3-L1 adipocytes with affinity-purified anti-p235 antibodies documented a punctate peripheral pattern. In COS cells, the expressed p235 N-terminal but not the C-terminal region displayed a vesicular pattern similar to that in 3T3-L1 adipocytes that became diffuse upon Zn2+ chelation or FYVE finger truncation. A recombinant protein comprising the N-terminal but not the C-terminal region of the molecule was found to bind 2.2 mole equivalents of Zn2+. Determination of the lipid kinase activity in the p235 immunoprecipitates derived from 3T3-L1 adipocytes or from COS cells transiently expressing p235 revealed that p235 displayed unique preferences for PI substrate over already phosphorylated PI. In conclusion, the mouse p235 protein determines an important novel class of phosphoinositide kinases that seems to be targeted to specific intracellular loci by a Zn-dependent mechanism.

Subtype-specific translocation of diacylglycerol kinase alpha and gamma and its correlation with protein kinase C.

Shirai Y, Segawa S, Kuriyama M, Goto K, Sakai N, Saito N
J Biol Chem. 2000; 275: 24760-6

We examined the translocation of diacylglycerol kinase (DGK) alpha and gamma fused with green fluorescent protein in living Chinese hamster ovary K1 cells (CHO-K1) and investigated temporal and spatial correlations between DGK and protein kinase C (PKC) when both kinases are overexpressed. DGKalpha and gamma were present throughout the cytoplasm of CHO-K1 cells. Tetradecanoylphorbol 13-acetate (TPA) induced irreversible translocation of DGKgamma, but not DGKalpha, from the cytoplasm to the plasma membrane. The (TPA)-induced translocation of DGKgamma was inhibited by the mutation of C1A but not C1B domain of DGKgamma and was not inhibited by staurosporine. Arachidonic acid induced reversible translocation of DGKgamma from the cytoplasm to the plasma membrane, whereas DGKalpha showed irreversible translocation to the plasma membrane and the Golgi network. Purinergic stimulation induced reversible translocation of both DGKgamma and alpha to the plasma membrane. The timing of the ATP-induced translocation of DGKgamma roughly coincided with that of PKCgamma re-translocation from the membrane to the cytoplasm. Furthermore, re-translocation of PKCgamma was obviously hastened by co-expression with DGKgamma and was blocked by an inhibitor of DGK (R59022). These results indicate that DGK shows subtype-specific translocation depending on extracellular signals and suggest that PKC and DGK are orchestrated temporally and spatially in the signal transduction.

Diacylglycerol kinase gamma is one of the specific receptors of tumor-promoting phorbol esters.

Shindo M, Irie K, Ohigashi H, Kuriyama M, Saito N
Biochem Biophys Res Commun. 2001; 289: 451-6

Diacylglycerol kinase (DGK) and protein kinase C (PKC) are two different enzyme families that interact with diacylglycerol. Both enzymes contain cysteine-rich C1 domains with a zinc finger-like structure. Most of the C1 domains of PKCs show strong phorbol-12,13-dibutyrate (PDBu) binding with nanomolar dissociation constants (K(d)'s). However, there has been no experimental evidence that phorbol esters bind to the C1 domains of DGKs. We focused on DGK gamma because its C1A domain has a high degree of sequence homology to those of PKCs, and because DGK gamma translocates from the cytoplasm to the plasma membrane following 12-O-tetradecanoylphorbol-13-acetate treatment similar to PKCs. Two C1 domains of DGK gamma (DGK gamma-C1A and DGK gamma-C1B) were synthesized and tested for their PDBu binding along with whole DGK gamma (Flag-DGK gamma) expressed in COS-7 cells. DGK gamma-C1A and Flag-DGK gamma showed strong PDBu binding affinity, while DGK gamma-C1B was completely inactive. Scatchard analysis of DGK gamma-C1A and Flag-DGK gamma gave K(d)'s of 3.1 and 4.4 nM, respectively, indicating that the major PDBu binding site of DGK gamma is C1A. This is the first evidence that DGK gamma is a specific receptor of tumor-promoting phorbol esters.

Synthesis and phorbol ester binding of the cysteine-rich domains of diacylglycerol kinase (DGK) isozymes. DGKgamma and DGKbeta are new targets of tumor-promoting phorbol esters.

Shindo M, Irie K, Masuda A, Ohigashi H, Shirai Y, Miyasaka K, Saito N
J Biol Chem. 2003; 278: 18448-54

Diacylglycerol kinase (DGK) and protein kinase C (PKC) are two distinct enzyme families associated with diacylglycerol. Both enzymes have cysteine-rich C1 domains (C1A, C1B, and C1C) in the regulatory region. Although most PKC C1 domains strongly bind phorbol esters, there has been no direct evidence that DGK C1 domains bind phorbol esters. We synthesized 11 cysteine-rich sequences of DGK C1 domains with good sequence homology to those of the PKC C1 domains. Among them, only DGKgamma-C1A and DGKbeta-C1A exhibited significant binding to phorbol 12,13-dibutyrate (PDBu). Scatchard analysis of rat-DGKgamma-C1A, human-DGKgamma-C1A, and human-DGKbeta-C1A gave K(d) values of 3.6, 2.8, and 14.6 nm, respectively, suggesting that DGKgamma and DGKbeta are new targets of phorbol esters. An A12T mutation of human-DGKbeta-C1A enhanced the affinity to bind PDBu, indicating that the beta-hydroxyl group of Thr-12 significantly contributes to the binding. The K(d) value for PDBu of FLAG-tagged whole rat-DGKgamma (4.4 nm) was nearly equal to that of rat-DGKgamma-C1A (3.6 nm). Moreover, 12-O-tetradecanoylphorbol 13-acetate induced the irreversible translocation of whole rat-DGKgamma and its C1B deletion mutant, not the C1A deletion mutant, from the cytoplasm to the plasma membrane of CHO-K1 cells. These results indicate that 12-O-tetradecanoylphorbol 13-acetate binds to C1A of DGKgamma to cause its translocation.

Molecular cloning and characterization of PKC iota, an atypical isoform of protein kinase C derived from insulin-secreting cells.

Selbie LA, Schmitz-Peiffer C, Sheng Y, Biden TJ
J Biol Chem. 1993; 268: 24296-302

The protein kinase C (PKC) family of serine-threonine kinases comprises at least eight members. These are differentially expressed, show varying affinities for activators such as Ca2+ and lipid species, and are therefore thought to play wide-ranging roles in the regulation of such cellular processes as differentiation, growth, and secretion. The aim of this study was to identify new PKC isoforms in the insulin-secreting cell line RINm5F that might be activated by the alterations in lipid metabolism that accompany nutrient-stimulated insulin release. Fragments of cDNA, derived from RINm5F cell mRNA, were amplified by the polymerase chain reaction using degenerate oligonucleotide primers corresponding to highly conserved regions in the catalytic domains of all known PKCs. A novel sequence generated by this approach was subsequently used to screen cDNA libraries. The entire 587-amino acid coding region of a new PKC isoform, PKC iota, was deduced from two overlapping clones isolated from a human kidney cDNA library. The amino acid sequence of PKC iota showed greatest homology to PKC zeta, with 72% identity overall rising to 84% in the catalytic domain. In contrast, the homology of PKC iota to the other isoforms was less pronounced, with < 53% identity even in the highly conserved catalytic region. Further similarities between PKC zeta and PKC iota included a highly conserved pseudosubstrate sequence, the absence of an apparent Ca(2+)-binding region, and the presence of only one cysteine-rich, zinc finger-like domain. Northern blot analysis, using the full-length PKC iota clone as a probe, revealed a single 4.6-kilobase transcript present predominantly in lung and brain, but also expressed at lower levels in many tissues including pancreatic islets. In CHO-K1 cells stably expressing the PKC iota cDNA under the human beta-actin promoter, the protein was detected as a 65-kDa band by Western blotting using an antibody to the COOH terminus of PKC zeta (conserved in PKC iota). Extracts of transfected CHO-K1 cells also displayed a significantly increased kinase activity using myelin basic protein as a substrate. The results suggest that PKC iota should be included in the atypical subgroup of PKCs whose definitive member is PKC zeta. As such, PKC iota is unlikely to be activated by the diacylglycerol that is derived from phosphoinositide hydrolysis, but might be a target for novel lipid activators that are elevated during nutrient-stimulated insulin secretion.

Diacylglycerol kinase is phosphorylated in vivo upon stimulation of the epidermal growth factor receptor and serine/threonine kinases, including protein kinase C-epsilon.

Schaap D, van der Wal J, van Blitterswijk WJ, van der Bend RL, Ploegh HL
Biochem J. 1993; 289: 875-81

In signal transduction, diacylglycerol (DG) kinase attenuates levels of the second messenger DG by converting it to phosphatidic acid. A previously cloned full-length human 86 kDa DG kinase cDNA was expressed as fusion protein in Escherichia coli, to aid in the generation of DG-kinase-specific monoclonal antibodies suitable for immunoprecipitation experiments. To investigate whether phosphorylation of DG kinase is a possible mechanism for its regulation, COS-7 cells were transiently transfected with the DG kinase cDNA and phosphorylation of the expressed DG kinase was induced by various stimuli. Activation of both cyclic AMP-dependent protein kinase and protein kinase C (PKC) resulted in phosphorylation of DG kinase on serine residues in vivo, and both kinases induced this phosphorylation within the same tryptic phosphopeptide, suggesting that they may exert similar control over DG kinase. No phosphorylation was observed upon ionomycin treatment, intended to activate Ca2+/calmodulin-dependent kinases. Co-transfections of DG kinase with either PKC-alpha or PKC-epsilon cDNA revealed that both protein kinases, when stimulated, are able to phosphorylate DG kinase. For PKC-epsilon, DG kinase is the first in vivo substrate identified. Stimulation with epidermal growth factor (EGF) of COS-7 cells transfected with both DG kinase and EGF-receptor cDNA results mainly in phosphorylation of DG kinase on tyrosine. Since the EGF receptor has an intrinsic tyrosine kinase activity, this finding implies that DG kinase may be a direct substrate for the activated EGF receptor.

Consensus sequences for ATP-binding sites in protein kinases do not apply to diacylglycerol kinases.

Schaap D, van der Wal J, van Blitterswijk WJ
Biochem J. 1994; 304: 661-2

Dynamics of diacylglycerol kinase zeta translocation in living T-cells. Study of the structural domain requirements for translocation and activity.

Santos T, Carrasco S, Jones DR, Merida I, Eguinoa A
J Biol Chem. 2002; 277: 30300-9

The diacylglycerol kinases (DGK) regulate diacylglycerol-based signals by phosphorylating this key lipid intermediate to phosphatidic acid. Here, we have investigated the spatial and temporal regulation of diacylglycerol kinase zeta (DGK zeta) in living Jurkat T-cells expressing a muscarinic type I receptor. Using real time confocal videomicroscopy, we show the rapid translocation of a green fluorescent protein-tagged enzyme from the cytosol to the plasma membrane following receptor stimulation. The generation of a panel of truncations, deletions, and point mutations of the enzyme allowed us to examine the requirements of the different structural motifs for both activity and receptor-regulated translocation. The data show that DGK zeta has strict requirements for intact zinc fingers and the conserved catalytic domain for full enzymatic activity. Protein kinase C-driven myristoylated alanine-rich C kinase substrate domain phosphorylation and intact zinc fingers are in turn essential for plasma membrane translocation. DGK zeta does not translocate to the membrane following stimulation of the endogenous T-cell receptor, and our data demonstrate that the specificity in terms of receptor response is provided by the regulatory motifs present at the C-terminal domain of the protein. This is the first report that shows in vivo DGK zeta translocation in response to agonist stimulation and establishes the role of the different domains in enzymatic activity and the selectivity of the response to receptors.

T cell activation in vivo targets diacylglycerol kinase alpha to the membrane: a novel mechanism for Ras attenuation.

Sanjuan MA, Pradet-Balade B, Jones DR, Martinez-A C, Stone JC, Garcia-Sanz JA, Merida I
J Immunol. 2003; 170: 2877-83

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol to produce phosphatidic acid, leading to decreased and increased levels, respectively, of these two lipid messengers that play a central role in T cell activation. Nine DGK isoforms, grouped into five subtypes, are found in higher organisms; all contain a conserved C-terminal domain and at least two cysteine-rich motifs of unknown function. In this study, we have researched in vivo the regulation of DGK alpha, using a transgenic mouse model in which injection of an antigenic peptide activates the majority of peripheral T cells. We demonstrate that DGK alpha, highly expressed in resting T lymphocytes, is subject to complex control at the mRNA and protein levels during in vivo T cell activation. Subcellular fractionation of T lymphocytes shortly after in vivo engagement of the TCR shows rapid translocation of cytosolic DGK alpha to the membrane fraction. At early time points, DGK alpha translocation to the membrane correlates with rapid translocation of Ras guanyl nucleotide-releasing protein (RasGRP), a nucleotide exchange activator for Ras that associates to the membrane through a diacylglycerol-binding domain. To demonstrate a causal relationship between DGK alpha activity and RasGRP relocation to the membrane, we determined RasGRP translocation kinetics in a T cell line transiently transfected with constitutive active and dominant-negative DGK alpha mutants. We show that membrane localization of DGK alpha is associated with a negative regulatory signal for Ras activation by reversing RasGRP translocation. This study is the first demonstration of in vivo regulation of DGK alpha, and provides new insight into the functional role of a member of this family of lipid kinases in the regulation of the immune response.

Role of diacylglycerol kinase alpha in the attenuation of receptor signaling.

Sanjuan MA, Jones DR, Izquierdo M, Merida I
J Cell Biol. 2001; 153: 207-20

Diacylglycerol kinase (DGK) is suggested to attenuate diacylglycerol-induced cell responses through the phosphorylation of this second messenger to phosphatidic acid. Here, we show that DGKalpha, an isoform highly expressed in T lymphocytes, translocates from cytosol to the plasma membrane in response to two different receptors known to elicit T cell activation responses: an ectopically expressed muscarinic type I receptor and the endogenous T cell receptor. Translocation in response to receptor stimulation is rapid, transient, and requires calcium and tyrosine kinase activation. DGKalpha-mediated phosphatidic acid generation allows dissociation of the enzyme from the plasma membrane and return to the cytosol, as demonstrated using a pharmacological inhibitor and a catalytically inactive version of the enzyme. The NH(2)-terminal domain of the protein is shown to be responsible for receptor-induced translocation and phosphatidic acid-mediated membrane dissociation. After examining induction of the T cell activation marker CD69 in cells expressing a constitutively active form of the enzyme, we present evidence of the negative regulation that DGKalpha exerts on diacylglycerol-derived cell responses. This study is the first to describe DGKalpha as an integral component of the signaling cascades that link plasma membrane receptors to nuclear responses.

[The primary structure of diacylglycerol kinase and regulation of its activity]

Sakane F, Yamada K, Kanoh H
Tanpakushitsu Kakusan Koso. 1991; 36: 290-8

Different effects of sphingosine, R59022 and anionic amphiphiles on two diacylglycerol kinase isozymes purified from porcine thymus cytosol.

Sakane F, Yamada K, Kanoh H
FEBS Lett. 1989; 255: 409-13

Porcine thymus cytosol contains two immunologically distinct forms of diacylglycerol kinase (DGK) [Yamada, K. and Kanoh, H. (1988) Biochem. J. 255, 601-608]. These 2 DGK species, having apparent molecular masses of 80 and 150 kDa, were purified from the thymus cytosol. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the 150-kDa DGK gave 2 polypeptide bands of 50 and 75 kDa, whereas the 80-kDa DGK yielded a single protein band. The 80-kDa DGK was markedly activated by 10-20 microM sphingosine as well as by the known anionic activators such as phosphatidylserine and deoxycholate. In contrast, the 150-kDa DGK was fully active in the absence of the anionic activators and was strongly inhibited by sphingosine (IC50, 20 microM). The putative DGK inhibitor R59022 inhibited the 80-kDa DGK (IC50, 10 microM), but had little effect on the 150-kDa form. It is therefore clear that in the thymus cytosol there are at least 2 DGK isozymes operating under different control mechanisms.

Porcine 80-kDa diacylglycerol kinase is a calcium-binding and calcium/phospholipid-dependent enzyme and undergoes calcium-dependent translocation.

Sakane F, Yamada K, Imai S, Kanoh H
J Biol Chem. 1991; 266: 7096-100

We attempted to assess the regulatory role of EF-hand motifs recently detected in the primary structure of porcine 80-kDa diacylglycerol kinase (DGK) (Sakane, F., Yamada, K., Kanoh, H., Yokoyama, C., and Tanabe, T. (1990) Nature 344, 345-348). By using 80-kDa DGK purified from porcine thymus cytosol, we found that this isozyme indeed bound 2 mol Ca2+ per mol enzyme with high affinity (apparent dissociation constant, kd = 0.3 microM). The Ca2+ binding was cooperative with a Hill coefficient of 1.4. We next studied the effect of 1 x 10(-5) M Ca2+ on the kinetic properties of DGK employing a beta-octyl glucoside mixed micellar assay system. In the absence of Ca2+, phosphatidylserine, so far used as an enzyme activator in various assay systems, was rather inhibitory, and Ca2+ alone activated enzyme to a limited extent. However, phosphatidylserine plus Ca2+ markedly activated the enzyme, giving approximately 4-fold higher Vmax and 10-fold less Km values for ATP. In contrast, the apparent Km values for diacylglycerol were not significantly affected (approximately 3 mol %). Furthermore, by immunoblotting using anti-80 kDa DGK antibodies we found that the soluble DGK in the homogenate of porcine thymocytes was translocated to membranes in a Ca2(+)-dependent manner. Indeed we noted the presence of a 33-residue amphipathic alpha-helix in the DGK sequence, which may account for the protein-lipid interaction. The results demonstrate that Ca2+ plays a key role in the regulation of DGK action by controlling enzyme interaction with membrane phospholipids.

The C-terminal part of diacylglycerol kinase alpha lacking zinc fingers serves as a catalytic domain.

Sakane F, Kai M, Wada I, Imai S, Kanoh H
Biochem J. 1996; 318: 583-90

All mammalian diacylglycerol kinase (DGK) isoenzymes so far cloned consist of four conserved regions, namely, C1, C2 (tandem EF-hand structures), C3 (tandem cysteine-rich zinc finger sequences) and the C-terminal C4 domains. To determine the catalytic domain we expressed in COS-7 cells various truncation mutants of pig DGK alpha and assessed their enzyme activities. We found that the C4 domain lacking the whole N-terminal region including the zinc fingers possessed DGK activity that was dependent on the concentrations of diacylglycerol and ATP very similarly, as did the wild-type DGK alpha. Furthermore the DGK activity of the wild-type DGK and that expressed by the C4 domain were similarly activated by anionic amphiphiles such as phosphatidylserine, phosphatidylinositol and deoxycholate. It was also shown that a DGK mutant consisting of the zinc fingers and the C4 domain has enzymological properties very similar to those expressed by the C4 domain alone. We also confirmed that the intact DGKs alpha, beta and gamma expressed in COS-7 cells displayed no detectable phorbol ester binding. These results show that the C4 domain of DGK is the catalytic region that is responsible for the enzyme activities sensitive to different activators. We cannot exclude the possibility that the N-terminal portion including the zinc fingers can still interact with diacylglycerol and activators without affecting the enzyme activity measured in vitro. However, it is quite likely that the DGK zinc fingers do not serve as diacylglycerol-binding sites, in contrast with those present in other proteins such as protein kinases C and n-chimaerin. Site-directed mutagenesis of all six putative ATP binding sites (Lys248, Lys383, Lys395, Lys483, Lys492, and Lys554) did not significantly affect the enzyme activity. We therefore suggest that DGK does not contain a typical P-loop of ATP binding sites.

Alternative splicing of the human diacylglycerol kinase delta gene generates two isoforms differing in their expression patterns and in regulatory functions.

Sakane F, Imai S, Yamada K, Murakami T, Tsushima S, Kanoh H
J Biol Chem. 2002; 277: 43519-26

Diacylglycerol kinase (DGK) plays an important role in signal transduction through modulating the balance between two signaling lipids, diacylglycerol and phosphatidic acid. DGKdelta (type II isozyme) contains a pleckstrin homology domain at the N terminus and a sterile alpha motif domain at the C terminus. We identified another DGKdelta isoform (DGKdelta2, 135 kDa) that shared the same sequence with DGKdelta previously cloned (DGKdelta1, 130 kDa) except for the 52 residues N-terminally extended. Analysis of panels of human normal and tumor tissue cDNAs revealed that DGKdelta2 was ubiquitously expressed in all normal and tumor tissues examined, whereas the transcript of DGKdelta1 was detected only in ovary and spleen, and in a limited set of tumor-derived cells. The expression of DGKdelta2 was induced by treating cells with epidermal growth factor and tumor-promoting phorbol ester. In contrast, the levels of mRNA and protein of DGKdelta1 were suppressed by phorbol ester treatment. It thus becomes clear that the two DGKdelta isoforms are expressed under distinct regulatory mechanisms. DGKdelta1 was translocated through its pleckstrin homology domain from the cytoplasm to the plasma membrane in response to phorbol ester stimulation, whereas DGKdelta2 remained in the cytoplasm even after stimulation. Further experiments showed that the delta2-specific N-terminal sequence blocks the phorbol ester-dependent translocation of this isoform. Co-immunoprecipitation analysis of differently tagged DGKdelta1 and DGKdelta2 proteins showed that they were able to form homo- as well as hetero-oligomers. Taken together, alternative splicing of the human DGKdelta gene generates at least two isoforms, differing in their expressions and regulatory functions.

The regulatory role of EF-hand motifs of pig 80K diacylglycerol kinase as assessed using truncation and deletion mutants.

Sakane F, Imai S, Yamada K, Kanoh H
Biochem Biophys Res Commun. 1991; 181: 1015-21

To elucidate the regulatory function of EF-hand motifs of pig 80K diacylglycerol (DG) kinase, we constructed and expressed several truncation and deletion mutants of the enzyme in E. coli or COS-7 cells. The bacterially expressed EF-hand region could bind Ca2+ and was suggested to undergo conformational change like calmodulin. A mutant enzyme lacking EF-hands lost Ca(2+)-binding activity, but could be fully activated by phosphatidylserine (PS) or deoxycholate in the absence of Ca2+. The full activation of the wild-type enzyme by PS, on the other hand, was totally dependent on Ca2+. Further, the wild-type enzyme expressed in COS-7 cells was exclusively soluble, whereas the EF-hand-deleted mutant was considerably associated with the membranes. The results suggest that under Ca(2+)-free condition, the EF-hand masks the PS-binding site of the DG kinase, and that the Ca(2+)-binding results in the exposure of the PS-binding site through the conformational change of the EF-hand region.

A diacylglycerol-protein kinase C-RasGRP1 pathway directs Ras activation upon antigen receptor stimulation of T cells.

Roose JP, Mollenauer M, Gupta VA, Stone J, Weiss A
Mol Cell Biol. 2005; 25: 4426-41

Ras GTPases are on/off switches regulating numerous cellular responses by signaling to various effector molecules. In T lymphocytes, Ras can be activated by two Ras exchange factors, SOS and RasGRP1, which are recruited through the adapters Grb2 and LAT and via the second-messenger diacylglycerol (DAG), respectively. Mitogen-activated protein (MAP) kinase phosphorylation patterns induced by active Ras can vary and contribute to distinct cellular responses. The different consequences of Ras activation by either guanine exchange factor are unknown. DAG also recruits and activates the kinase protein kinase Ctheta (PKCtheta) turning on the Erk MAP kinase pathway, but the biochemical mechanism responsible is unclear. We generated T-cell clones deficient in phorbol myristate acetate (a surrogate for DAG)-induced Ras activation. Analysis of a RasGRP1-deficient Jurkat T-cell clone and RasGRP1 RNA interference in wild-type cells revealed that RasGRP1 is required for optimal, antigen receptor-triggered Ras-Erk activation. RasGRP1 relies on its DAG-binding domain to selectively activate Erk kinases. Activation of Erk correlates with the phosphorylation of threonine residue 184 in RasGRP1. This phosphorylation event requires the activities of novel PKC kinases. Conversely, active PKCtheta depends on RasGRP1 sufficiency to effectively trigger downstream events. Last, DAG-PKC-RasGRP1-driven Ras-Erk activation in T cells is a unique signaling event, not simply compensated for by SOS activity.

Cloning and expression of cDNA encoding protein synthesis elongation factor-2 kinase.

Redpath NT, Price NT, Proud CG
J Biol Chem. 1996; 271: 17547-54

A cDNA from rat skeletal muscle encoding calcium/calmodulin-dependent eukaryotic elongation factor-2 kinase (eEF-2K) has been cloned and sequenced, and the amino acid sequence of the protein has been deduced. The kinase is composed of 724 amino acids and has a predicted molecular mass of 81,499 Da. The cDNA was judged to be full-length, as the protein, expressed in rabbit reticulocyte lysate or wheat germ extract, migrated upon SDS-PAGE with the same apparent molecular weight as the purified kinase and possessed eEF-2K activity. eEF-2K contains all of the 12 catalytic subdomains present in the majority of protein kinases, but they are atypical and display only limited homology with other kinases. A putative calmodulin-binding domain is present C-terminal to the catalytic domain as is a putative pseudosubstrate sequence. Two antipeptide antibodies raised against sequences derived from a partial rabbit cDNA clone, cross-reacted with purified eEF-2K, and one also immunoprecipitated eEF-2K activity from cell extracts. Northern blot analysis demonstrated that eEF-2K mRNA is expressed in a number of different tissues and that it may exist in multiple forms.

Inhibition of diacylglycerol kinase by the antitumor agent calphostin C. Evidence for similarity between the active site of diacylglycerol kinase and the regulatory site of protein kinase C.

Redman C, Lefevre J, MacDonald ML
Biochem Pharmacol. 1995; 50: 235-41

Calphostin C is an anti-tumor agent that binds to the regulatory domain of protein kinase C and inhibits the binding of phorbol dibutyrate. Recent studies suggest that there may be structural similarities between protein kinase C (PKC) and diacylglycerol kinase (DGK). Both enzymes bind diacylglycerol and phosphatidylserine, and sequencing of the 80 kDa diacylglycerol kinase shows that it contains zinc finger-like sequences, similar to those occurring in PKC. Similarities in some enzymatic properties of PKC and DGK led us to examine whether regulatory-site inhibitors of PKC also might inhibit DGK. For these studies, the membrane-bound DGK was partially purified from porcine testis membranes. Calphostin C inhibited DGK with an IC50 in the micromolar range. The inhibition of DGK by calphostin C was competitive with respect to diacylglycerol and was not affected by the presence or absence of phosphatidylserine. Other inhibitors of protein kinase C were without effect, with the exception of Adriamycin, which inhibited at millimolar concentrations. Staurosporine, which binds to the catalytic domain of protein kinase C, did not inhibit DGK. The results suggest that there are functional similarities between the substrate binding site of DGK and the regulatory site of protein kinase C.

Cloning and characterization of mouse deoxyguanosine kinase. Evidence for a cytoplasmic isoform.

Petrakis TG, Ktistaki E, Wang L, Eriksson S, Talianidis I
J Biol Chem. 1999; 274: 24726-30

Deoxyguanosine kinase (dGK) is a nuclear gene product that catalyzes the phosphorylation of purine deoxyribonucleosides and their analogues. The human enzyme is located predominantly in the mitochondria, as shown by biochemical fractionation studies and in situ localization of the overexpressed recombinant protein. Here we describe the cloning of mouse dGK cDNA and the identification of a novel amino-terminally truncated isoform that corresponds to about 14% of the total dGK mRNA population in mouse spleen. In situ fluorescence assays suggest that the new isoform cannot translocate into the mitochondria and thus may represent a cytoplasmic enzyme. Expression of mouse dGK mRNA was highly tissue-specific and differed from the tissue distribution observed in humans. Recombinant mouse dGK showed similar specific activity and substrate specificity as compared with the human enzyme. The broad specificity, restricted tissue distribution, and location of mouse dGK in multiple cellular compartments raise new considerations with respect to the role of the individual deoxynucleoside kinases in nucleotide metabolism.

Molecular cloning and characterization of a protein tyrosine phosphatase enriched in testis, a putative murine homologue of human PTPMEG.

Park KW, Lee EJ, Lee S, Lee JE, Choi E, Kim BJ, Hwang R, Park KA, Baik J
Gene. 2000; 257: 45-55

Protein tyrosine phosphorylation is regulated by protein tyrosine kinase and protein tyrosine phosphatase activities. These two counteracting proteins are implicated in cell growth and transformation. Using polymerase chain reaction with degenerate primers, we have identified a novel mouse protein tyrosine phosphatase (PTP). This cDNA contains a single open reading frame of the predicted 926 amino acids. Those predicted amino acids showed significant identity with human megakaryocyte protein-tyrosine phosphatase by 91% in nucleotide sequences and 94% in amino acid sequences. We have identified that expression of this PTP is highly enriched in the testis in mouse and human and has been termed here as a 'testis-enriched phosphatase' (TEP). Northern analysis detected two mRNA species of 3.7 and 3.2kb for this PTP in mouse testis and the expression of TEP is regulated during development. The recombinant phosphatase domain possesses protein tyrosine phosphatase activity when expressed in Escherichia coli. Immunohistochemical analysis of the cellular localization of TEP on mouse testis sections showed that this PTP is specifically expressed in spermatocytes and spermatids within seminiferous tubules, suggesting an important role in spermatogenesis.

Identification of mRNAs for the various diacylglycerol kinase isoforms in neutrophils from patients with localized aggressive periodontitis.

Oyaizu K, Kantarci A, Maeda H, Batista EL Jr, Hasturk H, Murayama Y, Badwey JA, Van Dyke TE
J Periodontal Res. 2003; 38: 488-95

BACKGROUND: Diacylglycerol kinase (DGK) metabolizes diacylglycerol (DAG), an endogenous activator of protein kinase C, to phosphatidic acid. We have previously reported increased DAG in neutrophils from patients with localized aggressive periodontitis (LAP) associated with reduced DGK activity. This reduction could be related to a mutation, post-translational modification, differential expression, or lack of expression of a particular isoform(s). OBJECTIVE: The aim of this study was to identify the mRNAs for DGK isoforms in normal and LAP neutrophils. METHODS: The alpha-, gamma-, and delta-isoforms of DGK were identified by polymerase chain reaction (PCR) using specific oligonucleotide primers for each isoform. The PCR products were isolated and sequenced for comparison to published sequences to confirm the validity of the PCR reaction. Total RNA was isolated from LAP and normal neutrophils, and northern blotting and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to examine the level of mRNA for each isoform. RESULTS: No major differences were found in the isoform pattern between resting normal and LAP neutrophils. However, the levels of mRNA for the alpha- and gamma-isoforms of DGK were increased in normal neutrophils while slightly decreased in LAP cells upon stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP). CONCLUSION: These data suggest that alterations in the mRNAs for the various isoforms of DGK during cell stimulation and the involvement of DGK that is expressed in multiple forms are subject to a variety of regulatory/control mechanisms and these mechanisms may explain the role of the 'primed' neutrophil phenotype associated with LAP.

Diacylglycerol kinase alpha activity promotes survival of CD4+ 8+ double positive cells during thymocyte development.

Outram SV, Crompton T, Merida I, Varas A, Martinez-A C
Immunology. 2002; 105: 391-8

The diacylglycerol kinases (DGK) form a family of isoenzymes that catalyse the conversion of diacylglycerol (DAG) to phosphatidic acid (PA), both powerful second messengers in the cell. DGKalpha is expressed in brain, peripheral T cells and thymocytes and has been shown to translocate to the nuclear matrix upon T-cell receptor (TCR) engagement. Here, we show that high level expression of DGKalpha is induced following a signal transmitted through the pre-TCR and the protein tyrosine kinase, lck. Activity of DGKalpha contributes to survival in CD4+ 8+ (DP) thymocytes as pharmacological inhibition of DGK activity results in death of this cell population both in cell suspension and thymic explants. DGKalpha promotes survival in these thymocytes through a Bcl-regulated pathway. A consequence of inhibition of DGKalpha is the specific down-regulation of Bcl-xl, whereas in transgenic mice that over-express Bcl-2, death induced by the inhibitor is partially blocked. Thus we report a novel activity of DGKalpha in survival of thymocytes immediately after entry into the DP stage in development.

A phorbol ester receptor/protein kinase, nPKC eta, a new member of the protein kinase C family predominantly expressed in lung and skin.

Osada S, Mizuno K, Saido TC, Akita Y, Suzuki K, Kuroki T, Ohno S
J Biol Chem. 1990; 265: 22434-40

Protein kinase C (PKC)-related cDNA clones isolated from mouse epidermis cDNA library encoded a 78-kDa protein, nPKC eta. nPKC eta contains a characteristic cysteine-rich repeat sequence (C1 region) and a protein kinase domain sequence (C3 region), both of which are conserved among PKC family members. However, nPKC eta lacks a putative Ca2+ binding region (C2 region) that is seen in conventional PKCs (alpha, beta I, beta II, gamma), but not in novel PKCs (nPKC delta, -epsilon, -zeta). nPKC eta shows the highest sequence similarity to nPKC epsilon (59.4% identity). The similarity extends to the NH2-terminal sequence (E region) which corresponds to one of the divergent regions (D1 region). Northern blot analysis showed that the mRNA for nPKC eta is highly expressed in the lung and skin but, in contrast to other members of the PKC family, only slightly expressed in the brain. nPKC eta expressed in COS cells shows phorbol ester binding activity with a similar affinity to nPKC epsilon. Antiserum raised against a COOH-terminal peptide of nPKC eta identified an 82-kDa protein in mouse lung extract as well as in an extract from COS cells transfected with the nPKC eta-cDNA expression plasmid. Autophosphorylation of nPKC eta immunoprecipitated with the specific antiserum was observed, indicating that nPKC eta is a protein kinase. These results clearly demonstrate the existence and the possible importance of nPKC eta as a member of the phorbol ester receptor/protein kinase, PKC, family.

Protein kinase C zeta subspecies from rat brain: its structure, expression, and properties.

Ono Y, Fujii T, Ogita K, Kikkawa U, Igarashi K, Nishizuka Y
Proc Natl Acad Sci U S A. 1989; 86: 3099-103

The primary structure of the zeta subspecies of rat brain protein kinase C was deduced from its overlapping cDNAs. The zeta subspecies of protein kinase C consists of 592 amino acid residues with the calculated molecular mass of 67,740 Da and has regulatory and protein kinase domains in its amino- and carboxyl-terminal halves, respectively. Although all members of the protein kinase C family so far identified have a tandem repeat of the characteristic cysteine-rich zinc-finger-like sequence in the regulatory domain, the zeta subspecies contains only one set of this sequence. Northern (RNA)-blot hybridization analysis indicated that two major RNA transcripts of the zeta subspecies with different lengths may be generated by the use of different polyadenylylational signals. The enzyme was expressed in COS-7 cells by transfection with the cDNA construct encoding its whole sequence. It showed an approximate molecular mass of 64,000 Da upon SDS/PAGE. The enzyme activity was significantly dependent on phospholipid but was independent of the presence of Ca2+ or diacylglycerol, when assayed with calf thymus H1 histone as a phosphate acceptor protein. The zeta subspecies expressed in COS-7 cells did not appear to show binding activity of phorbol ester. The structural and biochemical properties indicate that the zeta subspecies is related to, but distinct from, other subspecies of protein kinase C. Perhaps, this subspecies belongs to another entity of the enzyme family.

Identification and characterization of PKNbeta, a novel isoform of protein kinase PKN: expression and arachidonic acid dependency are different from those of PKNalpha.

Oishi K, Mukai H, Shibata H, Takahashi M, Ona Y
Biochem Biophys Res Commun. 1999; 261: 808-14

The cDNA clone encoding a novel isoform of protein kinase PKN, termed PKNbeta, was isolated from a HeLa cDNA library. PKNbeta had high sequence homology with PKNalpha, originally isolated PKN, especially in the repeats of charged amino acid-rich region with leucine-zipper like sequences (CZ region/HR1), in the carboxyl-terminal catalytic domain, and in approximately 130 amino acid stretch (D region/HR2), located between CZ region/HR1 and the catalytic domain. However, the amino acid sequence of PKNbeta differed from that of PKNalpha in the region immediately amino-terminal to the catalytic domain, which contained two distinct proline-rich sequences consistent with the class II consensus sequence, PXXPXR, for binding to SH3 domain. Distribution of PKNbeta differed from that of PKNalpha in the following two respects: (1) Northern blotting indicated that PKNbeta mRNA could not be detected in human adult tissues, but was expressed abundantly in human cancer cell lines; (2) immunochemical analysis indicated that PKNbeta localized in nucleus and perinuclear Golgi apparatus, and was almost absent in cytoplasmic region in NIH3T3 cells. Recombinant PKNbeta expressed in COS7 cells displayed autophosphorylation and peptide kinase activity, but was found to be significantly less responsive to arachidonic acid than PKNalpha. The identification of this novel isoform underscores the diversity of PKN signaling pathway.

A novel insulin receptor substrate protein, xIRS-u, potentiates insulin signaling: functional importance of its pleckstrin homology domain.

Ohan N, Bayaa M, Kumar P, Zhu L, Liu XJ
Mol Endocrinol. 1998; 12: 1086-98

A novel Xenopus insulin receptor substrate cDNA was isolated by hybridization screening using the rat insulin receptor substrate-1 (IRS-1) cDNA as a probe. The xIRS-u cDNA encodes an open reading frame of 1003 amino acids including a putative amino-terminal pleckstrin homology (PH) domain and phosphotyrosine-binding (PTB) domain. The carboxy terminus of xIRS-u contains several potential Src homology 2 (SH2)-binding sites, five of which are in the context of YM/LXM (presumptive binding sites for phosphatidylinositol 3-kinase). It also contains a putative binding site for Grb2 (YINID). Pair-wise amino acid sequence comparisons with the previously identified xIRS-1 and the four members of the mammalian IRS family (1 through 4) indicated that xIRS-u has similar overall sequence homology (33-45% identity) to all mammalian IRS proteins. In contrast, the previously isolated xIRS-1 is particularly similar (67% identical) to IRS-1 and considerably less similar (31-46%) to the other IRS family members (2 through 4). xIRS-u is also distinct from xIRS-1, having an overall sequence identity of 47%. These sequence analyses suggest that xIRS-u is a novel member of the IRS family rather than a Xenopus homolog of an existing member. Microinjection of mRNA encoding a Myc-tagged xIRS-u into Xenopus oocytes resulted in the expression of a 120-kDa protein (including 5 copies of the 13-amino acid Myc tag). The injection of xIRS-u mRNA accelerated insulin-induced MAP kinase activation with a concomitant acceleration of insulin-induced oocyte maturation. An aminoterminal deletion of the PH domain (xIRS-u deltaPH) significantly reduced the ability of xIRS-u to potentiate insulin signaling. In contrast to the full-length protein, injection of xIRS-u (1-299), which encoded the PH and PTB domain, or xIRS-u (1-170), which encoded only the PH domain, blocked insulin signaling in Xenopus oocytes. Finally, xIRS-u (119-299), which had a truncated PH domain and an intact PTB domain, had no effect on insulin signaling. This is the first report that the PH domain of an IRS protein can function in a dominant negative manner to inhibit insulin signaling.

Membrane-associated diacylglycerol kinase activity is increased by noradrenaline, but not by angiotensin II, in arterial smooth muscle.

Ohanian J, Heagerty AM
Biochem J. 1994; 300: 51-6

In rat small arteries, noradrenaline stimulates the sustained production of arachidonoyl-phosphatidic acid, whereas there is only a slight and transient increase with angiotensin II [Ohanian, Ollerenshaw, Collins and Heagerty (1990) J. Biol. Chem. 265, 8921-8928]. Diacylglycerol kinase (DGK) is the enzyme responsible for generating phosphatidic acid from 1,2-diacylglycerol (DAG). To investigate whether agonists influence DGK activity, we have studied this enzyme in both particulate and soluble fractions prepared from rat small arteries. Soluble DGK activity was inhibited by octyl glucoside. Therefore a deoxycholate assay was used for this fraction, whereas an octyl glucoside mixed-micelle assay was used to examine particulate fractions. Particulate DGK selectively phosphorylated long-chain DAG at a rate 2.5-3-fold higher than that for the synthetic substrate dioctanoylglycerol. In contrast, the substrate preference of the soluble isoenzyme(s) was: dioctanoylglycerol > arachidonoyl-DAG= dioleoylglycerol. Stimulation of intact arteries with noradrenaline (15 microM) increased membrane-associated DGK activity 3-fold, transiently. Angiotensin II (100 nM) stimulation did not alter the DGK activity of this fraction. The activity of the soluble DGK was increased by both agonists, but only transiently. These results demonstrate that rat small arteries contain a membrane-associated DGK which metabolizes arachidonoyl-containing substrate. Also, the activity of this enzyme is regulated differentially by vasoconstrictor hormones. It is concluded that modulation of DGK activity may represent one point at which agonists using the same signal-transduction pathway may tailor the cellular response.

Diacylglycerol kinase activity in intact small arteries.

Ohanian J, Heagerty AM
Biochem Soc Trans. 1993; 21: 349-349

LIMK-1 and LIMK-2, two members of a LIM motif-containing protein kinase family.

Nunoue K, Ohashi K, Okano I, Mizuno K
Oncogene. 1995; 11: 701-10

We previously isolated human cDNA encoding LIM-kinase (LIMK), a putative protein kinase which contains two repeats of the LIM motif at the N-terminus and a protein kinase consensus sequence at the C-terminus. Using as a probe a cDNA fragment of human LIMK, we isolated from a rat brain cDNA library cDNA clones encoding two distinct protein kinases (termed LIMK-1 and LIMK-2) related to human LIMK. LIMK-1 shares with human LIMK 95% of the total 647 amino acids and is probably a rat equivalent of human LIMK. LIMK-2 has an overall sequence and a domain structure similar to that of human LIMK and rat LIMK-1, but overall identity is 50-51% at the amino acid level. Like human LIMK, the protein kinase domains of rat LIMK-1 and -2 contain a characteristic sequence DLNSHN in subdomain VIB and a highly basic insert between subdomain VII and VIII. LIMK-1 and -2 are therefore closely related but distinct members of a novel LIM-containing protein kinase subfamily. Several forms of LIMK-2 transcripts encoding proteins that are N-terminally modified and/or C-terminally truncated are generated by alternative splicing or alternative initiation. Northern blot analysis revealed the expression of LIMK-1 mRNA predominantly in the brain and the expression of LIMK-2 mRNA in various tissues in the rat. Antibody raised against LIMK-1 specifically immunoprecipitated and identified in Rat2 fibroblast cells a 72 kDa protein, which has no detectable autophosphorylating activity but is capable of phosphorylating serine and threonine residues of myelin basic protein, by in vitro kinase reaction. As the LIMK family kinases have unique structural features, they are likely to have specific functions in previously uncharacterized signaling pathways.

Diacylglycerol kinase activity in purified basolateral membranes of kidney tubules. I. Evidence for coupling with phospholipase C.

Nogaroli L, Silva OF, Bonilha TA, Moreno PA, Bernardo RR, Vieyra A, Einicker-Lamas M
Int J Biochem Cell Biol. 2005; 37: 79-90

The diacylglycerol kinase (DGK) catalyzes the phosphorylation of diacylglycerol (DAG) yielding phosphatidic acid (PA) signaling molecules which are involved in the modulation of different cell responses. The aim of this work was to characterize the DGK activity associated to the basolateral membranes (BLM) of kidney proximal tubules, in a native preparation that preserves the membrane microenvironment. The Arrhenius plot of DGK activity was non-linear, indicating a complex influence of the lipid environment of the native membrane. The formation of PA was strongly impaired by U73122, an inhibitor of PLC, whereas remained unmodified when exogenous DAG or PLC were added. The Mg.ATP2- complex is the true phosphoryl-donor substrate, and the very narrow peak of activation at pH 7.0 suggests that amino acids that dissociate at this pH, i.e. hystidine residues, play a role by acting in the coordination of the Mg2+ atoms. The renal DGK is almost completely blocked by 0.1 mM sphingosine, but it is insensitive to micromolar free Ca2+ concentrations and to R59499, the most potent inhibitor of the classical DGKs. Taken as a whole, these data suggest that the DGK isoform present in BLM of proximal tubules is different from those included in the type I family, and that membranous PLC could be the main source of DAG for DGK catalysis.

Cloning, expression, and localization of 230-kDa phosphatidylinositol 4-kinase.

Nakagawa T, Goto K, Kondo H
J Biol Chem. 1996; 271: 12088-94

A phosphatidylinositol (PI) 4-kinase cDNA was cloned from a rat brain cDNA library. This cDNA encoded a protein of 2041 amino acids with a calculated molecular weight of 231,317. The deduced amino acid sequence shared the identity of 52.3 and 34.4% in the presumed catalytic domain with two yeast PI 4-kinases, STT4 and PIK1, respectively, and showed 31.7% identity to p110alpha subunit of rat PI 3-kinase in the same domain. In addition, a 3' half coding region of the present cDNA was 89.6% identical to and its deduced amino acid sequence was 98.2% identical to the sequence for P14Kalpha, a recently reported human PI 4-kinase of type II, suggesting that P14Kalpha is an alternative form of the present PI 4-kinase molecule. The present cDNA contained sequences encoding the ankyrin repeat domain, lipid kinase unique domain, pleckstrin homology domain, presumed lipid kinase/protein kinase homology domain, proline-rich region, and SH3 domain. By examining PI kinase activity in transfected COS-7 cells using the epitope tag immunoprecipitation as well as the conventional way, the product phosphatidylinositol phosphate was identified as phosphatidylinositol 4-phosphate but not phosphatidylinositol 3-phosphate. This PI 4-kinase activity was markedly enhanced in the presence of Triton X-100 but relatively insensitive to inhibition by adenosine. By epitope tag immunohistochemistry, the immunoreactivity for this PI 4-kinase molecule was largely localized in close association with the membranes of the Golgi vesicles and vacuoles. By in situ hybridization analysis, the expression of mRNA for this PI 4-kinase was evident throughout the gray matter of entire brain with higher expression intensity in fetal brain. These data imply that this novel PI 4-kinase is involved in some processes essential to neuronal differentiation and maturation including the synaptogenesis and synaptic plasticity.

cDNA cloning of a novel protein containing two zinc-finger domains that may function as a transcription factor for the human heme-oxygenase-1 gene.

Muraosa Y, Takahashi K, Yoshizawa M, Shibahara S
Eur J Biochem. 1996; 235: 471-9

Heme oxygenase 1 is an essential enzyme in heme catabolism that cleaves heme to form biliverdin, iron, and carbon monoxide. The human heme-oxygenase-1 gene is transcriptionally activated through the cis-regulatory element (MTE), GTCATATGAC (positions -156 to -147), during 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced differentiation of myelomonocytic cell lines, such as THP-1, to macrophages. MTE is responsible for the myelomonocytic-specific induction of heme-oxygenase-1 gene expression, and is bound by ubiquitous and myelomonocytic cell-line-restricted proteins. In this study, we cloned the cDNA segments coding for a portion of a protein that binds to MTE by a Southwestern procedure from a THP-1 cDNA expression library; we subsequently isolated putative full-length cDNAs by a conventional hybridization procedure. The deduced protein, termed MTB-Zf, consists of 1482 amino acid residues, has a molecular mass of about 162 kDa, and contains the two widely separated zinc-finger domains located near the N- and C-termini. MTB-Zf possesses other structural features characteristic of transcription factors, including a long stretch of acidic amino acids (amino acids 67 - 95), a proline-rich region (positions 733-849), a region rich in basic amino acids (positions 1161-1247), and a leucine repeat-like region (positions 486-514). We show that a portion of MTB-Zf, including an N-terminal zinc-finger domain, binds in vitro to MTE and that the transient coexpression of MTB-Zf cDNA leads to transactivation of the heme-oxygenase-1 gene promoter. Since the 6.5 kb MTB-Zf is expressed in various human cell lines of different lineages, MTB-Zf may represent a ubiquitous MTE-binding protein. Furthermore, the MTB-Zf gene has been mapped to human chromosome 1p35-36.1 by fluorescence in situ hybridization, a region which is frequently deleted in various solid tumors, including neurogenic tumors. We found remarkable differences in the expression patterns of MTB-Zf mRNA and two other hybridizable mRNAs of 5kb and 8.5 kb when human brain and primary brain tumors were compared. Both MTB-Zf and the 8.5-kb mRNAs were abundantly expressed in the five primary brain tumors examined, but only the 5-kb mRNA was detectable in the human brain. These results suggest that MTB-Zf is a transcription factor and may also play an important role in cell growth or differentiation.

Identification and characterization of two splice variants of human diacylglycerol kinase eta.

Murakami T, Sakane F, Imai S, Houkin K, Kanoh H
J Biol Chem. 2003; 278: 34364-72

Diacylglycerol kinase (DGK) participates in regulating the intracellular concentrations of two bioactive lipids, diacylglycerol and phosphatidic acid. DGK eta (eta 1, 128 kDa) is a type II isozyme containing a pleckstrin homology domain at the amino terminus. Here we identified another DGK eta isoform (eta 2, 135 kDa) that shared the same sequence with DGK eta 1 except for a sterile alpha motif (SAM) domain added at the carboxyl terminus. The DGK eta 1 mRNA was ubiquitously distributed in various tissues, whereas the DGK eta 2 mRNA was detected only in testis, kidney, and colon. The expression of DGK eta 2 was suppressed by glucocorticoid in contrast to the marked induction of DGK eta 1. DGK eta 2 was shown to form through its SAM domain homo-oligomers as well as hetero-oligomers with other SAM-containing DGKs (delta 1 and delta 2). Interestingly, DGK eta 1 and DGK eta 2 were rapidly translocated from the cytoplasm to endosomes in response to stress stimuli. In this case, DGK eta 1 was rapidly relocated back to the cytoplasm upon removal of stress stimuli, whereas DGK eta 2 exhibited sustained endosomal association. The experiments using DGK eta mutants suggested that the oligomerization of DGK eta 2 mediated by its SAM domain was largely responsible for its sustained endosomal localization. Similarly, the oligomerization of DGK eta 2 was suggested to result in negative regulation of its catalytic activity. Taken together, alternative splicing of the human DGK eta gene generates at least two isoforms with distinct biochemical and cell biological properties responding to different cellular metabolic requirements.

Molecular cloning and characterization of a novel member of the MAP kinase superfamily.

Miyata Y, Akashi M, Nishida E
Genes Cells. 1999; 4: 299-309

BACKGROUND: Members of the MAP kinase superfamily play important roles in a wide variety of signal transduction pathways, and several members have been identified. However, the diversity and complexity of cellular responses in mammalian systems may imply existence of hitherto unidentified members of the MAP kinase superfamily. RESULTS: We report the molecular cloning and characterization of a novel member of the MAP kinase superfamily. We isolated full-length mouse and human cDNAs that encode complete open reading frames of a novel protein kinase, termed MOK. MOK consists of 419 (human) and 420 (mouse) amino acids, with a calculated molecular weight of 48kDa. MOK contains all of the protein serine/threonine kinase consensus motifs and shows a modest similarity to members of the MAP kinase superfamily and MAK and MAK-related kinase (MRK). In addition, MOK possesses a Thr-Glu-Tyr (TEY) motif in the activation loop domain, as do classical MAP kinases. MOK is widely expressed in normal tissues and organs and localizes to the cytoplasm. MOK is able to phosphorylate several known MAP kinase substrates and to undergo autophosphorylation. A mutation in the TEY motif to AEF abolished the kinase activity of MOK, and the treatment of cells with a phosphatase inhibitor, okadaic acid, enhanced the kinase activity of MOK, suggesting the existence of an upstream kinase. Phorbol ester TPA was found to stimulate the kinase activity of MOK, whereas serum stimulation, osmotic shock, or anisomycin treatment did not significantly activate MOK. CONCLUSION: These results indicate that MOK is distantly related to members of known subfamilies of the MAP kinase superfamily and can therefore be classified as a novel member.

Human sphingosine kinase: molecular cloning, functional characterization and tissue distribution.

Melendez AJ, Carlos-Dias E, Gosink M, Allen JM, Takacs L
Gene. 2000; 251: 19-26

Sphingosine-1-phosphate (SPP), the product of sphingosine kinase, is an important signaling molecule with intra- and extracellular functions. The cDNA for the mouse sphingosine kinase has recently been reported. In this paper we describe the cloning, expression and characterization of the human sphingosine kinase (huSPHK1). Sequence analysis comparison revealed that this kinase is evolutionarily very conserved, having a high degree of homology with the murine enzyme, and presenting several conserved regions with bacteria, yeast, plant, and mammalian proteins. Expressed huSPHK1 cDNA specifically phosphorylates D-erythro-sphingosine and, to a lesser extent, D, L-erythro-dihydrosphingosine, and not at all the 'threo' isoforms of dihydrosphingosine; hydroxy-ceramide or non-hydroxy-ceramide; diacylglycerol (DAG); phosphatidylinositol (PI); phosphatidylinositol-4-phosphate (PIP); or phosphatidylinositol-4, 5-bisphosphate (PIP(2)). huSPHK1 shows typical Michaelis-Menten kinetics (V(max)=56microM and K(m)=5microM). The kinase is inhibited by D,L-threo-dihydrosphingosine (K(i)=3microM), and by N, N-dimethyl-sphingosine (K(i)=5microM). Northern blots indicate highest expression in adult lung and spleen, followed by peripheral blood leukocyte, thymus and kidney, respectively. It is also expressed in brain and heart. In addition, database searches with the stSG2854 sequence indicate that huSPHK1 is also expressed in endothelial cells, retinal pigment epithelium, and senescent fibroblasts.

Nuclear transportation of diacylglycerol kinase gamma and its possible function in the nucleus.

Matsubara T, Shirai Y, Miyasaka K, Murakami T, Yamaguchi Y, Ueyama T, Kai M, Sakane F, Kanoh H, Hashimoto T, Kamada S, Kikkawa U, Saito N
J Biol Chem. 2006; 281: 6152-64

Diacylglycerol kinases (DGKs) convert diacylglycerol (DG) to phosphatidic acid, and both lipids are known to play important roles in lipid signal transduction. Thereby, DGKs are considered to be a one of the key players in lipid signaling, but its physiological function remains to be solved. In an effort to investigate one of nine subtypes, we found that DGKgamma came to be localized in the nucleus with time in all cell lines tested while seen only in the cytoplasm at the early stage of culture, indicating that DGKgamma is transported from the cytoplasm to the nucleus. The nuclear transportation of DGKgamma didn't necessarily need DGK activity, but its C1 domain was indispensable, suggesting that the C1 domain of DGKgamma acts as a nuclear transport signal. Furthermore, to address the function of DGKgamma in the nucleus, we produced stable cell lines of wild-type DGKgamma and mutants, including kinase negative, and investigated their cell size, growth rate, and cell cycle. The cells expressing the kinase-negative mutant of DGKgamma were larger in size and showed slower growth rate, and the S phase of the cells was extended. These findings implicate that nuclear DGKgamma regulates cell cycle.

Drosophila retinal degeneration A gene encodes an eye-specific diacylglycerol kinase with cysteine-rich zinc-finger motifs and ankyrin repeats.

Masai I, Okazaki A, Hosoya T, Hotta Y
Proc Natl Acad Sci U S A. 1993; 90: 11157-61

The Drosophila visual mutant, carrying the retinal degeneration A gene (rdgA), has photoreceptor cells that degenerate within a week after eclosion. Morphological studies suggested that this mutant harbors abnormalities in membrane turnover of the photoreceptor cells. Biochemically, the rdgA mutant lacks an eye-specific and membrane-associated diacylglycerol kinase (DGK; EC 2.7.1.107) activity in a gene-dosage-dependent manner, suggesting that rdgA gene encodes a DGK. We report the molecular cloning and characterization of a DGK gene, which maps to the rdgA locus. This gene, designated as DGK2, has a single open reading frame that encodes 1454 amino acids. Like porcine DGK, DGK2 has two cysteine-rich zinc-finger motifs as well as a DGK catalytic domain. The DGK2 protein contains four ankyrin-like repeats at the C-terminal region, suggesting that DGK2 is likely anchored to the membrane or cytoskeleton. Northern blot analysis and tissue in situ hybridization to adult sections revealed that DGK2 is expressed exclusively in the adult retina and that the amount of its mRNA is reduced in some of the rdgA mutant alleles. Furthermore, in two rdgA alleles, rdgA1 and rdgA2, nonsense and missense mutations occur within their DGK2 gene, respectively. Thus, we conclude that rdgA encodes an eye-specific DGK, the absence of which leads to rhabdomere degeneration due to defective phospholipid turnover.

Molecular cloning of a Drosophila diacylglycerol kinase gene that is expressed in the nervous system and muscle.

Masai I, Hosoya T, Kojima S, Hotta Y
Proc Natl Acad Sci U S A. 1992; 89: 6030-4

We have isolated a Drosophila melanogaster diacylglycerol kinase (DGK, EC 2.7.1.107) homologue by using a porcine DGK cDNA probe and we have characterized its structure and expression. The DGK cDNA has a single open reading frame that encodes 791 amino acids. The Drosophila and porcine DGKs share a similar carboxyl-terminal region, a putative catalytic domain, which is divided into two separate domains in Drosophila. The DGK gene was mapped to the cytogenetic position 43F1, and its DGK mRNA is abundant both in embryo and in adult fly. By in situ hybridization to sections of adult flies, we demonstrated that the mRNA is present predominantly in the nervous system and muscles, including compound eyes, brain cortex, fibrillar muscle, and tubular muscle. In a 10- to 11-hr embryo, the DGK gene is expressed abundantly in a limited number of cells in the procephalic region and in the ventral nerve cord. The pattern of temporal and spatial expression suggests that the DGK protein has an important function in the adult nervous system and muscle and during the development of the embryonic nervous system.

Cloning and expression of the heterodimeric deoxyguanosine kinase/deoxyadenosine kinase of Lactobacillus acidophilus R-26.

Ma GT, Hong YS, Ives DH
J Biol Chem. 1995; 270: 6595-601

Two uniquely paired deoxynucleoside kinases, deoxycytidine kinase/deoxyadenosine kinase (dCK/dAK) and deoxyguanosine kinase/deoxyadenosine kinase (dGK/dAK) are required, together with thymidine kinase (TK), for deoxynucleotide synthesis in Lactobacillus acidophilus R-26. Using polymerase chain reaction-generated probes based on N-terminal amino acid sequences, we have cloned tandem genes for 25- and 26-kDa polypeptides, whose derived amino acid sequences and size correspond to wild-type Lactobacillus enzyme subunits. Expression in Escherichia coli uses a single endogenous promoter and yields active dGK/dAK (approximately 3% of extracted protein) closely resembling wild-type dGK/dAK in specificity, kinetics, heterotropic activation, and end product inhibition. Alignment of cloned genes reveals 65% identity in their DNA sequences and 61% identity in derived amino acid sequences. Comparison with herpes-viral TKs reveals three conserved regions: glycine- and arginine-rich ATP-binding motifs and a D/E-R-S/H motif at the putative TK deoxynucleoside site. Greater homology, however, is seen upon multiple alignment of dGK with mammalian deoxycytidine kinases, yielding the consensus sequence-D/E-R-S-I/V-Y-x-D-.dGK also shares a sequence (-Y-D-P-T-I/L-E-D-S/Y-Y-) required for GTP hydrolysis by p21ras.

Association of diacylglycerol kinase zeta with protein kinase C alpha: spatial regulation of diacylglycerol signaling.

Luo B, Prescott SM, Topham MK
J Cell Biol. 2003; 160: 929-37

Activation of PKC depends on the availability of DAG, a signaling lipid that is tightly and dynamically regulated. DAG kinase (DGK) terminates DAG signaling by converting it to phosphatidic acid. Here, we demonstrate that DGKzeta inhibits PKCalpha activity and that DGK activity is required for this inhibition. We also show that DGKzeta directly interacts with PKCalpha in a signaling complex and that the binding site in DGKzeta is located within the catalytic domain. Because PKCalpha can phosphorylate the myristoylated alanine-rich C-kinase substrate (MARCKS) motif of DGKzeta, we tested whether this modification could affect their interaction. Phosphorylation of this motif significantly attenuated coimmunoprecipitation of DGKzeta and PKCalpha and abolished their colocalization in cells, indicating that it negatively regulates binding. Expression of a phosphorylation-mimicking DGKzeta mutant that was unable to bind PKCalpha did not inhibit PKCalpha activity. Together, our results suggest that DGKzeta spatially regulates PKCalpha activity by attenuating local accumulation of signaling DAG. This regulation is impaired by PKCalpha-mediated DGKzeta phosphorylation.

Protein kinase C alpha phosphorylates and negatively regulates diacylglycerol kinase zeta.

Luo B, Prescott SM, Topham MK
J Biol Chem. 2003; 278: 39542-7

Diacylglycerol kinase (DGK) terminates diacylglycerol (DAG) signaling by phosphorylating DAG to produce phosphatidic acid, which also has signaling properties. Thus, precise control of DGK activity is essential for proper signal transduction. We demonstrated previously that a peptide corresponding to the myristoylated alanine-rich C kinase substrate (MARCKS) phosphorylation site domain (PSD) in DGK zeta was phosphorylated in vitro by an active fragment of protein kinase C (PKC). In the present study, we tested full-length DGK zeta and found that PKC alpha phosphorylated DGK zeta on serines within the MARCKS PSD in vitro and in vivo. DGK zeta also coimmunoprecipitated with PKC alpha, suggesting that they reside in a regulated signaling complex. We then tested whether phosphorylation affected DAG kinase activity. We found that a mutant (DGK zeta S/D) in which serines within the MARCKS PSD were altered to aspartates (to mimic phosphorylation) had lower activity compared with wild-type DGK zeta or a control mutant (DGK zeta S/N) in which the same serines were changed to asparagines. Furthermore, activation of PKC alpha by phorbol 12-myristate 13-acetate inhibited the activity of wild-type DGK zeta, but not DGK zeta S/D, in human embryonic kidney 293 cells. These results suggest that by phosphorylating the MARCKS PSD, PKC alpha attenuates DGK zeta activity. Supporting this, we found that cells expressing DGK zeta S/D had higher DAG levels and grew more rapidly compared with cells expressing DGK zeta S/N that could not be phosphorylated. Taken together, these results indicate that PKC alpha phosphorylates DGK zeta in cells, and this phosphorylation inhibits its kinase activity to remove cellular DAG, thereby affecting cell growth.

Primary structure and expression analysis of human UDP-N-acetyl-glucosamine-2-epimerase/N-acetylmannosamine kinase, the bifunctional enzyme in neuraminic acid biosynthesis.

Lucka L, Krause M, Danker K, Reutter W, Horstkorte R
FEBS Lett. 1999; 454: 341-4

N-Acetylneuraminic acid is a main constituent of glycoproteins and gangliosides. In many membrane-bound receptors it is the target for external stimuli. The key enzyme for its biosynthesis is the bifunctional enzyme UDP-N-acetyl-glucosamine-2-epimerase/N-acetylmannosamine kinase, catalysing the first two steps of the biosynthesis in the cytosol. The rat enzyme was previously isolated and characterised. In this report we present the corresponding human cDNA sequence, compare it with the primary structure of the rodent enzyme, and report the analysis of its expression in different human tissues and cell lines.

Structure-activity relationship of diacylglycerol kinase theta.

Los AP, van Baal J, de Widt J, Divecha N, van Blitterswijk WJ
Biochim Biophys Acta. 2004; 1636: 169-74

Diacylglycerol kinase (DGK) phosphorylates the second messenger diacylglycerol (DAG) to phosphatidic acid (PA). Among the nine mammalian isotypes identified, DGKtheta is the only one with three cysteine-rich domains (CRDs) (instead of two) in its N-terminal regulatory region. We previously reported that DGKtheta binds to and is negatively regulated by active RhoA. We now report that RhoA strongly binds to the C-terminal catalytic domain, which would explain its inhibition of DGK activity. To help finding a physiological function of DGKtheta, we further determined its activity in vitro as a function of 15 different truncations and point mutations in the primary structure. Most of these alterations, located throughout the protein, inactivated the enzyme, suggesting that catalytic activity depends on all of its conserved domains. The most C-terminal CRD is elongated with a stretch of 15 amino acids that is highly conserved among DGK isotypes. Mutation analysis revealed a number of residues in this region that were essential for enzyme activity. We suggest that this CRD extension plays an essential role in the correct folding of the protein and/or in substrate presentation to the catalytic region of the protein.

Juvenile hormone diol kinase, a calcium-binding protein with kinase activity, from the silkworm, Bombyx mori.

Li S, Zhang QR, Xu WH, Schooley DA
Insect Biochem Mol Biol. 2005; 35: 1235-48

Juvenile hormone (JH) diol kinase (JHDK) is an important enzyme involved in the JH degradation pathway. Bombyx mori (Bommo)-JHDK cDNA (637bp) contains an open reading frame encoding a 183-amino acid protein, which reveals a high degree of identity to the two previously reported JHDKs. JHDK is similar to GTP-binding proteins with three conserved sequence elements involved in purine nucleotide binding, contains eight alpha-helices and three EF-hand motifs, and resembles the three-dimensional model of 2SCP and some other calcium-binding proteins. The Bommo-JHDK gene has only a single copy in the silkworm haploid genome, contains only one exon, and its 5'-upstream sequence does not have a JH response element. Although Bommo-JHDK is highly expressed in the gut of the silkworm, its mRNA expression remains at a constant level during larval development suggesting this enzyme is constitutive and not regulated by JH, at least at the transcriptional level. Recombinant Bommo-JHDK catalyzed the conversion of 10S-JH diol into JH diol phosphate, confirming its enzymatic function. Recombinant enzyme formed a dimer and had biochemical characteristics similar to other JHDKs. Bommo-JHDK, a calcium-binding protein with kinase activity, provides unique insights on how JH levels are regulated in the silkworm.

The isolation and characterization of cDNA encoding the mouse bifunctional ATP sulfurylase-adenosine 5'-phosphosulfate kinase.

Li H, Deyrup A, Mensch JR Jr, Domowicz M, Konstantinidis AK, Schwartz NB
J Biol Chem. 1995; 270: 29453-9

Biosynthesis of the activated sulfate donor, adenosine 3'-phosphate 5'-phosphosulfate, involves the sequential action of two enzyme activities: ATP sulfurylase, which catalyzes the formation of adenosine 5'-phosphosulfate (APS) from ATP and free sulfate, and APS kinase, which subsequently phosphorylates APS to produce adenosine 3'-phosphate 5'-phosphosulfate. Oligonucleotide primers were derived from a human infant brain-expressed sequence tag putatively encoding a portion of APS kinase. Using these primers, reverse transcriptase-polymerase chain reaction was performed on mRNA from neonatal normal mice resulting in amplification of a 127-bp DNA fragment. This fragment was subsequently used to screen a mouse brain lambda gt11 cDNA library, yielding a 2.2-kb clone. Primers were designed from the 5'-end of the 2.2-kb clone, and 5'-rapid amplification of cDNA ends was used to obtain the translation start site. Sequence from the overlapping clones was assembled into a 2475-bp composite sequence, which contains a single open reading frame that translates into a 624-deduced amino acid sequence. Northern blots of total RNA from neonatal mice yielded a single message species at approximately 3.3 kb. Southern blot of genomic DNA digested with several restriction enzymes suggested the gene is present as a single copy. Comparison against sequence data bases suggested the composite sequence was a fused sulfurylase-kinase product, since the deduced amino acid sequence showed extensive homology to known separate sequences of both ATP sulfurylase and APS kinase from several sources. The first 199 amino acids corresponded to APS kinase sequence, followed by 37 distinct amino acids, which did not match any known sequence, followed by 388 amino acids that are highly homologous to known ATP sulfurylase sequences. Finally, recombinant enzyme expressed in COS-1 cells exhibited both ATP sulfurylase and APS kinase activity.

Molecular cloning, complementary deoxyribonucleic acid structure and predicted full-length amino acid sequence of the hormone-inducible regulatory subunit of 3'-5'-cyclic adenosine monophosphate-dependent protein kinase from human testis.

Levy FO, Oyen O, Sandberg M, Tasken K, Eskild W, Hansson V, Jahnsen T
Mol Endocrinol. 1988; 2: 1364-73

In this study, we report the isolation and characterization of a full-length cDNA clone for the hormone-inducible regulatory subunit RII beta (formerly called RII51) of type II cAMP-dependent protein kinase from a human testis cDNA library. The cloned cDNA demonstrated tissue-specific expression of RII beta mRNA in human tissues, with the highest mRNA levels in testis and ovary. The isolated human cDNA clone was 3.3 kilobases (kb) in length and contained 166 base pairs (bp) of G/C-rich 5'-noncoding sequence, an open reading frame of 1254 bp and an A/T-rich 3'-nontranslated region containing 1836 bp followed by an 89 nucleotide long poly(A)-tail. The predicted protein contains 418 amino acids including the start methionine, and the estimated mol wt of human RII beta is 53,856. The nucleotide sequence within the open reading frame and the predicted amino acid sequence of human RII beta are highly conserved compared with partial rat RII beta sequences, displaying 91% and 97% similarity, respectively. Codon preference analysis of the cloned cDNA sequence indicated that the two cAMP-binding domains and the hinge region are highly conserved through evolution, whereas the dimerization domain displayed a codon preference pattern indicative of appearance at a later stage of evolution. The isolated human cDNA detected an FSH- and cAMP-inducible mRNA of 3.2 kb in rat Sertoli cells, thus confirming that the cloned cDNA represents the hormone-inducible regulatory subunit of cAMP-dependent protein kinase. This is the first report documenting the isolation of a full-length cDNA clone for the RII beta of cAMP-dependent protein kinase.

Structural and functional characterization of human NAD kinase.

Lerner F, Niere M, Ludwig A, Ziegler M
Biochem Biophys Res Commun. 2001; 288: 69-74

NADP is essential for biosynthetic pathways, energy, and signal transduction. Its synthesis is catalyzed by NAD kinase. Very little is known about the structure, function, and regulation of this enzyme from multicellular organisms. We identified a human NAD kinase cDNA and the corresponding gene using available database information. A cDNA was amplified from a human fibroblast cDNA library and functionally overexpressed in Escherichia coli. The obtained cDNA, slightly different from that deposited in the database, encodes a protein of 49 kDa. The gene is expressed in most human tissues, but not in skeletal muscle. Human NAD kinase differs considerably from that of prokaryotes by subunit molecular mass (49 kDa vs 30-35 kDa). The catalytically active homotetramer is highly selective for its substrates, NAD and ATP. It did not phosphorylate the nicotinic acid derivative of NAD (NAAD) suggesting that the potent calcium-mobilizing pyridine nucleotide NAADP is synthesized by an alternative route.

Distribution of distinct arachidonoyl-specific and non-specific isoenzymes of diacylglycerol kinase in baboon (Papio cynocephalus) tissues.

Lemaitre RN, King WC, MacDonald ML, Glomset JA
Biochem J. 1990; 266: 291-9

We investigated the diacyglycerol kinase species present in several baboon tissues using the substrates sn-1-stearoyl-2-arachidonoyl diacylglycerol and sn-1,2-didecanoyl diacylglycerol. Chromatography of octyl glucoside extracts of the baboon (Papio cynocephalus papio) tissues on hydroxyapatite columns revealed the presence of three diacylglycerol kinase species with different substrate preferences. One species markedly 'preferred' the substrate sn-1-stearoyl-2-arachidonoylglycerol, the two other species preferred sn-1,2-didecanoylglycerol. Measurement of the activity of the baboon brain diacylglycerol kinases toward diacylglycerols with a range of different fatty acid chains revealed a strict preference of the arachidonoyl diacylglycerol kinase for sn-1-acyl-2-arachidonoyl diacylglycerol, whereas the other enzymes showed no preference toward several long-chain-fatty-acid-containing diacylglycerols. The arachidonoyl diacylglycerol kinase was particularly abundant in brain and testis, whereas liver was practically devoid of this enzyme. The arachidonoyl diacylglycerol kinase from baboon brain was found to be predominantly associated with the particulate fraction and exhibited an apparent molecular mass of 130 kDa.

Molecular cloning of a diacylglycerol kinase isozyme predominantly expressed in rat retina.

Kohyama-Koganeya A, Watanabe M, Hotta Y
FEBS Lett. 1997; 409: 258-64

We have cloned and characterized a new diacylglycerol kinase (DGK) isozyme which is expressed in the retina and the brain of rat. The cDNA contains an open reading frame of 567 amino acid residues with a predicted protein of 64 kDa and shows very high homology to human DGK epsilon. The new DGK isozyme contains two distinctive zinc-finger structures and a putative catalytic domain. This DGK expressed predominantly in the inner and outer nuclear layers of retina. This expression pattern is different from those of the previously cloned DGKs including the human DGK epsilon, suggesting that this DGK isozyme has potential importance in visual functions as was the case in Drosophila retinal cells.

Cloning and expression of multiple protein kinase C cDNAs.

Knopf JL, Lee MH, Sultzman LA, Kriz RW, Loomis CR, Hewick RM, Bell RM
Cell. 1986; 46: 491-502

Three different protein kinase C related cDNA clones were isolated from a rat brain cDNA library and designated PKC-I, PKC-II, and PKC-III. These each encode very similar, but distinct, polypeptides that contain a region homologous with other protein kinases. COS cells transfected with either PKC-I or PKC-II specifically bind at least 5-fold more 3H-PDBu (phorbol ester) than control cells. An increase in Ca2+, phosphatidylserine, and diacylglycerol/phorbol-ester-dependent protein kinase activity is also observed in COS cells transfected with either PKC-I or PKC-II. The physiological implications of the discovery of three protein-kinase-C-related cDNAs are discussed.

Cloning and characterization of a glucocorticoid-induced diacylglycerol kinase.

Klauck TM, Xu X, Mousseau B, Jaken S
J Biol Chem. 1996; 271: 19781-8

Diacylglycerol kinase (DGK) plays a key role in cellular processes by regulating the intracellular concentration of the second messenger diacylglycerol. We screened a hamster DDT1 smooth muscle cell library and isolated a unique, glucocorticoid-inducible cDNA with substantial homology to known DGKs. DGK activity was increased in lysates of insect cells infected with recombinant baculovirus containing this cDNA. Antibodies raised against expressed sequences recognized a glucocorticoid-inducible 130-140-kDa protein on immunoblots of DDT1 cell lysates. Thus, this sequence appears to be a new member of the DGK family that we refer to as DGKeta. Homology to other DGKs was apparent in domains that are thought to be important for DGK function including the cysteine-rich motifs and potential catalytic domains. DGKeta shares substantial homology with DGKdelta including the N-terminal pleckstrin homology domain. The tissue distribution of DGKeta message (determined by ribonuclease protection assays) and protein (determined by immunoblots) was broader than reported for other DGKs, indicating that DGKeta may play a more general role in regulating cellular DG levels than other DGKs. Heterogeneity among DGK family members indicates that individual DGKs may have unique functions.

Molecular cloning of a cDNA encoding diacylglycerol kinase (DGK) in Arabidopsis thaliana.

Katagiri T, Mizoguchi T, Shinozaki K
Plant Mol Biol. 1996; 30: 647-53

Diacylglycerol kinase (DGK) synthesizes phosphatidic acid from diacylglycerol, an activator of protein kinase C (PKC), to resynthesize phosphatidylinositols. The structure of DGK has not been characterized in plants. We report the cloning of a cDNA, cATDGK1, encoding DGK from Arabidopsis thaliana. The cATDGK1 CDNA contains an open reading frame of 2184 bp, and encodes a putative protein of 728 amino acids with a predicted molecular mass of 79.4 kDa. The deduced ATDGK1 amino acid sequence exhibits significant similarity to that of rat, pig, and Drosophila DGKs. The ATDGK1 mRNA was detected in roots, shoots, and leaves. Southern blot analysis suggests that the ATDGK1 gene is a single-copy gene. The existence of DGK as well as phospholipase C suggests the existence of PKC in plants.

Diacylglycerol kinases: emerging downstream regulators in cell signaling systems.

Kanoh H, Yamada K, Sakane F
J Biochem (Tokyo). 2002; 131: 629-33

Diacylglycerol kinase (DGK) regulates signal transduction by modulating the balance between the two signaling lipids, diacylglycerol and phosphatidic acid. DGK and its homologs occur in a wide range of multicellular organisms and the mammalian DGK is known to consist of nine members with a considerable incidence of alternative splicing. Recent work has established that DGK serves as a key attenuator of diacylglycerol of signaling functions and that the mammalian isozymes are equipped with molecular machineries which enable them to act in specific intracellular sites and/or in signaling protein complexes.

[Diacylglycerol kinase--a novel enzyme having both zinc finger and E-F hand motifs]

Kanoh H, Sakane F, Yamada K
Tanpakushitsu Kakusan Koso. 1990; 35: 1558-63

Diacylglycerol kinase isozymes from brain and lymphoid tissues.

Kanoh H, Sakane F, Yamada K
Methods Enzymol. 1992; 209: 162-72

Diacylglycerol kinase and phosphatidic acid phosphatase--enzymes metabolizing lipid second messengers.

Kanoh H, Sakane F, Imai S, Wada I
Cell Signal. 1993; 5: 495-503

Molecular properties of enzymes involved in diacylglycerol and phosphatidate metabolism.

Kanoh H, Kai M, Wada I
J Lipid Mediat Cell Signal. 1996; 14: 245-50

Diacylglycerol and phosphatidate serve as key lipid intermediates of glycerolipid biosynthesis and also as the second messengers liberated in signal-stimulated cells. The molecular properties of enzymes involved in the metabolic processing of the two lipids are largely unknown. We have revealed by cDNA cloning the presence of a novel enzyme family of diacylglycerol kinase (DGK) having unique structural characteristics. Furthermore, we recently identified the plasma membrane-bound phosphatidate phosphatase (PAP) as a 35 kDa glycoprotein. Detailed studies on the two enzymes would contribute to understanding the molecular mechanisms underlying the control of both glycerolipid biosynthesis and signal transduction.

Molecular cloning of a diacylglycerol kinase isozyme predominantly expressed in human retina with a truncated and inactive enzyme expression in most other human cells.

Kai M, Sakane F, Imai S, Wada I, Kanoh H
J Biol Chem. 1994; 269: 18492-8

In order to clone novel diacylglycerol kinase (DGK) isozymes, we first obtained a DGK-related cDNA fragment by polymerase chain reaction using the human hepatoma cell line HepG2 mRNA and degenerated primers. The amplified fragment was subsequently used as a probe for screening the cDNA library from HepG2 cells. We obtained a cDNA clone coding for a novel DGK isozyme (designated DGK gamma) comprised of 791 amino acid residues. The amino acid sequence of DGK gamma was 52 and 62% identical to those of previously sequenced porcine 80-kDa and rat 90-kDa enzymes, respectively. DGK gamma, although initially cloned from the HepG2 cDNA libraries, was unexpectedly expressed in the human retina abundantly and to a much lesser extent in the brain. Other human tissues, including the liver and HepG2 cells, contained extremely low levels of DGK gamma mRNA. Furthermore, HepG2 cells and most of the human tissues except for the retina and brain expressed a truncated DGK gamma with an internal deletion of 25 amino acid residues (Ile451-Gly475). When transfected into COS-7 cells, the nontruncated cDNA gave phosphatidylserine-dependent DGK activity with no apparent specificity with regard to the acyl compositions of diacylglycerol. In contrast the truncated cDNA failed to give DGK activity in spite of the expression of its mRNA and enzyme protein in COS cells, thus demonstrating that the truncated DGK gamma is catalytically inactive. The sequence comparison of the three cloned DGKs revealed the presence of four highly conserved regions including the two sets each of EF-hand and zinc finger structures. Although the implication of the catalytically inactive form of DGK gamma remains unknown, this work further demonstrates the occurrence of multiple animal DGK isozymes with a conserved basic structure but with markedly different expression patterns depending on the cell types.

Domains, amino acid residues, and new isoforms of Caenorhabditis elegans diacylglycerol kinase 1 (DGK-1) important for terminating diacylglycerol signaling in vivo.

Jose AM, Koelle MR
J Biol Chem. 2005; 280: 2730-6

Diacylglycerol kinases (DGKs) inhibit diacylglycerol (DAG) signaling by phosphorylating DAG. DGK-1, the Caenorhabditis elegans ortholog of human neuronal DGK, inhibits neurotransmission to control behavior. DGK-1, like DGK, has three cysteine-rich domains (CRDs), a pleckstrin homology domain, and a kinase domain. To identify DGK domains and amino acid residues critical for terminating DAG signaling in vivo, we analyzed 20 dgk-1 mutants defective in DGK-1-controlled behaviors. We found by sequencing that the mutations included nine amino acid substitutions and seven premature stop codons that impair the physiological functions of DGK-1. All nine amino acid substitutions are in the second CRD, the third CRD, or the kinase domain. Thus, these domains are important for the termination of DAG signaling by DGK-1 in vivo. Seven of the substituted amino acid residues are present in all human DGKs and likely define key residues required for the function of all DGKs. An ATP-binding site mutation expected to inactivate the kinase domain retained very little physiological function, but we found two stop codon mutants predicted to truncate DGK-1 before its kinase domain that retained significantly more function. We detected novel splice forms of dgk-1 that can reconcile this apparent conflict, as they skip exons containing the stop codons to produce DGK-1 isoforms that contain the kinase domain. Two of these isoforms lack an intact pleckstrin homology domain and yet appear to have significant function. Additional novel isoform(s) account for all of the DGK-1 function necessary for one behavior, dopamine response.

Cloning and expression of human deoxyguanosine kinase cDNA.

Johansson M, Karlsson A
Proc Natl Acad Sci U S A. 1996; 93: 7258-62

A human cDNA sequence homologous to human deoxycytidine kinase (dCK; EC 2.7.1.74) was identified in the GenBank sequence data base. The longest open reading frame encoded a protein that was 48% identical to dCK at the amino acid level. The cDNA was expressed in Escherichia coli and shown to encode a protein with the same substrate specificity as described for the mitochondrial deoxyguanosine kinase (dGK; EC 2.7.1.113). The N terminus of the deduced amino acid sequence had properties characteristic for a mitochondrial translocation signal, and cleavage at a putative mitochondrial peptidase cleavage site would give a mature protein size of 28 kDa. Northern blot analysis determined the length of dGK mRNA to 1.3 kbp with no cross-hybridization to the 2.8-kbp dCK mRNA. dGK mRNA was detected in all tissues investigated with the highest expression levels in muscle, brain, liver, and lymphoid tissues. Alignment of the dGK and herpes simplex virus type 1 thymidine kinase amino acid sequences showed that five regions, including the substrate-binding pocket and the ATP-binding glycine loop, were also conserved in dGK. To our knowledge, this is the first report of a cloned mitochondrial nucleoside kinase and the first demonstration of a general sequence homology between two mammalian deoxyribonucleoside kinases. Our findings suggest that dCK and dGK are evolutionarily related, as well as related to the family of herpes virus thymidine kinases.

Selectivity of the diacylglycerol kinase inhibitor 3-[2-(4-[bis-(4-fluorophenyl)methylene]-1-piperidinyl)ethyl]-2, 3-dihydro-2-thioxo-4(1H)quinazolinone (R59949) among diacylglycerol kinase subtypes.

Jiang Y, Sakane F, Kanoh H, Walsh JP
Biochem Pharmacol. 2000; 59: 763-72

Diacylglycerol kinases (DGKs) attenuate diacylglycerol-induced protein kinase C activation during stimulated phosphatidylinositol turnover. This reaction also initiates phosphatidylinositol resynthesis. Two agents, 3-(2-(4-[bis-(4-fluorophenyl)methylene]-1-piperidinyl)ethyl)-2,3-dihydro -2-thioxo-4(1H)quinazolinone (R59949) and 6-(2-(4-[(4-fluorophenyl)phenylmethylene]-1-piperidinyl)ethyl)-7-m ethyl-5H-thiazolo(3,2-a)pyrimidin-5-one (R59022), inhibit diacylglycerol phosphorylation in several systems. To examine the mechanism of this effect, we developed a mixed micelle method suitable for in vitro study of DGK inhibition. Animal cells express multiple DGK isoforms. In a survey of DGK isotypes, these agents selectively inhibited Ca2+-activated DGKs. R59949 was the more selective of the two. To map the site of interaction with the enzyme, a series of DGKalpha deletion mutants were prepared and examined. Deletion of the Ca2+-binding EF hand motif, which is shared by Ca2+-activated DGKs, had no effect on inhibition. Consistent with this observation, inhibition kinetics were noncompetitive with Ca2+. A construct expressing only the catalytic domain was also inhibited by R59949. Studies of substrate kinetics demonstrated that MgATP potentiated R59949 inhibition, indicating synergy of inhibitor and MgATP binding. These results indicate that R59949 inhibits DGKalpha by binding to its catalytic domain.

A domain with homology to neuronal calcium sensors is required for calcium-dependent activation of diacylglycerol kinase alpha.

Jiang Y, Qian W, Hawes JW, Walsh JP
J Biol Chem. 2000; 275: 34092-9

Diacylglycerol kinases (DGKs) phosphorylate diacylglycerol produced during stimulus-induced phosphoinositide turnover and attenuate protein kinase C activation. Diacylglycerol kinase alpha is an 82-kDa DGK isoform that is activated in vitro by Ca(2+). The DGK alpha regulatory region includes tandem C1 protein kinase C homology domains and Ca(2+)-binding EF hand motifs. It also contains an N-terminal recoverin homology (RVH) domain that is related to the N termini of the recoverin family of neuronal calcium sensors. To probe the structural basis of Ca(2+) regulation, we expressed a series of DGK alpha deletions spanning its regulatory domain in COS-1 cells. Deletion of the RVH domain resulted in loss of Ca(2+)-dependent activation. Further deletion of the EF hands resulted in a constitutively active enzyme, suggesting that sequences in or near the EF hands are sufficient for autoinhibition. Binding of Ca(2+) to the EF hands protected sites within both the RVH domain and EF hands from trypsin cleavage and increased the phenyl-Sepharose binding of a recombinant DGK alpha fragment that included both the RVH domain and EF hands. These observations suggested that Ca(2+) elicits a concerted conformational change of these two domains. A cationic amphiphile, octadecyltrimethylammonium chloride, also activated DGK alpha. As with Ca(2+), this activation required the RVH domain. However, this agent did not protect the EF hands and RVH domain from trypsin cleavage. These findings indicate that the EF hands and RVH domain act as a functional unit during Ca(2+)-induced DGK alpha activation.

Cloning and characterization of diacylglycerol kinase iota splice variants in rat brain.

Ito T, Hozumi Y, Sakane F, Saino-Saito S, Kanoh H, Aoyagi M, Kondo H, Goto K
J Biol Chem. 2004; 279: 23317-26

Diacylglycerol kinase (DGK) catalyzes phosphorylation of a second messenger diacylglycerol (DG) to phosphatidic acid in cellular signal transduction. Previous studies have revealed that DGK consists of a family of isozymes including our rat clones. In this study we isolated from rat brain cDNA library the cDNA clones for a rat homologue of DGKiota (rDGKiota-1) that contains two zinc finger-like sequences, the highly conserved DGK catalytic domain, a bipartite nuclear localization signal, and four ankyrin repeats at the carboxyl terminus. In addition, we found novel splice variants, which contain either insertion 1 (71 bp) or insertion 2 (19 bp) or both in the carboxyl-terminal portion. Each of the insertions causes a frameshift, and the resultant premature stop codons produce two truncated forms (termed rDGKiota-2 and -iota-3), the former lacking the ankyrin repeats at the carboxyl terminus and the latter lacking a part of the catalytic domain and the ankyrin repeats. Truncation of the carboxyl-terminal portion clearly exerts effects on the detergent solubility and enzymatic activity of the splice variants, although all three variants showed similar cytoplasmic localization in cDNA-transfected cultured neurons despite the continued presence of the nuclear localization signal sequence. Immunoblot analysis using anti-rDGKiota antibody raised against the common amino-terminal portion clearly shows that these rDGKiota variants are indeed expressed in the brain. These results suggest that the carboxyl-terminal truncated forms of rDGKiota-2 and -iota-3 that exhibit reduced enzymatic activities might show a dominant negative effect against the intact rDGKiota-1, and that the modulation of signal transduction by the splice variants may play some roles in the physiologic and/or pathologic conditions of neurons.

Type I phosphatidylinositol-4-phosphate 5-kinases. Cloning of the third isoform and deletion/substitution analysis of members of this novel lipid kinase family.

Ishihara H, Shibasaki Y, Kizuki N, Wada T, Yazaki Y, Asano T, Oka Y
J Biol Chem. 1998; 273: 8741-8

Type I phosphatidylinositol 4-phosphate (PtdIns(4)P) 5-kinases (PIP5K) catalyze the synthesis of phosphatidylinositol 4, 5-bisphosphate, an essential lipid molecule in various cellular processes. Here, we report the cloning of the third member (PIP5Kgamma) and the characterization of members of the type I PIP5K family. Type I PIP5Kgamma has two alternative splicing forms, migrating at 87 and 90 kDa on SDS-polyacrylamide gel electrophoresis. The amino acid sequence of the central portion of this isoform shows approximately 80% identity with those of the alpha and beta isoforms. Northern blot analysis revealed that the gamma isoform is highly expressed in the brain, lung, and kidneys. Among three isoforms, the beta isoform has the greatest Vmax value for the PtdIns(4)P kinase activity and the gamma isoform is most markedly stimulated by phosphatidic acid. By analyzing deletion mutants of the three isoforms, the minimal kinase core sequence of these isoforms were determined as an approximately 380-amino acid region. In addition, carboxyl-terminal regions of the beta and gamma isoforms were found to confer the greatest Vmax value and the highest phosphatidic acid sensitivity, respectively. It was also discovered that lysine 138 in the putative ATP binding motif of the alpha isoform is essential for the PtdIns(4)P kinase activity. As was the case with the alpha isoform reported previously (Shibasaki, Y., Ishihara, H., Kizuki, N., Asano, T., Oka, Y., Yazaki, Y. (1997) J. Biol. Chem. 272, 7578-7581), overexpression of either the beta or the gamma isoform induced an increase in short actin fibers and a decrease in actin stress fibers in COS7 cells. Surprisingly, a kinase-deficient substitution mutant also induced an abnormal actin polymerization, suggesting a role of PIP5Ks via structural interactions with other molecules.

Identification and characterization of a novel human type II diacylglycerol kinase, DGK kappa.

Imai S, Kai M, Yasuda S, Kanoh H, Sakane F
J Biol Chem. 2005; 280: 39870-81

Diacylglycerol kinase (DGK) plays an important role in signal transduction through modulating the balance between two signaling lipids, diacylglycerol and phosphatidic acid. Here we identified a tenth member of the DGK family designated DGK kappa. The kappa-isozyme (1271 amino acids, calculated molecular mass, 142 kDa) contains a pleckstrin homology domain, two cysteine-rich zinc finger-like structures, and a separated catalytic region as have been found commonly for the type II isozymes previously cloned (DGKdelta and DGKeta). The new DGK isozyme has additionally 33 tandem repeats of Glu-Pro-Ala-Pro at the N terminus. Reverse transcriptase-PCR showed that the DGK kappa mRNA is most abundant in the testis, and to a lesser extent in the placenta. DGK kappa, when expressed in HEK293 cells, was persistently localized at the plasma membrane even in the absence of cell stimuli. Deletion analysis revealed that the short C-terminal sequence (amino acid residues 1199-1268) is necessary and sufficient for the plasma membrane localization. Interestingly, DGK kappa, but not other type II DGKs, was specifically tyrosine-phosphorylated at Tyr78 through the Src family kinase pathway in H2O2-treated cells. Moreover, H2O2 selectively inhibited DGK kappa activity in a Src family kinase-independent manner, suggesting that the isozyme changes the balance of signaling lipids in the plasma membrane in response to oxidative stress. The expression patterns, subcellular distribution, and regulatory mechanisms of DGK kappa are distinct from those of DGKdelta and DGKeta despite high structural similarity, suggesting unique functions of the individual type II isozymes.

Nuclear localization of diacylglycerol kinase zeta in neurons.

Hozumi Y, Ito T, Nakano T, Nakagawa T, Aoyagi M, Kondo H, Goto K
Eur J Neurosci. 2003; 18: 1448-57

Diacylglycerol kinase (DGK) is involved in intracellular signal transduction as a regulator of levels of diacylglycerol which leads to protein kinase C activation. Previous studies have revealed that DGK consists of a family of isozymes in mammalian species and that most if not all of them show abundant expression in the central nervous system, suggesting the importance of this enzyme in neuronal function. Among the isozymes, DGK zeta (previously also known as DGK-IV for the rat clone) has unique structural features, such as four ankyrin-like repeats and a nuclear localization signal (NLS), and shows intense mRNA expression in neurons of the olfactory bulb, hippocampus and cerebral and cerebellar cortices (Goto, K. & Kondo, H. (1996), Proc. Natl Acad. Sci. USA, 93, 11196-11201). However, previous studies have given conflicting results about whether or not DGK zeta localizes to the nucleus in these cells. In this study, we have used immunohistochemistry with specific antibodies in brain tissues and cDNA transfection into primary cultured neurons to address this question. We have shown that, while DGK zeta is primarily a nuclear protein in neurons, it can also be cytoplasmic in some conditions, and the subcellular location depends not only on the cell type but also on the developmental state or growth conditions of the cell. In addition, we have used deletion mutants to show that nuclear transport of DGK zeta depends on a cooperative interaction between the NLS and the C-terminal region including ankyrin repeats in a manner which suggests that the NLS is a cryptic site whose exposure is regulated by the C-terminal region. Together, these results support the hypothesis that the localization of DGK zeta may be regulated by differential expression of these various proteins which interact with its C-terminal region.

Cloning of a novel human diacylglycerol kinase (DGKtheta) containing three cysteine-rich domains, a proline-rich region, and a pleckstrin homology domain with an overlapping Ras-associating domain.

Houssa B, Schaap D, van der Wal J, Goto K, Kondo H, Yamakawa A, Shibata M, Takenawa T, van Blitterswijk WJ
J Biol Chem. 1997; 272: 10422-8

Diacylglycerol kinase (DGK) attenuates levels of second messenger diacylglycerol in cells and produces another (putative) messenger, phosphatidic acid. We have previously purified a 110-kDa DGK from rat brain (Kato, M., and Takenawa, T. (1990) J. Biol. Chem. 265, 794-800). Here we report the cDNA cloning from human brain and retina cDNA libraries. The cDNA encodes a novel DGK isotype, termed DGKtheta, of 941 amino acids with an apparent molecular mass of 110 kDa. DGKtheta contains a C-terminal putative catalytic domain, which is present in all eukaryotic DGKs. In contrast to other DGK isotypes, DGKtheta contains three cysteine-rich domains instead of two. The third cysteine-rich domain is most homologous to the second one in other DGK isotypes. This particular sequence homology extends C-terminally beyond the typical cysteine/histidine core structure and is DGK-specific. DGKtheta furthermore contains various domains for protein-protein interaction, such as a proline- and glycine-rich domain with a putative SH3 domain-binding site and a pleckstrin homology domain with an overlapping Ras-associating domain. DGKtheta is expressed in the brain and, to a lesser extent, in the small intestine, duodenum, and liver. In situ hybridization of DGKtheta mRNA in adult rat brain reveals high expression in the cerebellar cortex and hippocampus. DGKtheta activity in COS cell lysates is optimal toward diacylglycerols containing an unsaturated fatty acid at the sn-2 position.

Testisin, a new human serine proteinase expressed by premeiotic testicular germ cells and lost in testicular germ cell tumors.

Hooper JD, Nicol DL, Dickinson JL, Eyre HJ, Scarman AL, Normyle JF, Stuttgen MA, Douglas ML, Loveland KA, Sutherland GR, Antalis TM
Cancer Res. 1999; 59: 3199-205

We have cloned and characterized a cDNA encoding a new human serine proteinase, testisin, that is abundantly expressed only in the testis and is lost in testicular tumors. The testisin cDNA was identified by homology cloning using degenerate primers directed at conserved sequence motifs within the catalytic regions of serine proteinases. It is 1073 nucleotides long, including 942 nucleotides of open reading frame and a 113-nucleotide 3' untranslated sequence. Northern and dot blot analyses of RNA from a range of normal human tissues revealed a 1.4-kb mRNA species that was present only in testis, which was not detected in eight of eight testicular tumors. Testisin cDNA is predicted to encode a protein of 314 amino acids, which consists of a 19-amino acid (aa) signal peptide, a 22-aa proregion, and a 273-aa catalytic domain, including a unique 17-aa COOH-terminal hydrophobic extension that is predicted to function as a membrane anchor. The deduced amino acid sequence of testisin shows 44% identity to prostasin and contains features that are typical of serine proteinases with trypsin-like substrate specificity. Antipeptide antibodies directed against the testisin polypeptide detected an immunoreactive testisin protein of Mr 35,000-39,000 in cell lysates from COS-7 cells that were transiently transfected with testisin cDNA. Immunostaining of normal testicular tissue showed that testisin was expressed in the cytoplasm and on the plasma membrane of premeiotic germ cells. No staining was detected in eight of eight germ cell-derived testicular tumors. In addition, the testisin gene was localized by fluorescence in situ hybridization to the short arm of human chromosome 16 (16p13.3), a region that has been associated with allellic imbalance and loss of heterozygosity in sporadic testicular tumors. These findings demonstrate a new cell surface serine proteinase, loss of which may have a direct or indirect role in the progression of testicular tumors of germ cell origin.

Molecular cloning and expression of cDNA encoding human 3'-phosphoadenylylsulfate:galactosylceramide 3'-sulfotransferase.

Honke K, Tsuda M, Hirahara Y, Ishii A, Makita A, Wada Y
J Biol Chem. 1997; 272: 4864-8

We have isolated a cDNA clone encoding human 3'-phosphoadenylylsulfate:galactosylceramide 3'-sulfotransferase (EC 2.8.2.11). Degenerate oligonucleotides, based on amino acid sequence data for the purified enzyme, were used as primers to amplify fragments of the gene from human renal cancer cell cDNA by the polymerase chain reaction method. The amplified cDNA fragment was then used as probe to screen a human renal cancer cell cDNA library. The isolated cDNA clone contained an open reading frame encoding 423 amino acids including all of the peptides that were sequenced. The deduced amino acid sequence predicts a type II transmembrane topology and contains two potential N-glycosylation sites. There is no significant homology between this sequence and either the sulfotransferases cloned to date or other known proteins. Northern blot analysis demonstrated that a 1.9-kilobase mRNA was unique to renal cancer cells. When the cDNA was inserted into the expression vector pSVK3 and transfected into COS-1 cells, galactosylceramide sulfotransferase activity in the transfected cells increased from 8- to 16-fold over that of controls, and the enzyme product, sulfatide, was expressed on the transformed cells.

Identification of four novel human phosphoinositide 3-kinases defines a multi-isoform subfamily.

Ho LK, Liu D, Rozycka M, Brown RA, Fry MJ
Biochem Biophys Res Commun. 1997; 235: 130-7

Phosphoinositide (PI) 3-kinases have critical roles in diverse cellular signalling processes and in protein trafficking. This suggests that like other intracellular signalling molecules, e.g., phospholipase C and protein kinase C, there might be a large family of PI 3-kinase isoforms with the individual members having discrete signalling roles. Reverse transcription-polymerase chain reaction methods, using degenerate oligonucleotide primers against the lipid kinase consensus region, revealed eight sequences from human cDNA containing a high degree of identity to the family of PI 3-kinases. The sequences obtained included the previously described p110 alpha, p110 beta, and p110 gamma isoforms and HsVps34. Additionally, we have identified four novel sequences which are related to PI 3-kinases. Three of the novel sequences would appear to form a distinct sub-family of PI 3-kinases. We report the expression of these novel PI 3-kinases in human tissues and in cells derived from normal breast.

Interaction of gamma 1-syntrophin with diacylglycerol kinase-zeta. Regulation of nuclear localization by PDZ interactions.

Hogan A, Shepherd L, Chabot J, Quenneville S, Prescott SM, Topham MK, Gee SH
J Biol Chem. 2001; 276: 26526-33

Syntrophins are modular adapter proteins that link ion channels and signaling proteins to dystrophin and its homologues. A yeast two-hybrid screen of a human brain cDNA library using the PDZ domain of gamma 1- syntrophin, a recently identified brain-specific isoform, yielded overlapping clones encoding the C terminus of diacylglycerol kinase-zeta (DGK-zeta), an enzyme that converts diacylglycerol into phosphatidic acid. In biochemical assays, the C terminus of DGK-zeta, which contains a consensus PDZ-binding motif, was found to be necessary and sufficient for association with gamma 1-syntrophin. When coexpressed in HeLa cells, DGK-zeta and gamma 1-syntrophin formed a stable complex that partitioned between the cytoplasm and nucleus. DGK-zeta translocates from the cytosol to the nucleus, a process negatively regulated by protein kinase C phosphorylation. We found that DGK-zeta recruits gamma 1-syntrophin into the nucleus and that the PDZ-binding motif is required. Disrupting the interaction altered the intracellular localization of both proteins; DGK-zeta accumulated in the nucleus, whereas gamma 1-syntrophin remained in the cytoplasm. The level of endogenous syntrophins in the nucleus of HeLa cells also reflected the amount of nuclear DGK-zeta. In the brain, DGK-zeta and gamma 1-syntrophin were colocalized in cell bodies and dendrites of cerebellar Purkinjie neurons and other neuronal cell types, suggesting that their interaction is physiologically relevant. Moreover, coimmunoprecipitation and pull-down experiments from brain extracts and cells suggest that DGK-zeta, gamma 1-syntrophin, and dystrophin form a ternary complex. Collectively, our results suggest that gamma 1-syntrophin participates in regulating the subcellular localization of DGK-zeta to ensure correct termination of diacylglycerol signaling.

The identification of several stearoyl-arachidonyl selective diacylglycerol kinases in the particulate fraction of porcine testes.

Hodgkin M, Gardner S, Wakelam M
Biochem Soc Trans. 1993; 21: 490-490

Molecular cloning and characterization of murine and human N-acetylglucosamine kinase.

Hinderlich S, Berger M, Schwarzkopf M, Effertz K, Reutter W
Eur J Biochem. 2000; 267: 3301-8

N-Acetylglucosamine is produced by the endogenous degradation of glycoconjugates and by the degradation of dietary glycoconjugates by glycosidases. It enters the pathways of aminosugar metabolism by the action of N-acetylglucosamine kinase. In this study we report the isolation and characterization of a cDNA clone encoding the murine enzyme. An open reading frame of 1029 base pairs encodes 343 amino acids with a predicted molecular mass of 37.3 kDa. The deduced amino-acid sequence contains matches of the sequences of eight peptides derived from tryptic cleavage of rat N-acetylglucosamine kinase. The recombinant murine enzyme was functionally expressed in Escherichia coli BL21 cells, where it displays N-acetylglucosamine kinase activity as well as N-acetylmannosamine kinase activity. The complete cDNA sequence of human N-acetylglucosamine kinase was derived from the nucleotide sequences of several expressed sequence tags. An open reading frame of 1032 base pairs encodes 344 amino acids and a protein with a predicted molecular mass of 37.4 kDa. Similarities between human and murine N-acetylglucosamine kinase were 86.6% on the nucleotide level and 91.6% on the amino-acid level. Amino-acid sequences of murine and human N-acetylglucosamine kinase show sequence similarities to other sugar kinases, and all five sequence motifs necessary for the binding of ATP by sugar kinases are present. Tissue distribution of murine N-acetylglucosamine kinase revealed an ubiquitous occurrence of the enzyme and a very high expression in testis. The size of the murine mRNA was 1.35 kb in all tissues investigated, with the exception of testis, where it was 1.45 kb mRNA of the murine enzyme was continuously expressed during mouse development. mRNA of the human enzyme was expressed in all investigated human tissues, as well as in cancer cell lines. In both the tissues and the cancer cell lines, the human mRNA was 1.35 kb in size.

Assignment of the gene for diacylglycerol kinase (DAGK) to human chromosome 12.

Hart TC, Champagne C, Zhou J, Van Dyke TE
Mamm Genome. 1994; 5: 123-4

A Drosophila gene encoding a protein with similarity to diacylglycerol kinase is expressed in specific neurons.

Harden N, Yap SF, Chiam MA, Lim L
Biochem J. 1993; 289: 439-44

A Drosophila gene encoding a protein with similarity to diacylglycerol kinase (DGK) was isolated by screening a genomic DNA library with a human DGK cDNA under low-stringency hybridization conditions. This putative Drosophila DGK gene (dDGK) maps to 43E on the right arm of chromosome 2. A dDGK cDNA with an open reading frame encoding a 517 amino acid protein was obtained in a screen of a 3-12-h embryonic cDNA library. In a 236-amino-acid overlap at their C-termini there is an identity of 52.5% between the dDGK protein and human DGK. The N-terminus of the Drosophila protein is not similar to human DGK, and contains clusters of polar amino acids. dDGK is transcribed in the embryonic, pupal and adult stages, with little expression during the larval stages. Transcripts of 1.7-2.2 kb, 3.5 kb, 3.7 kb and 6.6 kb are seen, although most of the smaller transcripts may be from genes with similarity to dDGK. In stage-16 and stage-17 embryos, dDGK transcripts are limited to the central nervous system and head. There is a particularly high level of expression in the cell bodies of the larval photoreceptor organ, and in the cell bodies of the ventral unpaired median neurons. The dDGK protein may be involved in regulating signal transduction in these specific neurons.

Molecular characterization and expression of heparan-sulfate 6-sulfotransferase. Complete cDNA cloning in human and partial cloning in Chinese hamster ovary cells.

Habuchi H, Kobayashi M, Kimata K
J Biol Chem. 1998; 273: 9208-13

Heparan-sulfate 6-sulfotransferase (HS6ST) catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 6 of the N-sulfoglucosamine residue of heparan sulfate. The enzyme was purified to apparent homogeneity from the serum-free culture medium of Chinese hamster ovary (CHO) cells (Habuchi, H., Habuchi, O., and Kimata, K. (1995) J. Biol Chem. 270, 4172-4179). From the amino acid sequence data of the purified enzyme, degenerate oligonucleotides were designed and used as primers for the reverse transcriptase-polymerase chain reaction using poly(A)+ RNA from CHO cells as a template. The amplified cDNA fragment was then used as a probe to screen a cDNA library of CHO cells. The cDNA clone thus obtained encoded a partial peptide sequence composed of 236 amino acid residues that included the sequences of six peptides obtained after endoproteinase digestion of the purified enzyme. This cDNA clone was applied to the screening of a human fetal brain cDNA library by cross-hybridization. The isolated cDNA clones contained a whole open reading frame that predicts a type II transmembrane protein composed of 401 amino acid residues. No significant amino acid sequence identity to any other proteins, including heparan-sulfate 2-sulfotransferases, was observed. When the cDNA for the entire coding sequence of the protein was inserted into a eukaryotic expression vector and transfected into COS-7 cells, the HS6ST activity increased 7-fold over the control. The FLAG fusion protein purified by anti-FLAG affinity chromatography showed the HS6ST activity alone. Northern blot analysis revealed the occurrence of a single transcript of 3.9 kilobases in both human fetal brain and CHO cells. The results, together with the ones from our recent cDNA analysis of heparan-sulfate 2-sulfotransferase (Kobayashi, M., Habuchi, H., Yoneda, M., Habuchi, O., and Kimata, K. (1997) J. Biol. Chem. 272, 13980-13985), suggest that at least two different gene products are responsible for 6- and 2-O-sulfations of heparan sulfate.

Cloning and sequence analysis of human and bovine corneal antigen (CO-Ag) cDNA: identification of host-parasite protein calgranulin C.

Gottsch JD, Stark WJ, Liu SH
Trans Am Ophthalmol Soc. 1997; 95: 111-25

PURPOSE: The primary structure of a cornea-associated antigen (CO-Ag) has been identified and has been implicated in the pathogenesis of Mooren's ulcer. The study designs were to isolate full-length clones encoding CO-Ag from a bovine and a human corneal cDNA library so that complete sequence analyses might further define the possible role of this protein in Mooren's ulcer. METHODS: DNA fragments of bovine and human CO-Ag were generated using unique oligonucleotide primers and reverse transcription polymerase chain reaction. These fragments were used as probes to obtain cDNA clones from a bovine and a human corneal cDNA libraries. The clones with the longest cDNA inserts were selected for sequence analyses. Human cDNA fragment was digested with Stu I and Hind III and cloned into a expression vector, pPROEXHT, at the same restriction enzyme sites. The plasmid was transformed into E. coli cells. Correct cloning and the full-length sequence of human CO-Ag were determined by sequencing the insert cDNA. RESULTS: The bovine cDNA insert sequence was 273 nucleotides in length for the entire mRNA coding region, 212 nucleotides in the 5' untranslated region, 83 nucleotides in the 3' untranslated region and a poly(A) tail. The DNA base sequence of this clone also contained a standard initiation codon, termination codon, and the polyadenylation signal. This cDNA predicts a protein which contains 91 amino acids with a molecular weight of 10,584 daltons. Plasmid expression vector, pPROEXHT-CO-Ag, was constructed that direct the synthesis of human CO-Ag in E. coli as fusion protein. Human CO-Ag fusion protein was purified to 90% pure with a yield of 17.2 mg per liter of the bacterial cell lysate. The nucleotide sequence of the CO-Ag cDNA insert was completely identical to human neutrophil calgranulin C. The deduced amino acid sequence was completely identical to a Ca(2+)-binding protein isolated on the surface of filarial nematodes. CONCLUSIONS: The isolation and analysis of cDNA clones containing the complete coding sequence of bovine and human CO-Ag proteins is reported. The proteins identified by deduced amino acid sequences demonstrate 100% sequence homology with human and bovine calgranulin C. Immune recognition of calgranulin C to a filarial nematode may lead to a hyperactive autoimmune response to CO-Ag in the cornea leading to a Mooren's ulcer.

Gene cloning, sequence, expression and in situ localization of 80 kDa diacylglycerol kinase specific to oligodendrocyte of rat brain.

Goto K, Watanabe M, Kondo H, Yuasa H, Sakane F, Kanoh H
Brain Res Mol Brain Res. 1992; 16: 75-87

A 3.1 kbp cDNA clone encoding diacylglycerol (DG) kinase of 80 kDa (80K-DG kinase) was isolated from a rat brain cDNA library. The deduced amino acid sequence was 82% homologous to previously identified porcine 80K-DG kinase and contained zinc finger-like sequences, E-F hand motifs and ATP-binding sites similar to the porcine counterpart. By in situ hybridization histochemistry of rat brain at postnatal week 3, the expression signals for 80K-DG kinase mRNA appeared predominantly on somata of discrete cells in the white matter, and the expression pattern was similar to that of the myelin-specific proteins. In immunohistochemistry using the antibody against bacterially expressed DG kinase-fusion protein, numerous fibrous or dot-like structures exhibiting the immunoreactivity were concentrated in the white matter and they were arranged to radiate in the cerebral cortex and the cerebellar granular layer in a pattern almost identical to that of oligodendrocytes. No neuronal cells exhibited the immunoreactivity. The present finding thus strongly suggests that 80K-DG kinase is expressed specifically in the oligodendrocytes, but not neurons, and may be involved in the myelin formation and metabolism. In addition, the intense hybridization signals and the immunoreactivity for this protein were detected in the entire medulla of the thymus and the periarterial lymphatic area of the splenic white pulp both of which represent T-cell-dependent areas.

Heterogeneity of diacylglycerol kinase in terms of molecular structure, biochemical characteristics and gene expression localization in the brain.

Goto K, Kondo H
J Lipid Mediat Cell Signal. 1996; 14: 251-7

Three different cDNA clones for diacylglycerol (DG) kinase were isolated from a rat brain cDNA library and designated DGK-I, DGK-II and DGK-III. These three encode distinct polypeptides with 58% identity to each other and contain EF-hand motifs, cysteine-rich zinc finger-like sequences and putative ATP-binding sites. A high kinase activity is shown in COS cells transfected with either one of the three cDNAs without substrate specificity among DG species, and the kinase activity is Ca-dependent. The activity for DGK-I is recovered dominantly in the soluble fraction of the cell, that for DGK-II in the particulate fraction; and that for DGK-III equally in both of the fractions. The difference in their expression localization is most noticeable: DGK-I is expressed in oligodendrocytes of the brain as well as T-lymphocytes in the thymus and spleen; DGK-II is expressed in neurons of the caudate-putamen, accumbens nucleus and olfactory tubercle; and DGK-III in the cerebellar Purkinje cells and granule cells. The functional significance of the discovery of three DG kinase isozymes is briefly discussed.

A 104-kDa diacylglycerol kinase containing ankyrin-like repeats localizes in the cell nucleus.

Goto K, Kondo H
Proc Natl Acad Sci U S A. 1996; 93: 11196-201

The cDNA corresponding to a fourth species of diacylglycerol (DG) kinase (EC 2.7.1.107) was isolated from cDNA libraries of rat retina and brain. This cDNA encoded a 929-aa, 104-kDa polypeptide termed DGK-IV. DGK-IV was different from previously identified mammalian DG kinase species, DGK-I, DGK-II, and DGK-III, in that it contained no EF-hand motifs but did contain four ankyrin-like repeats at the carboxyl terminus. These structural features of DGK-IV closely resemble the recently cloned, eye-specific DG kinase of Drosophila that is encoded by the retinal degeneration A (rdgA) gene. However, DGK-IV was expressed primarily in the thymus and brain with relatively low expression in the eye and intestine. Furthermore, the primary structure of the DGK-IV included a nuclear targeting motif, and immunocytochemical analysis revealed DGK-IV to localize in the nucleus of COS-7 cells transfected with the epitope-tagged cDNA, suggesting an involvement of DGK-IV in intranuclear processes.

Molecular cloning and expression of a 90-kDa diacylglycerol kinase that predominantly localizes in neurons.

Goto K, Kondo H
Proc Natl Acad Sci U S A. 1993; 90: 7598-602

A diacylglycerol kinase cDNA was isolated from a rat brain cDNA library. This cDNA encoded an 801-amino acid protein of 90,287 Da. This 90-kDa diacylglycerol kinase showed 58% identity in deduced amino acid sequence with a previously isolated rat 80-kDa diacylglycerol kinase. EF-hand motifs, cysteine-rich zinc-finger-like sequences, and putative ATP-binding sites were all conserved between the two kinase species. However, mRNA encoding the 90-kDa kinase was confined to restricted neuronal populations such as the caudate-putamen, the accumbens nucleus, and the olfactory tubercle. Further, the 90-kDa kinase was found to exhibit high phosphorylation activity for long-chain diacylglycerols and was mainly associated with the membrane fraction when the cDNA was transfected into COS-7 cells.

Diacylglycerol kinase in the central nervous system--molecular heterogeneity and gene expression.

Goto K, Kondo H
Chem Phys Lipids. 1999; 98: 109-17

Diacylglycerol (DAG) is one of the important second messengers, which serves as an activator of protein kinase C (PKC). DAG kinase (DGK) phosphorylates DAG to generate phosphatidic acid, thus DGK is considered to be a regulator of PKC activity through attenuation of DAG. Recent studies have revealed molecular structures of several DGK isozymes from mammalian species, and showed that most of the isozymes are expressed in the brain in various amounts. We have cloned four DGK isozyme cDNAs from rat brain library (DGK alpha, -beta, -gamma, and -zeta) (previously also designated DGK-I, -II, -III, and -IV, respectively) and examined their mRNA expressions in rat brain by in situ hybridization histochemistry. Interestingly, it is revealed that the mRNA for each isozyme is expressed in a distinct pattern in the brain; DGK alpha is expressed in oligodendrocytes, glial cells that form myelin; DGK beta in neurons of the caudate-putamen; DGK gamma predominantly in the cerebellar Purkinje cells; and DGK zeta in the cerebellar and cerebral cortices. Molecular diversity and distinct expression patterns of DGK isozymes suggest a physiological importance for the enzyme in brain function. Furthermore, functional implications of these DGK isozymes are briefly discussed.

Cloning and expression of a cytoskeleton-associated diacylglycerol kinase that is dominantly expressed in cerebellum.

Goto K, Funayama M, Kondo H
Proc Natl Acad Sci U S A. 1994; 91: 13042-6

A third species of diacylglycerol kinase (EC 2.7.1.107) cDNA was cloned from a rat brain cDNA library. The isolated cDNA encoded a 788-amino acid, 88-kDa polypeptide. This isozyme shared 58% identity with the previously isolated rat 80-kDa and 90-kDa diacylglycerol kinases. EF hand motifs, cysteine-rich zinc finger-like sequences, and putative ATP-binding site were all conserved among these isozymes. The 88-kDa diacylglycerol kinase was expressed specifically in brain and localized predominantly in cerebellar Purkinje cells. This isozyme was associated equally with particulate and supernatant fractions in cDNA-transfected COS-7 cells and dominantly with the particulate fraction in the brain. After Triton X-100 extraction, this isozyme remained in the detergent-insoluble cytoskeletal fraction of the brain and transfected COS-7 cells.

AtDGK2, a novel diacylglycerol kinase from Arabidopsis thaliana, phosphorylates 1-stearoyl-2-arachidonoyl-sn-glycerol and 1,2-dioleoyl-sn-glycerol and exhibits cold-inducible gene expression.

Gomez-Merino FC, Brearley CA, Ornatowska M, Abdel-Haliem ME, Zanor MI, Mueller-Roeber B
J Biol Chem. 2004; 279: 8230-41

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DAG) to generate phosphatidic acid (PA). Both DAG and PA are implicated in signal transduction pathways. DGKs have been widely studied in animals, but their analysis in plants is fragmentary. Here, we report the cloning and biochemical characterization of AtDGK2, encoding DGK from Arabidopsis thaliana. AtDGK2 has a predicted molecular mass of 79.4 kDa and, like AtDGK1 previously reported, harbors two copies of a phorbol ester/DAG-binding domain in its N-terminal region. AtDGK2 belongs to a family of seven DGK genes in A. thaliana. AtDGK3 to AtDGK7 encode approximately 55-kDa DGKs that lack a typical phorbol ester/DAG-binding domain. Phylogenetically, plant DGKs fall into three clusters. Members of all three clusters are widely expressed in vascular plants. Recombinant AtDGK2 was expressed in Escherichia coli and biochemically characterized. The enzyme phosphorylated 1,2-dioleoyl-sn-glycerol to yield PA, exhibiting Michaelis-Menten type kinetics. Estimated K(m) and V(max) values were 125 microm for DAG and 0.25 pmol of PA min(-1) microg(-1), respectively. The enzyme was maximally active at pH 7.2. Its activity was Mg(2+)-dependent and affected by the presence of detergents, salts, and the DGK inhibitor R59022, but not by Ca(2+). AtDGK2 exhibited substrate preference for unsaturated DAG analogues (i.e. 1-stearoyl-2-arachidonoyl-sn-glycerol and 1,2-dioleoyl-sn-glycerol). The AtDGK2 gene is expressed in various tissues of the Arabidopsis plant, including leaves, roots, and flowers, as shown by Northern blot analysis and promoter-reporter gene fusions. We found that AtDGK2 is induced by exposure to low temperature (4 degrees C), pointing to a role in cold signal transduction.

Arabidopsis AtDGK7, the smallest member of plant diacylglycerol kinases (DGKs), displays unique biochemical features and saturates at low substrate concentration: the DGK inhibitor R59022 differentially affects AtDGK2 and AtDGK7 activity in vitro and alters plant growth and development.

Gomez-Merino FC, Arana-Ceballos FA, Trejo-Tellez LI, Skirycz A, Brearley CA, Dormann P, Mueller-Roeber B
J Biol Chem. 2005; 280: 34888-99

Diacylglycerol kinase (DGK) regulates the level of the second messenger diacylglycerol and produces phosphatidic acid (PA), another signaling molecule. The Arabidopsis thaliana genome encodes seven putative diacylglycerol kinase isozymes (named AtDGK1 to -7), structurally falling into three major clusters. So far, enzymatic activity has not been reported for any plant Cluster II DGK. Here, we demonstrate that a representative of this cluster, AtDGK7, is biochemically active when expressed as a recombinant protein in Escherichia coli. AtDGK7, encoded by gene locus At4g30340, contains 374 amino acids with an apparent molecular mass of 41.2 kDa. AtDGK7 harbors an N-terminal catalytic domain, but in contrast to various characterized DGKs (including AtDGK2), it lacks a cysteine-rich domain at its N terminus, and, importantly, its C-terminal DGK accessory domain is incomplete. Recombinant AtDGK7 expressed in E. coli exhibits Michaelis-Menten type kinetics with 1,2-dioleoyl-sn-glycerol as substrate. AtDGK7 activity was affected by pH, detergents, and the DGK inhibitor R59022. We demonstrate that both AtDGK2 and AtDGK7 phosphorylate diacylglycerol molecular species that are typically found in plants, indicating that both enzymes convert physiologically relevant substrates. AtDGK7 is expressed throughout the Arabidopsis plant, but expression is strongest in flowers and young seedlings. Expression of AtDGK2 is transiently induced by wounding. R59022 at approximately 80 mum inhibits root elongation and lateral root formation and reduces plant growth, indicating that DGKs play an important role in plant development.

Isolation and characterization of the human diacylglycerol kinase gene.

Fujikawa K, Imai S, Sakane F, Kanoh H
Biochem J. 1993; 294: 443-9

The 80 kDa diacylglycerol kinase (DGK) is abundantly expressed in oligodendrocytes and lymphocytes but not to a detectable extent in other cells such as neurons and hepatocytes. As an initial attempt to delineate the mechanism of the transcriptional control of the DGK gene, we have cloned from a human genomic library a 22 kb genomic fragment. The genomic clone consists of the 5'-flanking region and 17 exons coding for approx. 53% of the total exons of human DGK, including those encoding EF-hand and zinc-finger regions. The translation initiation site is located in the second exon. S1 nuclease mapping and primer extension analysis of the human DGK mRNA identified a major transcription initiation site (position +1) at 264 bp upstream from the initiator ATG. In the 5'-flanking sequence we detected a single GC box at -35 but no canonical TATA and CAAT sequences. However, the sequence starting from the cap site (AGTTCCTGCCA) is very similar to the initiator element that specifies the transcription initiation site of some housekeeping genes. In addition, the 5'-upstream region contains several putative cis-elements. Jurkat and HepG2 cells were transfected with various 5'-deletion mutants of the upstream region fused to the structural gene of chloramphenicol acetyltransferase (CAT). The CAT assay revealed that among constructs containing up to 3.4 kb of the 5'-flanking region, a fragment of 263 bp from the transcription initiation site contains a basic promoter that is active in both types of cells. Moreover, the region between -263 and -850 contains a negative element that is active in HepG2 but not in Jurkat cells. This negative element may, at least in part, be responsible for the cell type-specific expression of the DGK gene.

Molecular cloning of the high affinity calcium-binding protein (calreticulin) of skeletal muscle sarcoplasmic reticulum.

Fliegel L, Burns K, MacLennan DH, Reithmeier RA, Michalak M
J Biol Chem. 1989; 264: 21522-8

A cDNA clone encoding the high affinity Ca2+-binding protein (HACBP) of rabbit skeletal muscle sarcoplasmic reticulum was isolated and sequenced. The cDNA encoded a protein of 418 amino acids, but a comparison of the deduced amino acid sequence with the NH2-terminal amino acid sequence of the purified protein indicates that a 17-residue NH2-terminal signal sequence was removed during synthesis. This was confirmed by studies of in vitro translation of mRNA encoding the protein. Structural predictions did not reveal any potential transmembrane segments in the protein. The COOH-terminal sequence of the high affinity Ca2+-binding protein, Lys-Asp-Glu-Leu, is the same as that proposed to be an endoplasmic reticulum retention signal (Munro, S., and Pelham, H. R. B. (1987) Cell 48, 899-907). All of these characteristics suggest that the protein is localized in the lumen of the sarcoplasmic reticulum. The mature protein of Mr 46,567 contains 109 acidic and 52 basic amino acids. Structural predictions suggest that the first half of the molecule forms a globular domain of 8 anti-parallel beta-strands with a helix-turn-helix motif at the extreme NH2 terminus. The next one-third of the sequence is proline-rich. This segment can be subdivided into a charged region which contains a 17-amino acid repeat, followed by a proline, serine, and threonine-rich segment extending from Pro-246 to Thr-316. Thirty-seven acidic residues are clustered within 56 amino acids at the COOH terminus of the protein. Although the protein binds 1 mol of Ca2+/mol with high affinity, no "EF-hand" consensus sequence was observed in the protein. The acidic COOH terminus, however, could account for the low affinity, high capacity Ca2+ binding observed in the protein. In agreement with other involved laboratories, we have chosen the name calreticulin for the protein.

Expression and characterization of a human cDNA that complements the temperature-sensitive defect in dolichol kinase activity in the yeast sec59-1 mutant: the enzymatic phosphorylation of dolichol and diacylglycerol are catalyzed by separate CTP-mediated kinase activities in Saccharomyces cerevisiae.

Fernandez F, Shridas P, Jiang S, Aebi M, Waechter CJ
Glycobiology. 2002; 12: 555-62

Dolichol kinase (DK) catalyzes the CTP-mediated phosphorylation of dolichol in eukaryotic cells, the terminal step in dolichyl monophosphate (Dol-P) biosynthesis de novo. In S. cerevisiae, the SEC59 gene encodes a protein essential for the expression of DK, an enzyme activity that is required for cell viability and normal rates of lipid intermediate synthesis and protein N-glycosylation. This study identifies a cDNA clone from human brain that encodes the mammalian homolog of DK (hDK1p). hDK1 is capable of complementing the growth defect, elevating DK activity, and consequently increasing Dol-P levels in vivo and restoring normal N-glycosylation of carboxypeptidase Y at the restrictive temperature in the temperature-sensitive mutant sec59-1. The CTP-mediated phosphorylation of diacylglycerol (DAG) is unaffected by either the temperature-sensitive mutation in the sec59-1 strain, overexpression of the SEC59 gene, or the mammalian homolog hDK1 under conditions that produced a loss or elevation in the level of DK activity. Additionally, overexpression of hDK1p in Sf-9 cells resulted in a 15-fold increase in DK activity but not DAG kinase activity in crude microsomal fractions. The cloned cDNA contains an open reading frame that would encode a protein with 538 amino acids and a molecular weight of 59,268 kDa. Consistent with this prediction, new polypeptides were detected with an apparent molecular weight of 59-60 kDa when His(6)-tagged constructs of hDK1 or the SEC59 gene were expressed in Sf-9 cells or the temperature-sensitive sec59-1 mutant cells, respectively. These results identify the first cDNA clone encoding a protein required for the expression of DK activity, possibly the catalytic subunit, in a mammalian cell, and establish that the phosphorylation of dolichol and DAG are catalyzed by separate kinase activities in yeast.

Lipid modulation of the activity of diacylglycerol kinase alpha- and zeta-isoforms: activation by phosphatidylethanolamine and cholesterol.

Fanani ML, Topham MK, Walsh JP, Epand RM
Biochemistry. 2004; 43: 14767-77

Diacylglycerol kinase (DGK) isoforms alpha and zeta were extracted from transfected cells that overexpressed these enzymes. We determined the lipid dependence of the binding of these isoforms to liposomes. The modulation by lipid of the rate of phosphorylation of diacylglycerol by these enzymes was also measured. Incorporation of phosphatidylethanolamine into the liposomes resulted in an increased partitioning of both isoforms of DGK to the membrane as well as an increased catalytic rate. We demonstrate that the increased catalytic rate is a consequence of both increased portioning of the enzyme to the membrane and increased catalytic activity of the membrane-bound form. DGKalpha, a calcium-dependent isoform, can be activated in a calcium-independent fashion in the presence of phosphatidylethanolamine. Similar effects are observed with cholesterol. In contrast, sphingomyelin inhibits the activity of both isoforms of DGK. Our results demonstrate that the translocation to membranes and activity of DGKalpha and DGKzeta are modulated by the composition and properties of the membrane. The enzymes are activated by the presence of lipids that promote the formation of inverted phases. However, the promotion of negative curvature is not the sole factor contributing to the lipid effects on enzyme binding and activity. A truncated form of DGKalphalacking both the E-F hand and the recoverin homology domain is constitutively active and is not further activated by any of the lipids tested or by calcium. However, a truncated form lacking only the recoverin homology domain is partially activated by either calcium or certain lipids.

Nuclear expression of diacylglycerol kinases: possible involvement in DNA replication.

Evangelisti C, Bortul R, Tabellini G, Papa V, Cocco L, Martelli AM
Eur J Histochem. 2006; 50: 9-13

The existence of intranuclear lipid-dependent signal transduction systems has been demonstrated by several independent groups. Remarkably, intranuclear lipid-dependent signal transduction pathways are regulated independently from their membrane/cytosolic counterparts. A sizable body of evidence suggests that nuclear lipid signaling controls critical biological functions such as cell proliferation, differentiation, and apoptosis. Diacylglycerol (DG) is a fundamental lipid second messenger which is produced in the nucleus. Since the levels of nuclear DG fluctuate during the cell cycle progression, it has been suggested that this lipid second messenger has important regulatory roles. Most likely, nuclear DG serves as a chemoattractant for some isoforms of protein kinase C that migrate to the nucleus in response to a variety of agonists. The nucleus also contains diacylglycerol kinases (DGKs), i.e. the enzymes that, by converting DG into phosphatidic acid (PA), terminate DG-dependent events. This review aims at highlighting the different isozymes of DGKs present within the nucleus as well as at discussing their potential functions with particular emphasis placed on DNA replication.

The alpha isoform of diacylglycerol kinase exhibits arachidonoyl specificity with alkylacylglycerol.

Epand RM, Kam A, Bridgelal N, Saiga A, Topham MK
Biochemistry. 2004; 43: 14778-83

We compared the diacylglycerol kinase (DGK) catalyzed phosphorylation of 1-O-hexanoyl-2-oleoylglycerol (HOG) with 1-O-hexanoyl-2-arachidonoylglycerol (HAG). We assayed the activity of DGKalpha and DGKzeta using a liposomal-based assay system. Liposomal assays show that the DGKalpha and, to a lesser extent, DGKzeta preferentially act on substrates containing an arachidonoyl group when this group is incorporated into alkylacylglycerols. The activity of DGKalpha was 82 times greater with HAG compared to HOG. DGKzeta is 10 times more active in catalyzing the phosphorylation of HAG compared to HOG. Although diacylglycerols were better substrates for both DGKalpha and DGKzeta than the alkylacylglycerols, no specificity was exhibited for arachidonoyl-containing diacylglycerols. However, this specificity for HAG over HOG is modulated by the phospholipid composition of the liposome. Addition of cholesterol and/or phosphatidylethanolamine partially reduces the substrate selectivity. We also analyzed the kinetic constants for the phosphorylation of both diacylglycerol and 1-alkyl-2-acylglycerol catalyzed by the alpha, epsilon, or zeta isoforms using a soluble Triton mixed micelle system. We found that all three isoforms of DGK can phosphorylate 1-alkyl-2-acylglycerols but generally at a lower rate than for the corresponding diacylglycerol. The specificity of DGKepsilon for diacylglycerols containing an arachidonoyl group was retained when the ester group in the C-1 position is replaced with an ether linkage. In contrast, DGKalpha and, to a lesser extent, DGKzeta had greater specificity for arachidonoyl-containing 1-alkyl-2-acylglycerols than for arachidonoyl-containing diacylglycerols. This demonstrates that the acyl chain specificity is affected by the structure of the lipid headgroup.

Fatty acids inhibit growth-factor-induced diacylglycerol kinase alpha activation in vascular smooth-muscle cells.

Du X, Jiang Y, Qian W, Lu X, Walsh JP
Biochem J. 2001; 357: 275-82

We have previously shown that unsaturated fatty acids amplify platelet-derived-growth-factor (PDGF)-induced protein kinase C (PKC) activation in vascular smooth-muscle cells (VSMCs). Diacylglycerol-induced PKC activation is normally terminated by diacylglycerol kinases (DGKs). We thus hypothesized that fatty acids act by inhibiting a DGK. Fractionation of VSMC extracts demonstrated that the DGK alpha isoform was the major DGK activity present. PDGF markedly increased the DGK activity of cultured cells. An inhibitor selective for the DGK alpha isoform, R59949 [3-[2-[4-(bis-(4-fluorophenyl)methylene]piperidin-1-yl)ethyl]-2,3-dihydro- 2-thioxo-4(1H)-quinazolinone], abolished the growth-factor-induced increase in DGK activity, but had little effect on basal activity. PDGF thus selectively activates DGKalpha. Epidermal growth factor and alpha-thrombin stimulated total DGK activity similarly to PDGF. Activation by epidermal growth factor was sensitive to R59949, again suggesting involvement of DGKalpha. However, the alpha-thrombin-induced activity was unaffected by this agent. Unsaturated fatty acids inhibited growth-factor-induced DGKalpha activation, but had no effect on basal activity. Fatty acids also amplified the PDGF-induced increase in cell diacylglycerol content. These results indicate that inhibition of DGKalpha contributes to fatty-acid-induced amplification of PKC activation. Increased levels of fatty acids in diabetes may thus contribute to chronic PKC activation associated with this disorder.

The cloning and sequence of the C isoform of PtdIns4P 5-kinase.

Divecha N, Truong O, Hsuan JJ, Hinchliffe KA, Irvine RF
Biochem J. 1995; 309: 715-9

In this study we describe the purification and sequencing of the C isoform of platelet PtdIns4P 5-kinase. Subsequently a cDNA was isolated from a human circulating-leucocyte library, which when sequenced was shown to contain all of the peptides identified in the purified protein. In addition, expression of this cDNA in bacteria led to the production of a protein which was recognized by specific monoclonal antibodies raised to the bovine brain enzyme [Brooksbank, Hutchings, Butcher, Irvine and Divecha (1993) Biochem. J. 291, 77-82] and also led to the appearance of PtdIns4P 5-kinase activity in the bacterial lysates. Interestingly, the cDNA showed no similarity to any of the previously cloned inositide kinases. A search of the DNA databases showed that two proteins from Saccharomyces cerevisiae shared close similarity to this enzyme, one of which, the mss4 gene product, has been implicated in the yeast inositol lipid pathway. These data suggest that the PtdIns4P 5-kinases are a new family of inositide kinases unrelated to the previously cloned phosphoinositide 3/4-kinases.

The cloning and characterization of a novel human diacylglycerol kinase, DGKiota.

Ding L, Traer E, McIntyre TM, Zimmerman GA, Prescott SM
J Biol Chem. 1998; 273: 32746-52

Diacylglycerol (DAG) plays a central role in both the synthesis of complex lipids and in intracellular signaling; diacylglycerol kinase (DGK) catalyzes the phosphorylation of DAG, which yields phosphatidic acid. A family of DGKs has been identified in multicellular organisms over the past few years, but the physiological function(s) of this diversity is not clear. One clue has come from the Drosophila DGK2, rdgA, since mutations in this gene cause retinal degeneration. We isolated a novel DGK, which we designated DGKiota, from human retina and brain libraries. DGKiota contains two cysteine-rich repeats, a region similar to the phosphorylation site domain of myristoylated alanine-rich C kinase substrate, a conserved catalytic domain, and four ankyrin repeats at its C terminus. By primary structure, it is most similar to human DGKzeta and Drosophila rdgA. An >12-kilobase mRNA for DGKiota was detected only in brain and retina among the tissues examined. In cells transfected with the DGKiota cDNA, we detected an approximately 130-kDa protein by immunoassay, and activity assays demonstrated that it encodes a functional DAG kinase. The protein was found to be in both the cytoplasm and nucleus with the localization controlled by PKC isoforms alpha and gamma. The gene encoding DGKiota was localized to human chromosome 7q32.3-33, which is known to be a locus for an inherited form of retinitis pigmentosa. These results have defined a novel isoform of DAG kinase, which may have important cellular functions in the retina and brain.

Alternative splicing of the human diacylglycerol kinase zeta gene in muscle.

Ding L, Bunting M, Topham MK, McIntyre TM, Zimmerman GA, Prescott SM
Proc Natl Acad Sci U S A. 1997; 94: 5519-24

Diacylglycerol can function as a second messenger, and one mechanism for the attenuation of this signal is its conversion to phosphatidic acid, which is catalyzed by diacylglycerol kinase (DGK). We screened a cDNA library from human skeletal muscle and isolated two DGKzeta cDNAs that differed from the 3.5-kb clone originally identified in endothelial cells. One transcript, which was 3.4 kb long, was shown to be nonfunctional; it had a 77-bp deletion that included the translation initiation site. The other was 4.1 kb long with a unique 5' sequence of 853 bp. We also isolated a genomic clone of DGKzeta and determined its organization and location; it contains 32 exons, spans approximately 50 kb of genomic sequence, and maps to chromosome 11p11.2. The protein encoded by the 4.1-kb transcript contains two cysteine-rich regions, a catalytic domain, and ankyrin repeats like the endothelial form of DGKzeta, as well as a unique N-terminal domain. The coding sequence was shown to be derived from alternative splicing of the DGKzeta gene. In cells transfected with the 4.1-kb clone, we detected a 130-kDa protein with an antibody to DGKzeta and demonstrated that it was localized predominantly in the nucleus. We conclude that alternative splicing generates tissue-specific variants of DGKzeta that share some properties but may have unique ones as well.

Membrane translocation of protein kinase Ctheta during T lymphocyte activation requires phospholipase C-gamma-generated diacylglycerol.

Diaz-Flores E, Siliceo M, Martinez-A C, Merida I
J Biol Chem. 2003; 278: 29208-15

Protein kinase C (PKC) is the only PKC isoform recruited to the immunological synapse after T cell receptor stimulation, suggesting that its activation mechanism differs from that of the other isoforms. Previous studies have suggested that this selective PKC recruitment may operate via a Vav-regulated, cytoskeletal-dependent mechanism, independent of the classical phospholipase C/diacylglycerol pathway. Here, we demonstrate that, together with tyrosine phosphorylation of PKC in the regulatory domain, binding of phospholipase C-dependent diacylglycerol is required for PKC recruitment to the T cell synapse. In addition, we demonstrate that diacylglycerol kinase alpha-dependent diacylglycerol phosphorylation provides the negative signal required for PKC inactivation, ensuring fine control of the T cell activation response.

Cloning and expression of a cDNA encoding human inositol 1,4,5-trisphosphate 3-kinase C.

Dewaste V, Pouillon V, Moreau C, Shears S, Takazawa K, Erneux C
Biochem J. 2000; 352: 343-51

Inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] 3-kinase catalyses the phosphorylation of Ins(1,4,5)P(3) to Ins(1,3,4,5)P(4). cDNAs encoding two isoenzymes of Ins(1,4,5)P(3) 3-kinase (3-kinases A and B) have been described previously. In the present study, we report the cloning of a full-length 2052 bp cDNA encoding a third human isoenzyme of the Ins(1,4,5)P(3) 3-kinase family, referred to as isoform C. This novel enzyme has a calculated molecular mass of 75. 207 kDa and a K(m) for Ins(1,4,5)P(3) of 6 microM. Northern-blot analysis showed the presence of a transcript of approx. 3.9 kb in various human tissues. Inositol trisphosphate 3-kinase C demonstrates enzymic activity when expressed in DH5alphaF' bacteria or COS-7 cells. Calcium alone decreases the Ins(1,4,5)P(3) 3-kinase activity of the 3-kinase C isoenzyme in transfected COS-7 cells. This inhibitory effect is reversed in the presence of calmodulin. The recombinant bacterial 3-kinase C can be adsorbed on calmodulin-Sepharose in the presence of calcium. The present data show that Ins(1,4,5)P(3) 3-kinase C: (i) shares a conserved catalytic domain of about 275 amino acids with the two other mammalian isoforms, (ii) could be purified on a calmodulin-Sepharose column and (iii) could be distinguished from the A and B isoenzymes by the effects of calcium and of calmodulin.

Dictyostelium discoideum has a single diacylglycerol kinase gene with similarity to mammalian theta isoforms.

De La Roche MA, Smith JL, Rico M, Carrasco S, Merida I, Licate L, Cote GP, Egelhoff TT
Biochem J. 2002; 368: 809-15

Diacylglycerol kinases (DGKs) phosphorylate the neutral lipid diacylglycerol (DG) to produce phosphatidic acid (PA). In mammalian systems DGKs are a complex family of at least nine isoforms that are thought to participate in down-regulation of DG-based signalling pathways and perhaps activation of PA-stimulated signalling events. We report here that the simple protozoan amoeba Dictyostelium discoideum appears to contain a single gene encoding a DGK enzyme. This gene, dgkA, encodes a deduced protein that contains three C1-type cysteine-rich repeats, a DGK catalytic domain most closely related to the theta subtype of mammalian DGKs and a C-terminal segment containing a proline/glutamine-rich region and a large aspargine-repeat region. This gene corresponds to a previously reported myosin II heavy chain kinase designated myosin heavy chain-protein kinase C (MHC-PKC), but our analysis clearly demonstrates that this protein does not, as suggested by earlier data, contain a protein kinase catalytic domain. A FLAG-tagged version of DgkA expressed in Dictyostelium displayed robust DGK activity. Earlier studies indicating that disruption of this locus alters myosin II assembly levels in Dictyostelium raise the intriguing possibility that DG and/or PA metabolism may play a role in controlling myosin II assembly in this system.

Gonadotropin-releasing hormone-induced activation of diacylglycerol kinase-zeta and its association with active c-src.

Davidson L, Pawson AJ, De Maturana RL, Freestone SH, Barran P, Millar RP, Maudsley S
J Biol Chem. 2004; 279: 11906-16

Gonadotropin-releasing hormone (GnRH)-induced receptor activation has been demonstrated to entrain a wide variety of signaling modalities. Most signaling pathways are concerned with the control of serine, threonine, or tyrosine-protein kinases, however, in the current article we demonstrate that in both a model cell line and in gonadotropes, GnRH additionally mediates the activation of lipid-directed kinases. We have shown that there is a functional connection between protein-tyrosine kinase modulation and lipid kinase activation. In HEK293 cells stably expressing the Type I mammalian GnRH receptor, we employed a proteomic approach to identify novel protein binding partners for GnRH-activated c-Src. Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry we identified a GnRH-induced association between c-Src and the lipid kinase, diacylglycerol kinase-zeta (DGK-zeta). Using reciprocal co-immunoprecipitation we show that there is a significant elevation of the association between catalytically active c-Src with DGK-zeta in both HEK293 cells and murine gonadotrope LbetaT2 cells. Employing lipid kinase assays we have shown that the catalytic activity of DGK-zeta is significantly heightened in both HEK293 and LbetaT2 cells by GnRH. In addition, we demonstrate that the activation of DGK-zeta exerts a functional role in the murine gonadotrope LbetaT2 cell line. Elevated expression of DGK-zeta resulted in a shortening of the time scale of ERK activation in these cells suggesting a potential role of endogenous DGK-zeta in controlling the induction of LHbeta transcription by ERK1/2.

A mammalian protein homologous to fructosamine-3-kinase is a ketosamine-3-kinase acting on psicosamines and ribulosamines but not on fructosamines.

Collard F, Delpierre G, Stroobant V, Matthijs G, Van Schaftingen E
Diabetes. 2003; 52: 2888-95

Fructosamine-3-kinase (FN3K) is an enzyme that appears to be responsible for the removal of fructosamines from proteins. In this study, we report the sequence of human and mouse cDNAs encoding proteins sharing 65% sequence identity with FN3K. The genes encoding FN3K and FN3K-related protein (FN3K-RP) are present next to each other on human chromosome 17q25, and they both have a similar 6-exon structure. Northern blots of mouse tissues RNAs indicate a high level of expression of both genes in bone marrow, brain, kidneys, and spleen. Human FN3K-RP was transfected in human embryonic kidney (HEK) cells, and the expressed protein was partially purified by chromatography on Blue Sepharose. Unlike FN3K, FN3K-RP did not phosphorylate fructoselysine, 1-deoxy-1-morpholino-fructose, or lysozyme glycated with glucose. In a more systematic screening for potential substrates for FN3K-RP, we found, however, that both enzymes phosphorylated ketosamines with a D-configuration in C3 (psicoselysine, 1-deoxy-1-morpholino-psicose, 1-deoxy-1-morpholino-ribulose, lysozyme glycated with allose-the C3 epimer of glucose, or with ribose). Tandem mass spectrometry and nuclear magnetic resonance analysis of the product of phosphorylation of 1-deoxy-1-morpholino-psicose by FN3K-RP indicated that this enzyme phosphorylates the third carbon of the sugar moiety. These results indicate that FN3K-RP is a ketosamine-3-kinase (ketosamine-3-kinase 2). This enzyme presumably plays a role in freeing proteins from ribulosamines or psicosamines, which might arise in a several step process, from the reaction of amines with glucose and/or glycolytic intermediates. This role is shared by fructosamine-3-kinase (ketosamine-3-kinase 1), which has, in addition, the unique capacity to phosphorylate fructosamines.

Characterization of two cytosolic diacylglycerol kinase forms.

Chen Q, Klemm N, Jeng I
J Neurochem. 1993; 60: 1212-9

Two forms of rat brain cytosolic diacylglycerol kinase (EC2.7.1.107) were separated by heparin-agarose column chromatography. These forms, designated DGK-I and DGK-II, were not interconvertible as determined by rechromatography. DGK-I and DGK-II had respective molecular masses of 88 and 180 kDa, as measured by Sepharose 6B chromatography. Both forms preferred diacylglycerol over monoacylglycerol and were insensitive to R59022. DGK-II, but not DGK-I, was activated by an activator substance prepared from chicken egg yolk. DGK-II was activated by a rat brain cytosolic activator and was exclusively sensitive to 5'-AMP-mediated inactivation. Further studies revealed that these two forms had the following distinct characteristics: (a) substrate specificity, (b) inhibition by heparin, (c) sensitivity to lysine-containing polyamino acids, and (d) responses to different phospholipids. In general, DGK-II was more responsive to various inhibitors and activators, making it a prime candidate for a regulatable enzyme.

Identification of a novel gene product, Sertoli cell gene with a zinc finger domain, that is important for FSH activation of testicular Sertoli cells.

Chaudhary J, Skinner MK
Endocrinology. 2002; 143: 426-35

Sertoli cells provide the cytoarchitectural support and microenvironment necessary for the process of spermatogenesis. A novel, ubiquitously expressed cDNA clone was isolated from Sertoli cells and termed Sertoli cell gene with a zinc finger domain (SERZ). A significant homology of SERZ was found with a mouse genomic sequence that suggested the presence of at least 10 exons. An open reading frame at the 5'-end of the cDNA, termed SERZ-alpha, had a cryptic basic helix-loop-helix (bHLH) domain, but no start codon. When a start codon was engineered into the 5'-end of the cDNA, an in vitro translation product of SERZ-alpha was obtained. The longest second open reading frame with an ATG start site at 304 bp from the 5'-end coded for a 308-amino acid SERZ-beta polypeptide. Motif analysis and BLAST search of SERZ-beta showed significant homology to the DHHC domain of conserved zinc finger proteins. A number of potential phosphorylation sites were observed in the SERZ-beta polypeptide sequence. The long 5'-untranslated region of SERZ-beta prompted an investigation of both potential alternate polypeptide products, SERZ-alpha and SERZ-beta. Both SERZ-alpha and SERZ-beta proteins were detected with specific antibodies to SERZ-beta and the 5'-end open reading frame SERZ-alpha in a Western blot analysis of total Sertoli cell proteins. The presence of the SERZ-beta polypeptide was also confirmed by in vitro translation of the cDNA, but SERZ-alpha was not translated in vitro in the absence of an engineered start codon. The expression pattern of SERZ mRNA was observed in all tissues examined. The transcript size of SERZ as determined by Northern blot analysis is approximately 2.7 kb. An antisense oligonucleotide to SERZ was found not to influence basal levels of transferrin promoter activation, but significantly blocked FSH-induced transferrin promoter activation. SERZ mRNA expression was not regulated by FSH treatment of Sertoli cell cultures. In summary, a novel gene product, SERZ, was identified that appears to have a role in maintaining Sertoli cell differentiated functions and mediating FSH actions. Translation of SERZ may give rise to two gene products; however, the SERZ-beta containing the zinc finger domain is probably the principal product of the SERZ gene.

Molecular cloning and expression of a cDNA encoding a novel isoenzyme of protein kinase C (nPKC). A new member of the nPKC family expressed in skeletal muscle, megakaryoblastic cells, and platelets.

Chang JD, Xu Y, Raychowdhury MK, Ware JA
J Biol Chem. 1993; 268: 14208-14

At least seven bacteriophage lambda clones encoding structurally related but unique polypeptides with PKC activity have been isolated from mammalian brain, epidermis, and lung cDNA libraries. The possibility that additional isoenzymes are expressed in human blood platelets or megakaryoblastoid human erythroleukemia cells was examined by polymerase chain reaction amplification of reverse transcribed RNA employing oligonucleotide primers corresponding to conserved peptide sequences. cDNAs encoding a novel PKC-related sequence, designated PKC-theta, and four (alpha, beta, delta, and eta) previously identified isoenzymes were isolated from reverse transcribed total RNA of human erythroleukemia cells and platelets. PKC-theta lacks a conserved region (C2) that is present in the calcium-dependent isoenzymes and therefore belongs to the group of novel, or nPKC, isoenzymes. Significantly increased [3H] phorbol 12,13-dibutyrate binding and cytoskeleton-associated calcium-independent PKC activity were found in COS cells expressing the transfected cDNA. Northern transfer analysis of mRNA from various human tissues revealed high level expression of PKC-theta in skeletal muscle, lung, and brain, and minimal expression in cardiac muscle, placenta, and liver. These findings extend the PKC family and suggest a novel approach to the study of diversity within this pathway of intracellular signal transduction.

Alternative splicing gives rise to two isoforms of Zfhep, a zinc finger/homeodomain protein that binds T3-response elements.

Cabanillas AM, Darling DS
DNA Cell Biol. 1996; 15: 643-51

We have previously isolated a cDNA for a transcription factor referred to as Zfhep (zinc finger homeodomain enhancer-binding protein) containing two separate zinc finger domains, ZD1 and ZD2, each of which binds DNA, and a homeodomain. The rat Zfhep cDNA lacks a 5'-methionine codon, present in some homologs from other species. Hence, the aim of this work was to isolate the 5'-end of the rat Zfhep cDNA. Zfhep-2 cDNA was isolated, having a total length of 2.5 kbp, including more than 1.1 kbp of novel sequence followed by 1.4 kbp identical to the Zfhep-1 clone. The 1.1 kbp of novel sequence contains multiple stop codons in all reading frames, suggesting that it represents the 5'-untranslated (5'-UT) region of the rat Zfhep-2 mRNA. However, the Zfhep-2 clone does not contain the extreme 5'-exon(s) of the Zfhep-1 coding sequence, possibly due to alternative splicing of Zfhep RNA. To distinguish between a splice junction versus an intron-exon junction, the polymerase chain reaction (PCR) with rat genomic DNA and junction-flanking primers from the Zfhep-2 sequence was conducted. No bands were amplified from the genomic DNA by two different pairs of primers, indicating that the Zfhep-2-specific sequence is not intronic. Ribonuclease protection assays were performed to investigate the expression of multiple Zfhep mRNAs. Two protected bands were detected, and both were identified in total RNA or mRNA of rat ovary, hindbrain, forebrain, heart, kidney, small intestine, and GH4C1 cells. Zfhep-2 represents about 20% of the Zfhep RNA in each tissue. Hence, two mRNAs are expressed in these tissues, confirming the alternative splicing. To confirm independently the presence of both Zfhep-2 and Zfhep-1 mRNAs, reverse transcriptase (RT)-PCR was done using primers that span the Zfhep splice site. Specific bands representing both RNAs were obtained. The Zfhep-2 PCR product was subcloned and DNA sequence analysis confirmed the absence of ATG codons near the 5'-end of the open reading frame. The theoretical translation of the Zfhep-2 clone predicts a smaller protein than Zfhep-1. In vitro translation in reticulocyte lysates showed that Zfhep-2 is about 40 kD smaller than Zfhep-1. Hence, Zfhep-2 apparently lacks most of the first zinc finger domain (ZD1) of Zfhep-1. Because the two zinc finger domains bind different DNA sequences, Zfhep-2 is predicted to bind to only a subset of genes recognized by Zfhep-1.

Molecular cloning and characterization of a novel human diacylglycerol kinase zeta.

Bunting M, Tang W, Zimmerman GA, McIntyre TM, Prescott SM
J Biol Chem. 1996; 271: 10230-6

Diacylglycerol (DAG) occupies a central position in the synthesis of complex lipids and also has important signaling roles. For example, DAG is an allosteric regulator of protein kinase C, and the cellular levels of DAG may influence a variety of processes including growth and differentiation. We previously demonstrated that human endothelial cells derived from umbilical vein express growth-dependent changes in their basal levels of diacylglycerol and diacylglycerol kinase activity (Whatley, R. E., Stroud, E. D., Bunting, M., Zimmerman, G. A., McIntyre, T. M., and Prescott, S. M. (1993) J. Biol. Chem. 268, 16130-16138). To further explore the role of diacylglycerol metabolism in endothelial responses, we used a degenerate reverse transcription-polymerase chain reaction method to identify diacylglycerol kinase isozymes expressed by human endothelial cells. We report the isolation of a 3.5-kilobase cDNA encoding a novel diacylglycerol kinase (hDGKzeta) with a predicted molecular mass of 103.9 kDa. Human DGK zeta contains two zinc fingers, an ATP binding site, and four ankyrin repeats near the carboxyl terminus. A unique feature, as compared with other diacylglycerol kinases, is the presence of a sequence homologous to the MARCKS phosphorylation site domain. From Northern blot analysis of multiple tissues, we observed that hDGKzeta mRNA is expressed at highest levels in brain. COS-7 cells transfected with the hDGKzeta cDNA express 117-kDa and 114-kDa proteins that react specifically with an antibody to a peptide derived from a unique sequence in hDGK zeta. The transfected cells also express increased diacylglycerol kinase activity, which is not altered in the presence of R59949, an inhibitor of human platelet DGK activity. The hDGKzeta displays stereoselectivity for 1,2-diacylglycerol species in comparison to 1,3-diacylglycerol, but does not exhibit any specificity for molecular species of long chain diacylglycerols.

Identification and cDNA cloning of a novel mammalian C2 domain-containing phosphoinositide 3-kinase, HsC2-PI3K.

Brown RA, Ho LK, Weber-Hall SJ, Shipley JM, Fry MJ
Biochem Biophys Res Commun. 1997; 233: 537-44

Phosphoinositide (PI) 3-kinases have been shown to have critical roles in signal transduction, cell transformation and intracellular protein trafficking. Reverse-transcription polymerase chain reaction methods, using degenerate primers derived from the lipid kinase consensus region, were utilised to identify PI 3-kinases in the normal human breast. Here we report the cDNA cloning of a novel human PI 3-kinase isoform, HsC2-PI3K. This PI 3-kinase is most closely related to the recently described C2 domain-containing family of PI 3-kinases which includes Drosophila PI3K_68D/cpk and murine cpk-m/p170. Sequence analysis suggests that HsC2-PI3K is a second distinct mammalian member of the C2 domain-containing PI 3-kinase family. Northern blot analysis of human tissues indicates that HsC2-PI3K is widely expressed. Fluorescence in situ hybridisation has mapped HsC2-PI3K to chromosome 1q32.

Divergent and convergent signaling by the diacylglycerol second messenger pathway in mammals.

Brose N, Betz A, Wegmeyer H
Curr Opin Neurobiol. 2004; 14: 328-40

Diacylglycerol is an essential second messenger in mammalian cells. The most prominent intracellular targets of diacylglycerol and the functionally analogous phorbol esters belong to the protein kinase C family, but at least five alternative types of high affinity diacylglycerol/phorbol ester receptors are known: protein kinase D, diacylglycerol kinases alpha, beta, and gamma, RasGRPs, chimaerins, and Munc13s. These function independently of protein kinase C isozymes, and form a network of signaling pathways in the diacylglycerol second messenger system that regulates processes as diverse as gene transcription, lipid signaling, cytoskeletal dynamics, intracellular membrane trafficking, or neurotransmitter release.

Nuclear diacylglycerol kinase-theta is activated in response to alpha-thrombin.

Bregoli L, Baldassare JJ, Raben DM
J Biol Chem. 2001; 276: 23288-95

Currently, there is substantial evidence that nuclear lipid metabolism plays a critical role in a number of signal transduction cascades. Previous work from our laboratory showed that stimulation of quiescent fibroblasts with alpha-thrombin leads to the production of two lipid second messengers in the nucleus: an increase in nuclear diacylglycerol mass and an activation of phospholipase D, which catalyzes the hydrolysis of phosphatidylcholine to generate phosphatidic acid. Diacylglycerol kinase (DGK) catalyzes the conversion of diacylglycerol to phosphatidic acid, making it an attractive candidate for a signal transduction component. There is substantial evidence that this activity is indeed regulated in a number of signaling cascades (reviewed by van Blitterswijk, W. J., and Houssa, B. (1999) Chem. Phys. Lipids 98, 95-108). In this report, we show that the addition of alpha-thrombin to quiescent IIC9 fibroblasts results in an increase in nuclear DGK activity. The examination of nuclei isolated from quiescent IIC9 cells indicates that DGK-theta and DGK-delta are both present. We took advantage of the previous observations that phosphatidylserine inhibits DGK-delta (reviewed by Sakane, F., Imai, S., Kai, M., Wada, I., and Kanoh, H. (1996) J. Biol. Chem. 271, 8394-8401), and constitutively active RhoA inhibits DGK-theta (reviewed by Houssa, B., de Widt, J., Kranenburg, O., Moolenaar, W. H., and van Blitterswijk, W. J. (1999) J. Biol. Chem. 274, 6820-6822) to identify the activity induced by alpha-thrombin. Constitutively active RhoA inhibited the nuclear stimulated activity, whereas phosphatidylserine did not have an inhibitory effect. In addition, a monoclonal anti-DGK-theta antibody inhibited the alpha-thrombin-stimulated nuclear activity in vitro. These results demonstrate that DGK-theta is the isoform responsive to alpha-thrombin stimulation. Western blot and immunofluorescence microscopy analyses showed that alpha-thrombin induced the translocation of DGK-theta to the nucleus, implicating that this translocation is at least partly responsible for the increased nuclear activity. Taken together, these data are the first to demonstrate an agonist-induced activity of nuclear DGK-theta activity and a nuclear localization of DGK-delta.

Man9-mannosidase from pig liver is a type-II membrane protein that resides in the endoplasmic reticulum. cDNA cloning and expression of the enzyme in COS 1 cells.

Bieberich E, Treml K, Volker C, Rolfs A, Kalz-Fuller B, Bause E
Eur J Biochem. 1997; 246: 681-9

Man9-mannosidase, one of three different alpha 1,2-exo-mannosidases known to be involved in N-linked oligosaccharide processing, has been cloned in lambda gt10, using a mixed-primed pig liver cDNA library. Three clones were isolated which allowed the reconstruction of a 2731-bp full-length cDNA. The cDNA construct contained a single open reading frame of 1977 bp, encoding a 659-residue polypeptide with a molecular mass of approximately 73 kDa. The Man9-mannosidase specificity of the cDNA construct was verified by the observation that all peptide sequences derived from a previously purified, catalytically active 49-kDa fragment were found within the coding region. The N-terminus of the 49-kDa fragment aligns with amino acid 175 of the translated cDNA, indicating that the catalytic activity is associated with the C-terminus. Transfection of COS 1 cells with the Man9-mannosidase cDNA gave rise to a > 30-fold over-expression of a 73-kDa protein whose catalytic properties, including substrate specificity, susceptibility towards alpha-mannosidase inhibitors and metal ion requirements, were similar to those of the 49-kDa enzyme fragment. Thus deletion of 174 N-terminal amino acids in the 73-kDa protein appears to have only marginal influence on the catalytic properties. Structural and hydrophobicity analysis of the coding region, as well as the results from tryptic degradation studies, point to pig liver Man9-mannosidase being a non-glycosylated type-II transmembrane protein. This protein contains a 48-residue cytosolic tail followed by a 22-residue membrane anchor (which probably functions as internal and non-cleavable signal sequence), a lumenal approximately 100-residue-stem region and a large 49-kDa C-terminal catalytic domain. As shown by immuno-fluorescence microscopy, the pig liver enzyme expressed in COS 1 cells, is resident in the endoplasmic reticulum, in contrast to COS 1 Man9-mannosidase from human kidney which is Golgi-located [Bieberich, E. & Bause, E. (1995) Eur. J. Biochem. 233, 644-649]. Localization of the porcine enzyme in the endoplasmic reticulum is consistent with immuno-electron-microscopic studies using pig hepatocytes. The different intracellular distribution of pig liver and human kidney Man9-mannosidase is, therefore, enzyme-specific rather than a COS-1-cell-typical phenomenon. Since we observe approximately 81% sequence similarity between the two alpha-mannosidases, we deduce that the localization in either endoplasmic reticulum or Golgi is likely to be sequence-dependent.

A novel A-isoform-like inositol 1,4,5-trisphosphate 3-kinase from chicken erythrocytes exhibits alternative splicing and conservation of intron positions between vertebrates and invertebrates.

Bertsch U, Haefs M, Moller M, Deschermeier C, Fanick W, Kitzerow A, Ozaki S, Meyer HE, Mayr GW
Gene. 1999; 228: 61-71

Based on the partial peptide sequence of inositol 1,4, 5-trisphosphate 3-kinase purified with 135 000-fold enrichment from chicken erythrocytes, cDNA-fragments were cloned by RT-PCR using degenerate oligonucleotides. Subsequent hybridization screening of an embryonic chicken cDNA library and 5'-RACE yielded a cDNA-contig of 2418 bp, encoding a 452 amino acid protein. The amino acid sequence shows the highest degree of homology with A-isoforms of inositol 1,4,5-trisphosphate 3-kinase (65% identities), whereas homology towards B and C isoforms was lower (57% and 52% amino acid identities respectively). These findings reveal a new tissue-specific pattern of A-isoform expression, a form which so far has only been found in brain and testes.Two overlapping lambda-genomic clones for chicken inositol 1,4,5-trisphosphate 3-kinase, isolated by hybridization screening, covered 18 499 bp of genomic sequence. This contig included four exons: three of them were present in all cDNA clones, whereas one was only represented in a single cDNA clone. In addition, the sequence of the latter differed from the other cDNAs by an in-frame deletion of 72 bp within the coding region for the catalytic domain of the enzyme. This divergent cDNA suggests the existence of alternative splice products, at least in embryonic tissue.A comparison of the position of introns, with the respective introns known from the corresponding gene from Caenorhabditis elegans, revealed a high degree of conservation of intron positions between vertebrates and invertebrates. Functional data for the enzyme suggests that the conserved exons represent defined functional protein modules.

Differentiation of HL-60 cells to granulocytes involves regulation of select diacylglycerol kinases (DGKs).

Batista EL Jr, Warbington M, Badwey JA, Van Dyke TE
J Cell Biochem. 2005; 94: 774-93

Diacylglycerol Kinases (DGKs) are a family of enzymes that regulate the levels of different pools of diacylglycerol (DAG), affecting DAG-mediated signal transduction. Since DAG is known to play several important regulatory roles in granulocyte physiology, we investigated the expression pattern of DGK isoforms throughout differentiation of HL-60 cells to granulocytes. HL-60 cells were incubated with 1.25% dimethyl-sulfoxide (DMSO) to initiate differentiation and total RNA isolated at different time points. DGK expression was assessed through Northern blot, end-point PCR, and real-time PCR. The non-selective inhibitors R59022 and R59949 were used to block DGK at different time points throughout differentiation. CD11b and GPI-80, reactive oxygen species (ROS) generation, changes in the cell cycle, and apoptosis were used as markers of differentiation. Of the nine isoforms of DGK evaluated (alpha, delta, epsilon, gamma, zeta, beta, theta;, iota, eta), only five (alpha, delta, epsilon, gamma, and zeta) were expressed in HL-60 cells. DGKalpha was virtually absent in non-differentiated cells, but was markedly upregulated throughout differentiation. The other isoforms (delta, epsilon, gamma, and zeta) were expressed in undifferentiated HL-60 cells but were substantially decreased throughout differentiation. Non-selective blocking of DGK with R59022 and R59949 led to acceleration of differentiation, reducing the time necessary to observe upregulation of CD11b, GPI-80 and generation of ROS by 50%. Likewise, the cell cycle was disrupted when DGK isoforms were inhibited. These results provide evidence that DGK levels are dynamically regulated throughout differentiation and that expression of DGKs play an important regulatory function during the differentiation of neutrophils.

Human protein NEFA, a novel DNA binding/EF-hand/leucine zipper protein. Molecular cloning and sequence analysis of the cDNA, isolation and characterization of the protein.

Barnikol-Watanabe S, Gross NA, Gotz H, Henkel T, Karabinos A, Kratzin H, Barnikol HU, Hilschmann N
Biol Chem Hoppe Seyler. 1994; 375: 497-512

The cDNA libraries constructed from the human acute lymphoblastic leukemia cell line KM3 in the expression vector lambda gt11, were screened with the anti-CALLA (common acute lymphoblastic leukemia antigen) mAb (monoclonal antibody) J5. The selected J5-positive clone I containing a partial cDNA insert was isolated and sequenced. For completing the cDNA sequence the cDNA libraries were further screened by hybridization with the DIG (digoxigenin)-labelled DNA probe derived from clone I, the 5'-end region was analysed by 5'-RACE (rapid amplification of cDNA ends) using a sequence specific primer. In total a 1639 bp cDNA sequence was determined. The cDNA sequence contains a 1260 bp open reading frame and the untranslated 3'- and 5'-end sides. The 420 residue amino acid sequence, deduced from the cDNA sequence, unexpectedly differs fundamentally from CALLA (CD10) although clones I and II were J5-positive in immuno screening. The mature protein corresponding to the cDNA was isolated and characterized from the KM3 cells using polyclonal antisera raised against the in vitro expressed polypeptide from clone I. The protein is expressed on plasma membrane, in cytosol and is secreted into culture medium, its relative molecular mass was determined to be 55 kDa on SDS-PAGE. The deduced amino acid sequence from cDNA was confirmed by peptide sequences. The new protein contains a basic amino acid rich putative DNA binding domain (b) with a potential nuclear targeting signal, two helix-loop-helix (HLH) motif regions, concurrently EF-hand motifs, an acidic amino acid rich region (a) between the EF-hands, and a leucine zipper (Z) motif. This DNA binding protein therefore is characterized by a linked motif "b/HLH/a/HLH/Z". The protein was designated NEFA: DNA binding/EF-hand/acidic amino acid rich region.

Isolation and molecular cloning of wortmannin-sensitive bovine type III phosphatidylinositol 4-kinases.

Balla T, Downing GJ, Jaffe H, Kim S, Zolyomi A, Catt KJ
J Biol Chem. 1997; 272: 18358-66

Agonist-sensitive phosphoinositide pools are maintained by recently-identified wortmannin (WT)-sensitive phosphatidylinositol (PI) 4-kinase(s) (Nakanishi, S., Catt, K. J., and Balla, T. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 5317-5321). Two loosely membrane-associated WT-sensitive type III PI 4-kinases were isolated from bovine adrenal cortex as [3H]WT-labeled 110- and 210-kDa proteins. Based on peptide sequences from the smaller enzyme, a 3. 9-kilobase pair (kb) cDNA with an open reading frame encoding a 90-kDa protein was isolated from a bovine brain cDNA library. Expression of this cDNA in COS-7 cells yielded a 110-kDa protein with WT-sensitive PI 4-kinase activity. Northern blot analysis of a human mRNA panel showed a single approximately 3.8-kb transcript. Peptide sequences obtained from the 210-kDa enzyme corresponded to those of a recently described rat 230-kDa PI 4-kinase. A 6.5-kb cDNA containing an open reading frame of 6129 nucleotides that encoded a 230-kDa protein, was isolated from brain cDNA. Northern blot analysis of human mRNA revealed a major 7.5-kb transcript. The molecular cloning of these novel WT-sensitive type III PI 4-kinases will allow detailed analysis of their signaling and other regulatory functions in mammalian cells.

Molecular cloning and characterization of PKC theta, a novel member of the protein kinase C (PKC) gene family expressed predominantly in hematopoietic cells.

Baier G, Telford D, Giampa L, Coggeshall KM, Baier-Bitterlich G, Isakov N, Altman A
J Biol Chem. 1993; 268: 4997-5004

Members of the protein kinase C (PKC) family of serine/threonine kinases play a key role in regulating the differentiation and growth of diverse cell types and, to date, the cloning of seven mammalian PKC genes encoding eight distinct isoforms has been reported. Here we describe the molecular cloning and deduced primary structure of a cDNA encoding a novel PKC isoform, termed PKC theta, which was isolated in the course of attempts to identify PKC genes that are expressed selectively in hematopoietic cells. Degenerate oligonucleotide primers corresponding to conserved sequence motifs, which distinguish the PKC family from other protein kinases, were employed in polymerase chain reactions (PCR) to amplify partial core sequences of putative PKC genes from a human peripheral blood lymphocyte-derived cDNA library. DNA sequencing of selected clones revealed several PKC-related sequences, including one that, on the basis of sequence comparison with known PKC isoforms, represented a novel PKC isoform. The complete cDNA sequence was determined by anchored PCR cloning and sequencing the entire coding sequence, using cDNA derived from a human leukemic T cell line (Jurkat). Included within this approximately 2.7-kilobase pair cDNA is an open reading frame of 2,118 nucleotides encoding a putative 82-kDa protein. The deduced primary structure contains consensus sequences characteristic of protein kinase catalytic domains and, based on its amino acid sequence and domain structure, is a member of the PKC family. PKC theta displays the highest homology to PKC delta, lacks the Ca(2+)-binding C2 domain and, thus, belongs to the subfamily of Ca(2+)-independent PKC enzymes which also includes the delta, epsilon, zeta, and eta isoforms. RNase protection assays and semiquantitative PCR analysis indicated that, although PKC theta transcripts are expressed ubiquitously, the highest levels are found in hematopoietic tissues and cell lines, including T cells and thymocytes. In contrast, the expression levels in the brain and testes are considerably lower, and no transcripts were detected in several human carcinoma cell lines. A rabbit antiserum raised against a unique (V3 domain) bacterially expressed PKC theta fragment immunoprecipitated specifically an 82-kDa protein from Jurkat cell lysates. Thus, PKC theta represents an additional member of the PKC family, and its predominant expression in hematopoietic cells suggests that it may play a role in signal transduction and growth regulatory pathways unique to these cells.

Insulin receptor substrate 1 binds two novel splice variants of the regulatory subunit of phosphatidylinositol 3-kinase in muscle and brain.

Antonetti DA, Algenstaedt P, Kahn CR
Mol Cell Biol. 1996; 16: 2195-203

We have identified two novel alternatively spliced forms of the p85alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase by expression screening of a human skeletal muscle library with phosphorylated baculovirus- produced human insulin receptor substrate 1. One form is identical to p85alpha throughout the region which encodes both Src homology 2 (SH2) domains and the inter-SH2 domain/p110 binding region but diverges in sequence from p85alpha on the 5' side of nucleotide 953, where the entire break point cluster gene and SH3 regions are replaced by a unique 34-amino-acid N terminus. This form has an estimated molecular mass of approximately 53 kDa and has been termed p85/AS53. The second form is identical to p85 and p85/AS53 except for a 24-nucleotide insert between the SH2 domains that results in a replacement of aspartic acid 605 with nine amino acids, adding two potential serine phosphorylation sites in the vicinity of the known serine autophosphorylation site (Ser-608). Northern (RNA) analyses reveal a wide tissue distribution of p85alpha, whereas p85/AS53 is dominant in skeletal muscle and brain, and the insert isoforms are restricted to cardiac muscle and skeletal muscle. Western blot (immunoblot) analyses using an anti-p85 polyclonal antibody and a specific anti-p85/AS53 antibody confirmed the tissue distribution of p85/AS53 protein and indicate a approximately 7-fold higher expression of p85/AS53 protein than of p85 in skeletal muscle. Both p85 and p85/AS53 bind to p110 in coprecipitation experiments, but p85alpha itself appears to have preferential binding to insulin receptor substrate 1 following insulin stimulation. These data indicate that the gene for the p85alpha regulatory subunit of PI 3-kinase can undergo tissue-specific alternative splicing. Two novel splice variants of the regulatory subunit of PI 3-kinase are present in skeletal muscle, cardiac muscle, and brain; these variants may have important functional differences in activity and may play a role in tissue-specific signals such as insulin-stimulated glucose transport or control of neurotransmitter secretion or action.

The cysteine-rich domain of human proteins, neuronal chimaerin, protein kinase C and diacylglycerol kinase binds zinc. Evidence for the involvement of a zinc-dependent structure in phorbol ester binding.

Ahmed S, Kozma R, Lee J, Monfries C, Harden N, Lim L
Biochem J. 1991; 280: 233-41

Diacylglycerol (DG) and its analogue phorbol 12-myristate 13-acetate (PMA) activate the ubiquitous phospholipid/Ca2(+)-dependent protein kinase, protein kinase C (PKC), and cause it to become tightly associated with membranes. DG is produced transiently as it is rapidly metabolized by DG kinase (DGK) to phosphatidic acid. Phorbol esters such as PMA are not metabolized and induced a prolonged membrane association of PKC. Until recently, PKC was the only known phorbol ester receptor. We have shown that a novel brain-specific cDNA, neuronal chimaerin (NC), expressed in Escherichia coli, binds phorbol ester with high affinity, stereospecificity and a phospholipid requirement [Ahmed, Kozma, Monfries, Hall, Lim, Smith & Lim (1990) Biochem. J. 272, 767-773]. The proteins NC, PKC and DGK possess a cysteine-rich domain with the motif HX11/12CX2CXnCX2CX4HX2CX6/7C (where n varies between 12 and 14). The partial motif, CX2CX13CX2C, is present in a number of transcription factors including the steroid hormone receptors and the yeast protein, GAL4, in which zinc plays a structural role of co-ordinating cysteine residues and is essential for DNA binding (protein-nucleic acid interactions). The cysteine-rich domain of NC and PKC is required for phospholipid-dependent phorbol is required for phospholipid-dependent phorbol ester binding, suggesting an involvement of this domain in protein-lipid interactions. We have expressed recombinant NC, PKC and DGK glutathione S-transferase and TrpE fusion proteins in E. coli to investigate the relationship between the cysteine-rich motif, HX11/12CX2CX10-14CX2CX4HX2CX6/7C, zinc and phorbol ester binding. The cysteine-rich domain of NC, PKC and DGK bound 65Zn2+ but only NC and PKC bound [3H]phorbol 12,13-dibutyrate. When NC and PKC were subjected to treatments known to remove metal ions from GAL4 and the human glucocorticoid receptor, phorbol ester binding was inhibited. These data provide evidence for the role of a zinc-dependent structure in phorbol ester binding.

Diacylglycerol kinase-zeta localization in skeletal muscle is regulated by phosphorylation and interaction with syntrophins.

Abramovici H, Hogan AB, Obagi C, Topham MK, Gee SH
Mol Biol Cell. 2003; 14: 4499-511

Syntrophins are scaffolding proteins that link signaling molecules to dystrophin and the cytoskeleton. We previously reported that syntrophins interact with diacylglycerol kinase-zeta (DGK-zeta), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show syntrophins and DGK-zeta form a complex in skeletal muscle whose translocation from the cytosol to the plasma membrane is regulated by protein kinase C-dependent phosphorylation of the DGK-zeta MARCKS domain. DGK-zeta mutants that do not bind syntrophins were mislocalized, and an activated mutant of this sort induced atypical changes in the actin cytoskeleton, indicating syntrophins are important for localizing DGK-zeta and regulating its activity. Consistent with a role in actin organization, DGK-zeta and syntrophins were colocalized with filamentous (F)-actin and Rac in lamellipodia and ruffles. Moreover, extracellular signal-related kinase-dependent phosphorylation of DGK-zeta regulated its association with the cytoskeleton. In adult muscle, DGK-zeta was colocalized with syntrophins on the sarcolemma and was concentrated at neuromuscular junctions (NMJs), whereas in type IIB fibers it was found exclusively at NMJs. DGK-zeta was reduced at the sarcolemma of dystrophin-deficient mdx mouse myofibers but was specifically retained at NMJs, indicating that dystrophin is important for the sarcolemmal but not synaptic localization of DGK-zeta. Together, our findings suggest syntrophins localize DGK-zeta signaling complexes at specialized domains of muscle cells, which may be critical for the proper control of lipid-signaling pathways regulating actin organization. In dystrophic muscle, mislocalized DGK-zeta may cause abnormal cytoskeletal changes that contribute to disease pathogenesis.