Secondary literature sources for the PP2Cc domain

For each of the hand selected primary papers associated with the PP2Cc 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.

Human blue-light photoreceptor hCRY2 specifically interacts with protein serine/threonine phosphatase 5 and modulates its activity.

Zhao S, Sancar A
Photochem Photobiol. 1997; 66: 727-31

Photolyase/blue-light photoreceptor family of proteins includes cyclobutane pyrimidine dimer photolyase, (6-4) photolyase and blue-light photoreceptors that were recently discovered in Arabidopsis thaliana, Sinapis alba and Chlamydomonas reinhardtii. Recently, we identified two human genes, hCRY1 and hCRY2, belonging to this family. The proteins encoded by these genes have no DNA repair activity and therefore were hypothesized to function in human blue-light response reactions. To identify downstream targets for these putative blue-light photoreceptors we searched for interacting proteins by the yeast two-hybrid method. We found that the tetratricopeptide repeat protein 1, Tpr1, and the protein serine/threonine phosphatase 5 (PP5) that contains the TPR motif specifically interacted with hCRY2. The effect of the hCRY2-PP5 interaction on the protein phosphatase activity was investigated. We found that hCRY2, but not the highly homologous (6-4) photolyase, inhibits the phosphatase activity of PP5. This inhibition may be on the pathway of blue-light signal transduction reaction in humans.

The catalytic subunits of Ser/Thr protein phosphatases from Caenorhabditis elegans.

Zeke T, Gergely P, Dombradi V
Comp Biochem Physiol B Biochem Mol Biol. 1998; 119: 317-24

The catalytic activities of protein phosphatase 1, 2A, 2B, and 2C were detected in crude extracts of Caenorhabditis elegans with different phosphoprotein substrates and specific inhibitors or activators. The enzymological properties of protein phosphatase 2B as well as those of the catalytic subunits of protein phosphatase 1 and protein phosphatase 2A were determined after partial purification. Gene fragments encoding the catalytic subunits of the protein phosphatase 1-2A-2B superfamily were amplified by polymerase chain reaction and were identified by DNA sequencing. Besides the homologs of protein phosphatase 1, 2B, and X, five protein phosphatase 1-type sequences and four novel protein phosphatase sequences were found. Our data, together with the results of the C. elegans genome project, suggest that this nematode contains an extensive family of Ser/Thr specific protein phosphatases including several up to now biochemically uncharacterized members.

Serine/threonine protein phosphatases and a protein phosphatase 1 inhibitor from Neurospora crassa.

Zapella PD, da-Silva AM, da-Costa-Maia JC, Terenzi HF
Braz J Med Biol Res. 1996; 29: 599-604

The major spontaneously active serine/threonine (Ser/Thr) protein phosphatase activities in N. crassa wild type (FGSC 424) were type-1 (PP1), type-2A (PP2A) and type-2C (PP2C). PP1 and PP2C predominantly dephosphorylated phosphorylase a and casein, respectively. PP2A acted on both substrates, but was two-fold more active against casein. PP1 activity was inhibited by protamine, heparin, okadaic acid (IC50 50 nM) and mammalian inhibitor-1 (IC50 2 nM). On the other hand. PP2A activity was inhibited by much lower concentrations of okadaic acid (IC50 0.2 nM) and also by protamine, but not by heparin or inhibitor-1. About 80% of total PP1 activity was associated with the particulate fraction and could be partially extracted with 0.5 M NaCl. Seventy and ninety percent of PP2A and PP2C activities, respectively, were found in the soluble fraction. In addition we have partially purified an acid and thermostable PP1 inhibitor which effectively inhibits both N. crassa and mammalian PP1.

Modulation of the growth of Plasmodium falciparum in vitro by protein serine/threonine phosphatase inhibitors.

Yokoyama D, Saito-Ito A, Asao N, Tanabe K, Yamamoto M, Matsumura T
Biochem Biophys Res Commun. 1998; 247: 18-23

To elucidate the physiological roles of the protein serine/threonine phosphatases of P. falciparum, first we identified and characterized phosphatase activities of Plasmodium falciparum enzymologically and pharmacologically. We have demonstrated that P. falciparum possesses phosphatase-1-like activities predominantly over phosphatase-2A-like activities, while erythrocytes possess mainly phosphatase-2A-like activities. Then, we examined the effects of okadaic acid and calyculin A, potent inhibitors of protein phosphatase 1 and 2A, on the growth of P. falciparum in vitro. Both of the drugs inhibited parasite growth dose dependently. The manner of growth inhibition by calyculin A and okadaic acid suggested that these drugs inhibit parasite growth mainly by inhibiting parasite phosphatase-1-like activities. Both drugs were shown to inhibit the growth of three different developmental stages of parasites--ring forms, trophozoites, and schizonts--and inhibit trophozoites the most. This is the first report on P. falciparum protein serine/threonine phosphatase activities, which are essential to regulate the erythrocytic stage of parasite growth.

Theoretical models of catalytic domains of protein phosphatases 1 and 2A with Zn2+ and Mn2+ metal dications and putative bioligands in their catalytic centers.

Wozniak-Celmer E, Oldziej S, Ciarkowski J
Acta Biochim Pol. 2001; 48: 35-52

The oligomeric metalloenzymes protein phosphatases dephosphorylate OH groups of Ser/Thr or Tyr residues of proteins whose actions depend on the phosphorus signal. The catalytic units of Ser/Thr protein phosphatases 1, 2A and 2B (PP1c, PP2Ac and PP2Bc, respectively), which exhibit about 45% sequence similarity, have their active centers practically identical. This feature strongly suggests that the unknown structure of PP2Ac could be successfully homology-modeled from the known structures of PP1c and/or PP2Bc. Initially, a theoretical model of PP1c was built, including a phosphate and a metal dication in its catalytic site. The latter was modeled, together with a structural hydroxyl anion, as a triangular pseudo-molecule (Zno or Mno), composed of two metal cations (double Zn2+ or Mn2+, respectively) and the OH- group. To the free PP1c two inhibitor sequences R29RRRPpTPAMLFR40 of DARPP-32 and R30RRRPpTPATLVLT42 of Inhibitor-1, and two putative substrate sequences LRRApSVA and QRRQRKpRRTI were subsequently docked. In the next step, a free PP2Ac model was built via homology re-modeling of the PP1c template and the same four sequences were docked to it. Thus, together, 20 starting model complexes were built, allowing for combination of the Zno and Mno pseudo-molecules, free enzymes and the peptide ligands docked in the catalytic sites of PP1c and PP2Ac. All models were subsequently subjected to 250-300 ps molecular dynamics using the AMBER 5.0 program. The equilibrated trajectories of the final 50 ps were taken for further analyses. The theoretical models of PP1c complexes, irrespective of the dication type, exhibited increased mobilities in the following residue ranges: 195-200, 273-278, 287-209 for the inhibitor sequences and 21-25, 194-200, 222-227, 261, 299-302 for the substrate sequences. Paradoxically, the analogous PP2Ac models appeared much more stable in similar simulations, since only their "prosegment" residues 6-10 and 14-18 exhibited an increased mobility in the inhibitor complexes while no areas of increased mobility were found in the substrate complexes. Another general observation was that the complexes with Mn dications were more stable than those with Zn dications for both PP1c and PP2Ac units.

A Mg(2+)-dependent, Ca(2+)-inhibitable serine/threonine protein phosphatase from bovine brain.

Wang Y, Santini F, Qin K, Huang CY
J Biol Chem. 1995; 270: 25607-12

The Mg(2+)-dependent serine/threonine protein phosphatases, also known as type 2C phosphatases (PP2C), belong to a gene family distinct from the other serine/threonine phosphatases and tyrosine phosphatases. Here we report the purification to apparent homogeneity of a novel Mg(2+)-dependent, Ca(2+)-inhibitable serine/threonine protein phosphatase from bovine brain. It is a type 2C enzyme in view of its Mg2+ requirement, resistance to okadaic acid and calyculin A, inability to use phosphorylase alpha as substrate, and a segment of amino acid sequence typical of all PP2C type phosphatases known to date. However, it differs from the other PP2C enzymes, particularly the mammalian PP2C alpha and -beta isoforms, in that its molecular weight, 76,000, is considerably larger and that it is inhibited by Ca2+, NaF, and polycations, but not by orthovanadate. The Ca2+ inhibition may not be related to its cellular regulation because of Ki values in the 20-90 microM range, but this property permits distinction of this enzyme from the other phosphatases. Although the precise physiological role of this phosphatase is not yet known, its ability to dephosphorylate a wide variety of phosphoproteins and its broad distribution, as shown by a survey of mouse tissues for its activity, suggest that it may serve an important cellular function.

Protein serine/threonine phosphatases and cell transformation.

Walter G, Mumby M
Biochim Biophys Acta. 1993; 1155: 207-26

Multiplicity of protein serine-threonine phosphatases in PC12 pheochromocytoma and FTO-2B hepatoma cells.

Wadzinski BE, Heasley LE, Johnson GL
J Biol Chem. 1990; 265: 21504-8

Protein purification and molecular cloning have defined five classes of protein serine-threonine phosphatase catalytic subunits referred to as types 1, 2A, 2B (calcineurin), 2C, and X. Protein serine-threonine phosphatases 1, 2A, 2B, and X appear to have significant sequence homologies, whereas the 2C enzyme is more divergent. We have used the polymerase chain reaction to define the multiplicity of the closely related types 1, 2A, 2B, and X phosphatase catalytic subunits in two clonal cell lines, rat PC12 pheochromocytoma and rat FTO-2B hepatoma. RNAs for all four related phosphatase types were expressed in both cell lines. In addition to the phosphatase X enzyme, four phosphatase 1, two phosphatase 2A, and three phosphatase 2B isoforms were identified in PC12 and FTO-2B cells. The results indicate a large multiplicity of protein serine-threonine phosphatases within clonal cells of different tissue origin, suggesting that their role in cell regulation will be as divergent as that for the protein serine-threonine kinases.

Protein phosphatase 2A: a panoply of enzymes.

Virshup DM
Curr Opin Cell Biol. 2000; 12: 180-5

Protein phosphatase 2A describes an extended family of intracellular protein serine/threonine phosphatases sharing a common catalytic subunit that regulates a variety of processes by means of diverse regulatory subunits. During the past year, studies have shown that protein phosphatase 2A influences events ranging from the initiation of DNA replication to vertebrate axis formation to apoptosis.

Protein serine/threonine phosphatases.

Villafranca JE, Kissinger CR, Parge HE
Curr Opin Biotechnol. 1996; 7: 397-402

In the past year, the three-dimensional structures of two serine/threonine phosphatases, protein phosphatase-1 and protein phosphatase-2b (calcineurin), have been determined. The new information puts previous sequence comparisons and mutagenesis studies into a detailed structural perspective. The active-site structure and catalytic mechanism appear to be common to a variety of phosphoesterase enzymes.

[The structure and regulatory mechanisms of protein phosphatase 2A]

Usui H, Nishito Y
Tanpakushitsu Kakusan Koso. 1998; 43: 945-51

Charged with meaning: the structure and mechanism of phosphoprotein phosphatases.

Taylor WP, Widlanski TS
Chem Biol. 1995; 2: 713-8

Many phosphatases require two metal ions for catalysis. New structural information on two serine/threonine phosphatases offers insight into how the metals contribute to catalysis. A comparison with the structures of protein tyrosine phosphatases, which do not use metal ions, shows that the only similarity at the active site is that of charge.

[Structure and function of mammalian protein phosphatase 2C]

Tamura S, Kobayashi T, Ohnishi M, Chida N, Hanada M
Tanpakushitsu Kakusan Koso. 1998; 43: 959-67

[Protein serine/threonine phosphatases]

Tamura S
Tanpakushitsu Kakusan Koso. 1998; 43: 935-6

[Protein phosphatase as a partner of protein kinase]

Takeda M
Tanpakushitsu Kakusan Koso. 1998; 43: 927-34

[Naturally occurring toxins with specific inhibitory activity against protein serine/threonine phosphatases 1 and 2A]

Takai A
Tanpakushitsu Kakusan Koso. 1998; 43: 1091-101

Purification and characterization of the catalytic subunit of protein phosphatase 1 from Neurospora crassa.

Szoor B, Dombradi V, Gergely P, Feher Z
Acta Biol Hung. 1997; 48: 289-302

Protein phosphorylation is a universal regulatory mechanism in eukaryotic cells. The phosphorylation state of proteins is affected by the antagonistic activities of protein kinases and phosphatases. Protein phosphatases (PPs) can be classified as serine/threonine and tyrosine specific phosphatases. Ser/Thr phosphatases are divided into four subclasses (PP1, PP2A, PP2B, PP2C) on the basis of their substrate specificity, metal ion dependence and inhibitor sensitivity. We were able to detect the activities of all four Ser/Thr protein phosphatases in the mycelial extract of Neurospora crassa. The catalytic subunit of PP1 was purified 1500-fold with a yield of 1.3% using ammonium sulfate-ethanol precipitation, DEAE-Sephacel, heparin-Sepharose and MonoQ FPLC chromatography. The protein product was nearly homogenous, as judged by SDS-polyacrylamide gel electrophoresis. The most important properties of the enzyme were the following: /1/ its molecular mass proved to be 35 kD, /2/ it was completely inhibited by inhibitor-2, microcystin and okadaic acid, /3/ it was bound to heparin-Sepharose, and /4/ its specific activity was 2000 mU/mg. These biochemical properties are very similar to those of the homologous enzyme from rabbit muscle and indicate a high level of conservation of PP1 structure during evolution.

Genetic analyses of yeast protein serine/threonine phosphatases.

Stark MJ, Black S, Sneddon AA, Andrews PD
FEMS Microbiol Lett. 1994; 117: 121-30

Protein phosphorylation is an important regulatory phenomenon in yeasts just as in other eukaryotic cells and controls a wide variety of cellular processes. The importance of protein phosphatases as well as protein kinases as key elements in such control is becoming increasingly clear. Over the past four years since the first yeast protein phosphatase gene was isolated, many more such genes have been described and the number of genes encoding protein phosphatase catalytic subunits in Saccharomyces cerevisiae has comfortably entered double figures. Given the genetic approaches available, yeasts offer powerful systems for addressing the cellular roles of these enzymes. This review summarises the results of genetic studies aimed at determining the functions of protein serine/threonine phosphatases in yeast.

Yeast protein serine/threonine phosphatases: multiple roles and diverse regulation.

Stark MJ
Yeast. 1996; 12: 1647-75

Since the isolation of the first yeast protein phosphatase genes in 1989, much progress has been made in understanding this important group of proteins. Yeast contain genes encoding all the major types of protein phosphatase found in higher eukaryotes and the ability to use genetic approaches will complement the wealth of biochemical information available from other systems. This review will summarize recent progress in understanding the structure, function and regulation of the PPP family of protein serine-threonine phosphatases, concentrating on the budding yeast Saccharomyces cerevisiae.

Gene cloning and expression and characterization of a toxin-sensitive protein phosphatase from the methanogenic archaeon Methanosarcina thermophila TM-1.

Solow B, Young JC, Kennelly PJ
J Bacteriol. 1997; 179: 5072-5

With oligonucleotides modelled after conserved regions within the protein-serine/threonine phosphatases (PPs) of the PP1/2A/2B superfamily, the gene for the archaeal protein phosphatase PP1-arch2 was identified, cloned, and sequenced from the methanogenic archaeon Methanosarcina thermophila TM-1. The DNA-derived amino acid sequence of PP1-arch2 exhibited a high degree of sequence identity, 27 to 31%, with members of the PP1/2A/2B superfamily such as PP1-arch1 from Sulfolobus solfataricus, PP1alpha from rats, PP2A from Saccharomyces cerevisiae, and PP2B from humans. The activity of the recombinant PP1-arch2 was sensitive to several naturally occurring microbial toxins known to potently inhibit eucaryal PP1 and PP2A, including microcystin-LR, okadaic acid, tautomycin, and calyculin A.

Purification of a fatty acid-stimulated protein-serine/threonine phosphatase from bovine brain and its identification as a homolog of protein phosphatase 5.

Skinner J, Sinclair C, Romeo C, Armstrong D, Charbonneau H, Rossie S
J Biol Chem. 1997; 272: 22464-71

An arachidonic acid-stimulated Ser/Thr phosphatase activity was detected in soluble extracts prepared from rat pituitary clonal GH4C1 cells, rat or bovine brain, and bovine heart. The enzyme activity was purified to homogeneity from bovine brain as a monomer with a Mr of 63,000 and a specific activity of 32 nmol of Pi released per min/mg of protein when assayed in the presence of 10 microM phosphocasein in the absence of lipid. Arachidonic acid stimulated activity 4-14-fold, with half-maximal stimulation at 50-100 microM, when assayed in the presence of a variety of phosphosubstrates including casein, reduced carboxamidomethylated and maleylated lysozyme, myelin basic protein, and histone. Oleic acid, linoleic acid, and palmitoleic acid also stimulated activity; however, saturated fatty acids and alcohol or methyl ester derivatives of fatty acids did not significantly affect activity. The lipid-stimulated phosphatase was identified as the bovine equivalent of protein phosphatase 5 or a closely related homolog by sequence analysis of proteolytic fragments generated from the purified enzyme. When recombinant rat protein phosphatase 5 was expressed as a cleavable glutathione S-transferase fusion protein, the affinity-purified thrombin-cleaved enzyme exhibited a specific activity and sensitivity to arachidonic acid similar to those of the purified bovine brain enzyme. These results suggest that protein phosphatase 5 may be regulated in vivo by a lipid second messenger or another endogenous activator.

Characterization of serine/threonine protein phosphatases in RINm5F insulinoma cells.

Sjoholm A, Honkanen RE, Berggren PO
Biosci Rep. 1993; 13: 349-58

This study investigates the occurrence and regulation of serine/threonine protein phosphatases (PPases) in insulin-secreting RINm5F insulinoma cells. PPases types 1 and 2A were identified in crude RINm5F cell homogenates by both enzymatic assay and Western blot analysis. We then characterized and compared the inhibitory actions of several compounds isolated from cyanobacteria, marine dinoflagellates and marine sponges, (viz. okadaic acid, microcystin-LR, calyculin-A and nodularin) cation-independent PPase activities in RINm5F cell homogenates. It was found that okadaic acid was the least potent inhibitor (IC50 approximately 10(-9) M, IC100 approximately 10(-6) M), while the other compounds exhibited IC50 values of approximately 5 x 10(-10) M and IC100 approximately 5 x 10(-9) M. The findings indicate that the inhibitory substances employed in this study may be used pharmacologically to investigate the role of serine/threonine PPases in RINm5F cell insulin secretion, a process that is likely to be regulated to a major extent by protein phosphorylation.

Inhibition of serine/threonine protein phosphatases by secretagogues in insulin-secreting cells.

Sjoholm A, Honkanen RE, Berggren PO
Endocrinology. 1995; 136: 3391-7

Reversible protein phosphorylation is considered to be an important and versatile mechanism by which cells transduce external signals into biological responses. Cellular levels of protein phosphorylation are determined by the balanced actions of both protein kinases and protein phosphatases (PPases). Compared with protein kinases, however, serine/threonine PPases have received less attention. In the present study, the effects of known insulin secretagogues and some intracellular second messengers on the activities of serine/threonine PPases in insulin-secreting RINm5F insulinoma cells were investigated. The stimulation of intact RINm5F cells with the insulin secretagogues L-arginine, L-glutamine, KCl, or ATP elicited time-dependent changes in PPase activities with an early (1 min) decrease in type 1-like and/or type 2A-like PPase activity that gradually returned to normal levels. Addition of cAMP, cGMP, or prostaglandins E2 and F1 alpha at widely different concentrations to RINm5F cell homogenates failed to affect PPase activities. In contrast, addition of physiological concentrations of adenine nucleotides, which are known to increase upon secretory stimulation, to cell homogenates inhibited type 2A-like and, to a lesser extent, type 1-like, PPase activity (ATP > ADP > AMP > adenosine). ATP and ADP IC50 values for type 2A-like PPase were approximately 75 and 250 microM, respectively. The inhibitory effect of ATP was reproduced and of comparable magnitude when purified PPases (types 1 and 2A) were used instead of RINm5F cell homogenates. It is concluded that insulin secretagogues cause time- and concentration-dependent inhibitory effects on RINm5F cell PPase activities, which may contribute to the increase in the phosphorylation state that occurs after stimulation of insulin release. Thus, inhibition of protein dephosphorylation may be a novel regulatory mechanism controlling the stimulus-secretion coupling in insulin-producing cells.

The serine, threonine, and/or tyrosine-specific protein kinases and protein phosphatases of prokaryotic organisms: a family portrait.

Shi L, Potts M, Kennelly PJ
FEMS Microbiol Rev. 1998; 22: 229-53

Inspection of the genomes for the bacteria Bacillus subtilis 168, Borrelia burgdorferi B31, Escherichia coli K-12, Haemophilus influenzae KW20, Helicobacter pylori 26695, Mycoplasma genitalium G-37, and Synechocystis sp PCC 6803 and for the archaeons Archaeoglobus fulgidus VC-16 DSM4304, Methanobacterium thermoautotrophicum delta H, and Methanococcus jannaschii DSM2661 revealed that each contains at least one ORF whose predicted product displays sequence features characteristic of eukaryote-like protein-serine/threonine/tyrosine kinases and protein-serine/threonine/tyrosine phosphatases. Orthologs for all four major protein phosphatase families (PPP, PPM, conventional PTP, and low molecular weight PTP) were present in the bacteria surveyed, but not all strains contained all types. The three archaeons surveyed lacked recognizable homologs of the PPM family of eukaryotic protein-serine/threonine phosphatases; and only two prokaryotes were found to contain ORFs for potential phosphatases from all four major families. Intriguingly, our searches revealed a potential ancestral link between the catalytic subunits of microbial arsenate reductases and the protein-tyrosine phosphatases; they share similar ligands (arsenate versus phosphate) and features of their catalytic mechanism (formation of arseno-versus phospho-cysteinyl intermediates). It appears that all prokaryotic organisms, at one time, contained the genetic information necessary to construct protein phosphorylation-dephosphorylation networks that target serine, threonine, and/or tyrosine residues on proteins. However, the potential for functional redundancy among the four protein phosphatase families has led many prokaryotic organisms to discard one, two, or three of the four.

Cyanobacterial PPP family protein phosphatases possess multifunctional capabilities and are resistant to microcystin-LR.

Shi L, Carmichael WW, Kennelly PJ
J Biol Chem. 1999; 274: 10039-46

The structural gene for a putative PPP family protein-serine/threonine phosphatase from the microcystin-producing cyanobacterium Microcystis aeruginosa PCC 7820, pp1-cyano1, was cloned. The sequence of the predicted gene product, PP1-cyano1, was 98% identical to that of the predicted product of an open reading frame, pp1-cyano2, from a cyanobacterium that does not produce microcystins, M. aeruginosa UTEX 2063. By contrast, PP1-cyano1 displayed less than 20% identity with other PPP family protein phosphatases from eukaryotic, archaeal, or other bacterial organisms. PP1-cyano1 and PP1-cyano2 were expressed in Escherichia coli and purified to homogeneity. Both enzymes exhibited divalent metal dependent phosphohydrolase activity in vitro toward phosphoserine- and phosphotyrosine-containing proteins and 3-phosphohistidine- and phospholysine-containing amino acid homopolymers. This multifunctional potential also was apparent in samples of PP1-cyano1 and PP1-cyano2 isolated from M. aeruginosa. Catalytic activity was insensitive to okadaic acid or the cyanobacterially produced cyclic heptapeptide, microcystin-LR, both potent inhibitors of mammalian PP1 and PP2A. PP1-cyano1 and PP1-cyano2 displayed diadenosine tetraphosphatase activity in vitro. Diadenosine tetraphosphatases share conserved sequence features with PPP family protein phosphatases. The diadenosine tetraphosphatase activity of PP1-cyano1 and PP1-cyano2 confirms that these enzymes share a common catalytic mechanism.

pp1-cyano2, a protein serine/threonine phosphatase 1 gene from the cyanobacterium Microcystis aeruginosa UTEX 2063.

Shi L, Carmichael WW
Arch Microbiol. 1997; 168: 528-31

Polymerase chain reaction (PCR) products similar to protein serine/threonine family I phosphatase genes were identified in five strains of cyanobacteria from three species. The gene for one of these protein phosphatase PCR products, pp1-cyano2 from Microcystis aeruginosa UTEX 2063, was cloned and sequenced. The deduced protein sequence PP1-cyano2 contains 264 amino acid residues ( approximately 30.3 kDa). In its N-terminal region, PP1-cyano2 had a GDXXHG(X)nGDXXDRG(X)nGNHE (nP23) sequence that is well-conserved in all protein serine/threonine family I phosphatases. Of 19 amino acid residues important for either metal binding, structure of the active site, or catalysis in eukaryotic PP1, 18 were present in PP1-cyano2. Reverse-transcription-PCR results showed that pp1-cyano2 was expressed under laboratory culture conditions.

Identification and characterization of a conserved family of protein serine/threonine phosphatases homologous to Drosophila retinal degeneration C.

Sherman PM, Sun H, Macke JP, Williams J, Smallwood PM, Nathans J
Proc Natl Acad Sci U S A. 1997; 94: 11639-44

The Drosophila retinal degeneration C (rdgC) gene encodes an unusual protein serine/threonine phosphatase in that it contains at least two EF-hand motifs at its carboxy terminus. By a combination of large-scale sequencing of human retina cDNA clones and searches of expressed sequence tag and genomic DNA databases, we have identified two sequences in mammals [Protein Phosphatase with EF-hands-1 and 2 (PPEF-1 and PPEF-2)] and one in Caenorhabditis elegans (PPEF) that closely resemble rdgC. In the adult, PPEF-2 is expressed specifically in retinal rod photoreceptors and the pineal. In the retina, several isoforms of PPEF-2 are predicted to arise from differential splicing. The isoform that most closely resembles rdgC is localized to rod inner segments. Together with the recently described localization of PPEF-1 transcripts to primary somatosensory neurons and inner ear cells in the developing mouse, these data suggest that the PPEF family of protein serine/threonine phosphatases plays a specific and conserved role in diverse sensory neurons.

Protein serine/threonine phosphatases--new avenues for cell regulation.

Shenolikar S
Annu Rev Cell Biol. 1994; 10: 55-86

The Bacillus subtilis regulator protein SpoIIE shares functional and structural similarities with eukaryotic protein phosphatases 2C.

Schroeter R, Schlisio S, Lucet I, Yudkin M, Borriss R
FEMS Microbiol Lett. 1999; 174: 117-23

Dephosphorylation of SpoIIAA-P by SpoIIE is strictly dependent on the presence of the bivalent metal ions Mn2+ or Mg2+. Replacement by Ala of one of the four Asp residues, invariant in all representatives of protein phosphatase 2C, completely abolished the SpoIIE phosphatase activity in vitro, whilst replacement of the Asp residues by another acidic amino acid, Glu, had varying effects on the activities of the resulting mutated proteins. D610E and D795E exhibited some residual activity while D628E and D745E were without enzymatic activity. The results suggest that the functional model in which metal-associated water molecules are involved in the dephosphorylation reaction catalyzed by human protein phosphatase 2C alpha can also be applied to the bacterial protein phosphatase 2C-like protein.

Regulation of gene expression by serine/threonine protein phosphatases.

Schonthal AH
Semin Cancer Biol. 1995; 6: 239-48

The activation of signal transduction pathways by extracellular stimuli, such as growth factors or hormones, ultimately results in changes in the expression of specific genes. The altered pattern of expression eventually determines the resulting cellular consequences, e.g. cell growth, division, or differentiation. It has been well established that the reversible phosphorylation of proteins is a major regulatory mechanism in these processes. However, while much has been learned about the role of kinases, the involvement of protein phosphatases is less clear and has only recently begun to be investigated in more detail. This review will present some of the new findings that demonstrate a crucial regulatory function of serine/threonine protein phosphatases (PPases) in gene regulatory processes.

Analyzing gene expression with the use of serine/threonine phosphatase inhibitors.

Schonthal AH
Methods Mol Biol. 1998; 93: 35-40

Regulatory interactions between the Reg1-Glc7 protein phosphatase and the Snf1 protein kinase.

Sanz P, Alms GR, Haystead TA, Carlson M
Mol Cell Biol. 2000; 20: 1321-8

Protein phosphatase 1, comprising the regulatory subunit Reg1 and the catalytic subunit Glc7, has a role in glucose repression in Saccharomyces cerevisiae. Previous studies showed that Reg1 regulates the Snf1 protein kinase in response to glucose. Here, we explore the functional relationships between Reg1, Glc7, and Snf1. We show that different sequences of Reg1 interact with Glc7 and Snf1. We use a mutant Reg1 altered in the Glc7-binding motif to demonstrate that Reg1 facilitates the return of the activated Snf1 kinase complex to the autoinhibited state by targeting Glc7 to the complex. Genetic evidence indicated that the catalytic activity of Snf1 negatively regulates its interaction with Reg1. We show that Reg1 is phosphorylated in response to glucose limitation and that this phosphorylation requires Snf1; moreover, Reg1 is dephosphorylated by Glc7 when glucose is added. Finally, we show that hexokinase PII (Hxk2) has a role in regulating the phosphorylation state of Reg1, which may account for the effect of Hxk2 on Snf1 function. These findings suggest that the phosphorylation of Reg1 by Snf1 is required for the release of Reg1-Glc7 from the kinase complex and also stimulates the activity of Glc7 in promoting closure of the complex.

Protein phosphatase 2C (PP2C) function in higher plants.

Rodriguez PL
Plant Mol Biol. 1998; 38: 919-27

In the past few years, molecular cloning studies have revealed the primary structure of plant protein serine/threonine phosphatases. Two structurally distinct families, the PP1/PP2A family and the PP2C family, are present in plants as well as in animals. This review will focus on the plant PP2C family of protein phosphatases. Biochemical and molecular genetic studies in Arabidopsis have identified PP2C enzymes as key players in plant signal transduction processes. For instance, the ABI1/ABI2 PP2Cs are central components in abscisic acid (ABA) signal transduction. Arabidopsis mutants containing a single amino acid exchange in ABI1 or ABI2 show a reduced response to ABA. Another member of the PP2C family, kinase-associated protein phosphatase (KAPP), appears to be an important element in some receptor-like kinase (RLK) signalling pathways. Finally, an alfalfa PP2C acts as a negative regulator of a plant mitogen-activated protein kinase (MAPK) pathway. Thus, the plant PP2Cs function as regulators of various signal transduction pathways.

Early inhibition of protein phosphatases preferentially blocks phorbol ester-stimulated mitogenic signalling in melanocytes: increase in specific tyrosine phosphoproteins.

Rieber M, Rieber MS
Biochem Biophys Res Commun. 1993; 192: 483-91

Inhibition of protein phosphatases has been suggested as an alternative mechanism of tumor promotion (H. Fujiki, Mol. Carcinog. 5:91, 1992). We have now used early melanocyte passages dependent on phorbol esters and serum for growth and later passages with partial phorbol ester independence, to investigate the role of protein phosphatases on melanocyte DNA synthesis. Neither okadaic acid, an inhibitor of ser/thr protein phosphatases, nor vanadate, an inhibitor of tyrosine phosphatases, can stimulate basal or serum-stimulated mitogenesis in contrast to phorbol esters. Moreover, both phosphatase inhibitors are able to suppress serum and phorbol ester-stimulated mitogenesis, if added within 4 hours of growth activation. Inhibition of mitogenesis by either inhibitor correlated with an early increase in a common set of tyrosine phosphoproteins, which included a major 33 Kd species. Our data suggest that protein phosphatase inhibitors are growth suppressors and antagonize phorbol ester effects in cells of melanocytic origin, implying an early requirement for protein phosphatase activity during mitogenic signalling in these cells.

Brain protein serine/threonine phosphatases.

Price NE, Mumby MC
Curr Opin Neurobiol. 1999; 9: 336-42

All of the known protein serine/threonine phosphatases are expressed in the brain. These enzymes participate in a variety of signaling pathways that modulate neuronal activity. The multifunctional activity of many serine/threonine phosphatases is achieved through their association with targeting proteins. Identification and analysis of targeting molecules has led to new insights into the functions of protein phosphatases in neuronal signaling. The recent use of transgenic mice has also increased our understanding of the physiological roles of these enzymes in the brain.

Phosphorylated synthetic peptides as tools for studying protein phosphatases.

Pinna LA, Donella-Deana A
Biochim Biophys Acta. 1994; 1222: 415-31

The expression of casein kinase 2alpha' and phosphatase 2A activity.

Perez M, Avila J
Biochim Biophys Acta. 1999; 1449: 150-6

Protein phosphatase 2A (PP2A) activity may be differentially regulated by the expression of proteins containing a related amino acid sequence motif such as the casein kinase 2alpha (CK2alpha) subunit or SV40 small t antigen (SVt). Expression of CK2alpha increases PP2A activity whereas SVt decreases its activity. In this work we have tested for the effect of the expression of a third protein containing a similar motif that could be involved in PP2A regulation, the catalytic casein kinase 2alpha' subunit. Our results show that despite the structural similarity of this protein with the other CK2 catalytic (alpha) subunit, the function of the two subunits with respect to the modulation of PP2A activity is quite different: CK2alpha increases whereas CK2alpha' slightly decreases PP2A activity.

Preliminary characterization of the role of protein serine/threonine phosphatases in the regulation of human lung mast cell function.

Peirce MJ, Cox SE, Munday MR, Peachell PT
Br J Pharmacol. 1997; 120: 239-46

1. Okadaic acid, a cell permeant inhibitor of protein serine/threonine phosphatases (PPs), attenuated the IgE-dependent release of mediators from human lung mast cells (HLMC). The concentration of okadaic acid required to inhibit by 50% (IC50) the IgE-dependent release of histamine was 0.2 microM. Okadaic acid also inhibited the IgE-mediated generation of prostaglandin D2 (PGD2) and sulphopeptidoleukotrienes (sLT) with IC50 values of 0.2 microM and 0.6 microM respectively. 2. The IgE-mediated generation of histamine, PGD2 and sLT was inhibited by okadaic acid and two analogues of okadaic acid, okadaol and okadaone, with the following rank order of activity; okadaic acid > okadaol > okadaone. This order of activity for the inhibition of mediator release parallels the activity of these compounds as inhibitors of isolated PPs. 3. Extracts of HLMC liberated 32P from radiolabelled glycogen phosphorylase and this PP activity was inhibited by the PP inhibitors (all at 3 microM), okadaic acid (73 +/- 4% inhibition, P < 0.0005), okadaol (26 +/- 7% inhibition, P < 0.05) and okadaone (8 +/- 7% inhibition, P = 0.52). The rank order of activity of okadaic acid > okadaol > okadaone parallels the activity of these compounds as inhibitors of isolated PPs. 4. Dephosphorylation of radiolabelled glycogen phosphorylase by extracts of HLMC was inhibited by 15 +/- 3% (P < 0.001) by a low (2 nM) concentration of okadaic acid and by 88 +/- 4% (P < 0.0005) by a higher (5 microM) concentration of okadaic acid. Because 2 nM okadaic acid may act selectively to inhibit PP2A whereas 5 microM okadaic acid inhibits both PP1 and PP2A, these data suggest that both PP1 and PP2A are present in HLMC. 5. Inhibitor 2, a PP1-selective inhibitor, attenuated (71 +/- 3% inhibition, P < 0.05) PP activity in extracts of HLMC suggesting that HLMC contain PP1 and that it may constitute 71% of the phosphorylase PP activity in extracts of HLMC. 6. Radiolabelled casein, a PP2A-restricted substrate, was dephosphorylated by extracts of purified HLMC and this activity was inhibited (81 +/- 8% inhibition, P < 0.005) by 2 nM okadaic acid suggesting that PP2A is resident in HLMC. 7. Collectively, these data suggest that both PP1 and PP2A are resident in HLMC. However, although the data suggest that okadaic acid regulates responses in HLMC by interacting with PPs, it has not been possible to determine whether either PP1 or PP2A or both PPs are involved in the okadaic acid-induced inhibition of mediator release from HLMC.

Protein phosphatases: function and regulation.

Parsadanian HK
Ukr Biokhim Zh. 1994; 66: 38-51

Thousands of proteins are expressed in a typical mammalian cell, of which a third are now thought to contain covalently bound phosphate. About 200 protein kinases and 100 protein phosphatases have already been identified. Whereas the classification, properties and regulation of protein kinases are largely studied, the information about the protein phosphatase is far to be completed. Relatively better is studied a group of serine/treonine protein phosphatases. It has been discovered that besides the control of the key enzymes of metabolic pathways, these phosphatases participate in the regulation of gene transcription and cell division in eukariots. In addition, the identification of tyrosine protein phosphatases points to a novel intracellular signalling pathways, controlling cell-cell communication, cell proliferation and signal transduction of receptors for peptide hormones and growth factors. The recent progress in these families of protein phosphatases is the topic of present review.

Physiologic importance of protein phosphatase inhibitors.

Oliver CJ, Shenolikar S
Front Biosci. 1998; 3: 96172-96172

Reversible protein phosphorylation is an important mode of regulation of cellular processes. While earlier studies focused on protein kinases, it is now apparent that protein phosphatases play an equally integral role in the control of cellular phosphoproteins. This review examines the role played by endogenous inhibitors of three major protein serine/threonine phosphatases, PP1, PP2A and PP2B in the control of cell physiology. The discussion highlights novel paradigms for signal transduction by protein phosphatase inhibitors that provide important avenues for signal amplification, the timing of physiological responses and cross-talk between distinct signal transduction pathways. New evidence also points to genetic abnormalities or altered expression of phosphatase inhibitors as potential mechanisms for human disease.Together, the data emphasize the physiological importance of protein phosphatase inhibitors and establish phosphatase regulation as a key feature of hormone signaling.

Localization of the mouse protein serine/threonine phosphatase 2C beta gene to chromosome 17E 4-5.

Ohnishi M, Nakagawara K, Mori M, Kobayashi T, Kato S, Sasahara Y, Kusuda K, Chida N, Kobayashi T, Yanagawa Y, Hiraga A, Takeuchi T, Tamura S
Genomics. 1996; 32: 134-6

Protein phosphatase 2C (PP2C) is one of four major classes of protein serine/threonine phosphatase and is considered to have a role in signal transduction of stress responses. It has two isotypes, alpha and beta, encoded by different genes. In this study, the mouse PP2C beta gene was mapped by in situ hybridization to chromosome 17E 4-5.

Characterization of PrpC from Bacillus subtilis, a member of the PPM phosphatase family.

Obuchowski M, Madec E, Delattre D, Boel G, Iwanicki A, Foulger D, Seror SJ
J Bacteriol. 2000; 182: 5634-8

We cloned the yloO gene and purified a His-tagged form of its product, the putative protein phosphatase YloO, which we now designate PrpC. This closely resembles the human protein phosphatase PP2C, a member of the PPM family, in sequence and predicted secondary structure. PrpC has phosphatase activity in vitro against a synthetic substrate, p-nitrophenol phosphate, and endogenous Bacillus subtilis proteins. The prkC and prpC genes are adjacent on the chromosome, and the phosphorylated form of PrkC is a substrate for PrpC. These findings suggest that PrkC and PrpC may function as a couple in vivo.

Alpha4 protein as a common regulator of type 2A-related serine/threonine protein phosphatases.

Nanahoshi M, Tsujishita Y, Tokunaga C, Inui S, Sakaguchi N, Hara K, Yonezawa K
FEBS Lett. 1999; 446: 108-12

The catalytic activity of the C subunit of serine/threonine phosphatase 2A is regulated by the association with A (PR65) and B subunits. It has been reported that the alpha4 protein, a yeast homolog of the Tap42 protein, binds the C subunit of serine/threonine phosphatase 2A and protein phosphatase 2A-related protein phosphatases such as protein phosphatase 4 and protein phosphatase 6. In the present study, we showed that alpha4 binds these three phosphatases and the association of alpha4 reduces the activities of these phosphatases in vitro. In contrast, PR65 binds to the C subunit of serine/threonine phosphatase 2A but not to protein phosphatase 4 and protein phosphatase 6. These results suggest that the alpha4 protein is a common regulator of the C subunit of serine/threonine phosphatase 2A and protein phosphatase 2A-related protein phosphatases.

Protein serine/threonine phosphatases as binding proteins for okadaic acid.

Nagao M, Shima H, Nakayasu M, Sugimura T
Mutat Res. 1995; 333: 173-9

Recently, many potent inhibitors of protein serine/threonine phosphatases (PPs) have been found. Some of them have proven to be tumor promoters in mouse skin two-step carcinogenesis and rat liver medium-term tests. Among these inhibitors, okadaic acid (OA) selectively inhibits PP2A, and its use has therefore been proposed to facilitate analysis of biological roles of this phosphatase. OA shows bimodal effects on in vitro transformation and, in addition to such epigenetic changes, also induces marked genetic changes. OA treatment for more than 1 week flattened NIH 3T3 transformants irreversibly, with loss of the transfected genes. It is also known to induce diphtheria toxin-resistant mutations in Chinese hamster lung cells and sister chromatid exchanges (SCEs) in Chinese hamster ovary cells and human lymphocytes. To analyze roles of protein phosphatases in gene stability, we isolated OA-resistant mutants. They were proven to have a mutation in the PP2A alpha catalytic subunit, in which cysteine 269 had been substituted for glycine; and it was demonstrated that this region interacts with OA. The recombinant mutant protein was 4 approximately 9-fold more resistant to OA than the wild type. Although the OA resistant mutants of CHO cells expressed high levels of P-glycoprotein, inhibition of PP2A itself was suggested to lead to SCE induction. However, the number of molecular species of PP which are known to be sensitive to OA continues to increase, and we have isolated cDNA for a novel type of OA sensitive PP. Our studies indicate that the fact that the roles of PP2A cannot be elucidated using only OA is of crucial importance.

Characterization of a Hank's type serine/threonine kinase and serine/threonine phosphoprotein phosphatase in Pseudomonas aeruginosa.

Mukhopadhyay S, Kapatral V, Xu W, Chakrabarty AM
J Bacteriol. 1999; 181: 6615-22

Pseudomonas aeruginosa is an opportunistic pathogen that causes infections in eye, urinary tract, burn, and immunocompromised patients. We have cloned and characterized a serine/threonine (Ser/Thr) kinase and its cognate phosphoprotein phosphatase. By using oligonucleotides from the conserved regions of Ser/Thr kinases of mycobacteria, an 800-bp probe was used to screen P. aeruginosa PAO1 genomic library. A 20-kb cosmid clone was isolated, from which a 4.5-kb DNA with two open reading frames (ORFs) were subcloned. ORF1 was shown to encode Ser/Thr phosphatase (Stp1), which belongs to the PP2C family of phosphatases. Overlapping with the stp1 ORF, an ORF encoding Hank's type Ser/Thr kinase was identified. Both ORFs were cloned in pGEX-4T1 and expressed in Escherichia coli. The overexpressed proteins were purified by glutathione-Sepharose 4B affinity chromatography and were biochemically characterized. The Stk1 kinase is 39 kDa and undergoes autophosphorylation and can phosphorylate eukaryotic histone H1. A site-directed Stk1 (K86A) mutant was shown to be incapable of autophosphorylation. A two-dimensional phosphoamino acid analysis of Stk1 revealed strong phosphorylation at a threonine residue and weak phosphorylation at a serine residue. The Stp1 phosphatase is 27 kDa and is an Mn(2+)-, but not a Ca(2+)- or a Mg(2+)-, dependent Ser/Thr phosphatase. Its activity is inhibited by EDTA and NaF, but not by okadaic acid, and is similar to that of PP2C phosphatase.

Purification of type 1 protein (serine/threonine) phosphatases by microcystin-Sepharose affinity chromatography.

Moorhead G, MacKintosh RW, Morrice N, Gallagher T, MacKintosh C
FEBS Lett. 1994; 356: 46-50

A microcystin (MC)-Sepharose column was prepared by addition of 2-aminoethanethiol to the alpha, beta-unsaturated carbonyl of the N-methyldehydroalanine residue of MC-LR, followed by reaction of the introduced amino group with N-hydroxysuccinimide-activated CH-Sepharose. The MC-Sepharose bound protein phosphatase-1 (PP1) with high capacity and purified human PP1 gamma in one step from E. coli extracts. It was also used to purify forms of PP1 bound to myofibrils from skeletal muscle. Two of these comprised PP1 complexed to N-terminal fragments of the M-subunit which enhance its myosin phosphatase activity, while the third comprised PP1 and an N-terminal fragment of the glycogen-binding (G)-subunit.

Human cytomegalovirus carries serine/threonine protein phosphatases PP1 and a host-cell derived PP2A.

Michelson S, Turowski P, Picard L, Goris J, Landini MP, Topilko A, Hemmings B, Bessia C, Garcia A, Virelizier JL
J Virol. 1996; 70: 1415-23

Human cytomegalovirus (CMV), a herpesvirus, is an important cause of morbidity and mortality in immunocompromised patients. When studying hyper-immediate-early events after contact between CMV virions and the cell membrane, we observed a hypophosphorylation of cellular proteins within 10 min. This can be explained in part by our finding that purified CMV contains serine/threonine protein phosphatase activities. Biochemical analyses indicate that this protein phosphatase activity has all characteristics of type 1 and 2A protein phosphatases (PP1 and PP2A). Specifically, PP1 accounts for approximately 30% and PP2A accounts for the remaining 70% of the phosphorylase phosphatase activity found. CMV produced in astrocytoma cells stably expressing an amino-terminally tagged PP2A catalytic subunit contained tagged enzyme, thus demonstrating the cellular origin of CMV-associated PP2A. PP2A is specifically found inside the virus, associated with the nucleocapsid fraction. Western blot (immunoblot) analysis of purified virus revealed the presence of the catalytic subunits of PP2A and PP1. Furthermore, the catalytic subunit of PP2A appears to be complexed to the regulatory subunits PR65 and PR55, which is also the most abundant configuration of this enzyme found in the host cells. Incubation of virus with okadaic acid before contact of CMV with cells prevented hypophosphorylation of cellular proteins, thus demonstrating the role of CMV-associated phosphatases in this phenomenon. CMV can thus transport an active enzyme from one cell to another.

Primary structure analysis proves that protein phosphatases 2C1 and 2C2 are isozymes.

McGowan CH, Campbell DG, Cohen P
Biochim Biophys Acta. 1987; 930: 279-82

The two recently discovered forms of protein phosphatase 2C, termed 2C1 and 2C2, were digested with CNBr or trypsin, and several peptides corresponding to two regions of the protein were sequenced. These studies revealed close homology between the two enzymes with 49 identities over the 62 residues that could be compared directly. The results establish that protein phosphatases 2C1 and 2C2 are the products of different genes. The C-terminus of protein phosphatase 2C2 has also been identified.

Molecular cloning and functional expression of a protein-serine/threonine phosphatase from the hyperthermophilic archaeon Pyrodictium abyssi TAG11.

Mai B, Frey G, Swanson RV, Mathur EJ, Stetter KO
J Bacteriol. 1998; 180: 4030-5

An open reading frame coding for a putative protein-serine/threonine phosphatase was identified in the hyperthermophilic archaeon Pyrodictium abyssi TAG11 and named Py-PP1. Py-PP1 was expressed in Escherichia coli, purified from inclusion bodies, and biochemically characterized. The phosphatase gene is part of an operon which may provide, for the first time, insight into a physiological role for archaeal protein phosphatases in vivo.

Characterization of protein serine/threonine phosphatases in rat pancreas and development of an endogenous substrate-specific phosphatase assay.

Lutz MP, Pinon DI, Miller LJ
Pancreas. 1994; 9: 418-24

Protein phosphatases have recently been recognized to represent an important, independently regulated portion of cellular signaling cascades. Although reversible phosphorylation of multiple pancreatic proteins has been described, suggesting a role for these enzymes, little is known about the characteristics of protein phosphatases in this organ. In this work, we have characterized and quantified the serine/threonine phosphatases present in pancreatic cytosol and plasma membranes. Using a sensitive and specific in vitro assay with standard substrates (phosphorylase a and phosphocasein), the predominant enzymes represented protein phosphatase 2A in cytosol and protein phosphatase 1 in plasma membranes, with both compartments having substantial amounts of both of these enzymes. Both compartments also had protein phosphatase 2B activity, whereas protein phosphatase 2C was only measurable in the plasma membrane fraction. Further, a novel assay was developed and validated in which the action of an endogenous protein phosphatase on a specific cellular phosphoprotein was studied. For this, we utilized as substrate the cholecystokinin receptor which had been phosphorylated in response to agonist stimulation of the intact acinar cell. This type of assay will be key for the analysis of the mediation and regulation of dephosphorylation events which actually occur in the cell.

Control and activity of type-1 serine/threonine protein phosphatase during the cell cycle.

Ludlow JW, Nelson DA
Semin Cancer Biol. 1995; 6: 195-202

The first part of this discourse will serve as a compendium of selected aspects of PP1 activity and control during the cell cycle. The latter sections will focus on the mammalian cell cycle dependent activity of PP1 on the negative growth regulatory product of the retinoblastoma susceptibility gene, pRB. The purpose here is to suggest one possible mechanism to explain how this phosphatase can effect cell cycle progression. An argument is made for PP1 playing an indirect role in regulating cell cycle progression by modulating the growth suppressive activity of pRB.

ABI1 of Arabidopsis is a protein serine/threonine phosphatase highly regulated by the proton and magnesium ion concentration.

Leube MP, Grill E, Amrhein N
FEBS Lett. 1998; 424: 100-4

The plant hormone abscisic acid (ABA) mediates various responses such as stomatal closure, maintenance of seed dormancy, and inhibition of plant growth. All three responses are regulated by the ABI1 gene product. The ABI1 protein (ABI1p) has been characterized as a protein serine/threonine phosphatase of type 2C that is highly affected in its activity by changes in the proton and magnesium ion concentrations. In the ABA-insensitive mutant abi1 of Arabidopsis thaliana a single amino acid exchange in the primary structure results in both a dominant insensitive phenotype and a strongly reduced protein phosphatase activity in vitro by possibly impairing metal ion coordination.

Isolation and cloning of a protein-serine/threonine phosphatase from an archaeon.

Leng J, Cameron AJ, Buckel S, Kennelly PJ
J Bacteriol. 1995; 177: 6510-7

A divalent metal ion-stimulated protein-serine/threonine phosphatase, PP1-arch, was purified approximately 1,000-fold from the extreme acidothermophilic archaeon Sulfolobus solfataricus (ATCC 35091). Purified preparations contained 40 to 70% of total protein as PP1-arch, as determined by assay-ing sodium dodecyl sulfate-polyacrylamide gels for protein phosphatase activity. The first 25 amino acids of the protein's sequence were identified, as well as an internal sequence spanning some 20 amino acids. Using this information, we cloned the gene for PP1-arch via the application of PCR and conventional cloning techniques. The gene for PP1-arch predicted a protein of 293 amino acids that bore striking resemblance to the members of the major family of protein-serine/threonine phosphatases from members of the domain Eucarya, the PP1/2A/2B superfamily. The core of the protein, spanning residues 4 to 275, possessed 29 to 31% identity with these eucaryal protein phosphatases. Of the 42 residues found to be absolutely conserved among the known eucaryal members of the PP1/2A/2B superfamily, 33 were present in PP1-arch. If highly conservative substitutions are included, this total reached 37. The great degree of sequence conservation between molecules from two distinct phylogenetic domains implies that the members of this enzyme superfamily had evolved as specialized, dedicated protein phosphatases prior to the divergence of members of the Archaea and Eucarya from one another.

A specific protein carboxyl methylesterase that demethylates phosphoprotein phosphatase 2A in bovine brain.

Lee J, Chen Y, Tolstykh T, Stock J
Proc Natl Acad Sci U S A. 1996; 93: 6043-7

Phosphoprotein phosphatase 2A (PP2A) is one of the four major protein serine/threonine phosphatases found in all eukaryotic cells. We have shown that the 36-kDa catalytic subunit of PP2A is carboxyl methylated in eukaryotic cells, and we have previously identified and purified a novel methyltransferase (MTase) that is responsible for this modification. Here, we describe a novel protein carboxyl methyl-esterase (MEase) from bovine brain that demethylates PP2A. The enzyme has been purified to homogeneity as a monomeric 46-kDa soluble protein. The MEase is highly specific for PP2A. It does not catalyze the demethylation of other protein or peptide methylesters. Moreover, MEase activity is dramatically inhibited by nanomolar concentrations of okadaic acid, a specific inhibitor of PP2A. From these results, we conclude that PP2A methylation is controlled by two specific enzymes, a MTase and a MEase. Since PP2A methylation is highly conserved in eukaryotes ranging from human to yeast, it is likely that this system plays an important role in phosphatase regulation.

Phosphorylase phosphatase: new horizons for an old enzyme.

Lee EY, Zhang L, Zhao S, Wei Q, Zhang J, Qi ZQ, Belmonte ER
Front Biosci. 1999; 4: 27085-27085

Protein phosphatase-1, originally studied as phosphorylase phosphatase, is one of the major ser/thr protein phosphatases. It has a long history and a complex enzymology. It consists of a catalytic subunit of 37 kDa, which is bound to a number of different regulatory or targeting subunits. These are believed to restrict its activity to its immediate microenvironment and thus define its specificity, as well as acting to regulate phosphatase activity. The existence of multiple protein phosphatase-1 binding proteins provides the mechanism whereby phosphatase-1 activity can be involved in a diverse range of cellular functions, and reflects a novel strategy for its evolutionary development.

Viral protein kinases and protein phosphatases.

Leader DP
Pharmacol Ther. 1993; 59: 343-89

Certain large DNA viruses (e.g. herpesviruses and poxviruses) encode proteins related to cellular protein-serine/threonine kinases, and Hepatitis B virus and vesicular stomatitis virus may encode structurally different protein kinases. Other viruses activate cellular protein kinases, e.g. interferon-induced eukaryotic initiation factor-2 kinase, growth factor-induced kinases and protein kinases that regulate mitosis. Protein phosphatases are encoded by vaccinia virus and bacteriophage lambda and must also play a role in viral infection--as do cellular protein phosphatases. The functions of many of these viral enzymes remain to be determined, but they represent possible new targets for anti-viral therapy.

Isoform-specific phosphorylation of fission yeast type 2C protein phosphatase.

Kobayashi T, Sadaie M, Ohnishi M, Wang H, Ikeda S, Hanada M, Yanagawa Y, Nakajima T, Tamura S
Biochem Biophys Res Commun. 1998; 251: 296-300

Protein phosphatase 2C (PP2C) is one of the four major protein serine/threonine phosphatases of eukaryotes and is implicated in the regulation of various cellular functions. With the goal of elucidating the mechanism responsible for regulating PP2C functions, we investigated the significance of phosphorylation of fission yeast Ptc1, Ptc2, and Ptc3, the yeast orthologs of mammalian PP2C. Both Ptc2 and Ptc3 but not Ptc1 were phosphorylated stoichiometrically by casein kinase II on serine residues at their carboxy-terminal regions. Mutational analysis of Ptc2 and Ptc3 revealed that serine residues of the conserved sequence (Ser-X-Ser-X-X-Glu/Asp) of these proteins were the phosphorylation sites. Interestingly, the activities of Ptc2 and Ptc3 were decreased 25 +/- 7.5% and increased 55 +/- 3.7%, respectively, by phosphorylation. In addition, the same site(s) of Ptc2 was phosphorylated when the protein was expressed in fission yeast cells. These results suggest that phosphorylation of PP2C plays important physiological roles in fission yeast cells.

Process formation of podocytes: morphogenetic activity of microtubules and regulation by protein serine/threonine phosphatase PP2A.

Kobayashi N, Reiser J, Schwarz K, Sakai T, Kriz W, Mundel P
Histochem Cell Biol. 2001; 115: 255-66

Podocytes possess major processes containing microtubules (MTs) and intermediate filaments and foot processes containing actin filaments (AFs) as core cytoskeletal elements. Although the importance of these cytoskeletal elements for maintaining podocyte processes was previously shown, so far no data are available concerning the developmental regulation of podocyte process formation. A conditionally immortalized mouse podocyte cell line, which can be induced to develop processes similar to those found in vivo, was treated with various reagents to disrupt cytoskeletal elements or to inhibit protein phosphatases. MTs colocalized with vimentin intermediate filaments but not with AFs. After AF disassembly, major processes were maintained, whereas after depolymerization of MTs, podocytes lost their processes, rounded up, and maintained only actin-based peripheral projections. Suppression of MT elongation by nanomolar vinblastine or inhibition of serine/threonine phosphatase PP2A with okadaic acid abolished process formation. PP2A was expressed in undifferentiated but not in differentiated podocytes. One- and two-dimensional western blot analyses revealed a dose-dependent increase in serine/threonine phosphorylation after okadaic acid treatment. Hence, morphogenetic activity of MTs induces podocyte process formation via serine/threonine protein dephosphorylation by PP2A. These results may open new avenues for understanding the signaling mechanism underlying podocyte cytoskeleton alterations during development and in glomerular diseases.

[Structural biology of PP1]

Kikuchi K
Tanpakushitsu Kakusan Koso. 1998; 43: 937-44

The role of protein phosphatases in the regulation of mitogen and stress-activated protein kinases.

Keyse SM
Free Radic Res. 1999; 31: 341-9

It is now established that a family of dual-specificity protein phosphatases are able to interact with mitogen and stress-activated protein kinases in a highly specific manner to differentially regulate these enzymes in mammalian cells. A role for these proteins in negative feedback regulation of MAP kinase activity is also supported by genetic and biochemical studies in yeasts and Drosophila. More recently it has become clear that other classes of protein phosphatase also play key roles in the regulated dephosphorylation of MAP kinases, including tyrosine-specific protein phosphatases and serine/threonine protein phosphatases. It is likely that a complex balance between upstream activators and these different classes of MAP kinase specific phosphatase are responsible for determining, at least in part, the magnitude and duration of MAP kinase activation and hence the physiological outcome of signalling.

Life among the primitives: protein O-phosphatases in prokaryotes.

Kennelly PJ, Potts M
Front Biosci. 1999; 4: 37285-37285

Prokaryotes contain at least five distinct families of protein O-phosphatases, including AceK, the chimeric isocitrate dehydrogenase kinase/phosphatase, and four protein phosphatase families first identified and characterized in Eukaryotes. The latter consist of the PPP and PPM families of protein-serine/threonine phosphatases, and the low molecular weight and conventional families of protein-tyrosine phosphatases. Prokaryotic protein O-phosphatases participate in the regulation of metabolic processes and the transduction of environmental signals. Certain pathogenic bacteria employ protein-tyrosine phosphatases as virulence factors, injecting them into host cells where they enzymatically perturb the phosphorylation state of proteins therein. While our understanding of protein O-phosphorylation events in Prokaryotes only now is emerging from its infancy, their phylogenetic diversity and malleability to genetic manipulation render these "simple'" organisms powerful vehicles for answering fundamental questions concerning the origins and evolution of this key biological regulatory mechanism.

Prokaryotic protein-serine/threonine phosphatases.

Kennelly PJ
Methods Mol Biol. 1998; 93: 1-21

Protein phosphatases: structures and implications.

Jia Z
Biochem Cell Biol. 1997; 75: 17-26

Protein phosphatases are signal transducing enzymes that dephosphorylate intracellular proteins phosphorylated on serine, threonine, and tyrosine residues. This brief review surveys recently determined structures of members of the protein tyrosine phosphatase and protein serine/threonine phosphatase families. In each family, characteristic and distinct structures confer different enzymatic mechanisms in catalyzing dephosphorylation reactions. Within each family, however, there exists remarkable similarity in active-site conformation and catalytic mechanism despite, in some cases, little or no sequence homology. The crystal structures also provide the basis for understanding the substrate binding specificity, inhibition by physiologically relevant compounds, and regulation of the protein phosphatases.

Isolation and characterization of par1(+) and par2(+): two Schizosaccharomyces pombe genes encoding B' subunits of protein phosphatase 2A.

Jiang W, Hallberg RL
Genetics. 2000; 154: 1025-38

Protein phosphatase 2A (PP2A) is one of the major serine/threonine phosphatases found in eukaryotic cells. We cloned two genes, par1(+) and par2(+), encoding distinct B' subunits of PP2A in fission yeast. They share 52% identity at the amino acid sequence level. Neither gene is essential but together they are required for normal septum positioning and cytokinesis, for growth at both high and low temperature, and for growth under a number of stressful conditions. Immunofluorescence microscopy revealed that Par2p has a cell-cycle-related localization pattern, being localized at cell ends during interphase and forming a medial ring in cells that are undergoing septation and cytokinesis. Our analyses also indicate that Par1p is more abundant than Par2p in the cell. Cross-organism studies showed that both par1(+) and par2(+) could complement the rts1Delta allele in Saccharomyces cerevisiae, albeit to different extents, in spite of the fact that neither contains a serine/threonine-rich N-terminal domain like that found in the S. cerevisiae homolog Rts1p. Thus, while Schizosaccharomyces pombe is more similar to higher eukaryotes with respect to its complement of B'-encoding genes, the function of those proteins is conserved relative to that of Rts1p.

Predicted secondary and supersecondary structure for the serine-threonine-specific protein phosphatase family.

Jenny TF, Gerloff DL, Cohen MA, Benner SA
Proteins. 1995; 21: 1-10

A bona fide consensus prediction for the secondary and supersecondary structure of the serine-threonine specific protein phosphatases is presented. The prediction includes assignments of active site segments, an internal helix, and a region of possible 3(10) helical structure. An experimental structure for a member of this family of proteins should appear shortly, allowing this prediction to be evaluated.

Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling.

Janssens V, Goris J
Biochem J. 2001; 353: 417-39

Protein phosphatase 2A (PP2A) comprises a family of serine/threonine phosphatases, minimally containing a well conserved catalytic subunit, the activity of which is highly regulated. Regulation is accomplished mainly by members of a family of regulatory subunits, which determine the substrate specificity, (sub)cellular localization and catalytic activity of the PP2A holoenzymes. Moreover, the catalytic subunit is subject to two types of post-translational modification, phosphorylation and methylation, which are also thought to be important regulatory devices. The regulatory ability of PTPA (PTPase activator), originally identified as a protein stimulating the phosphotyrosine phosphatase activity of PP2A, will also be discussed, alongside the other regulatory inputs. The use of specific PP2A inhibitors and molecular genetics in yeast, Drosophila and mice has revealed roles for PP2A in cell cycle regulation, cell morphology and development. PP2A also plays a prominent role in the regulation of specific signal transduction cascades, as witnessed by its presence in a number of macromolecular signalling modules, where it is often found in association with other phosphatases and kinases. Additionally, PP2A interacts with a substantial number of other cellular and viral proteins, which are PP2A substrates, target PP2A to different subcellular compartments or affect enzyme activity. Finally, the de-regulation of PP2A in some specific pathologies will be touched upon.

Anti-phosphatases take the stage.

Hunter T
Nat Genet. 1998; 18: 303-5

Photoreceptor serine/threonine protein phosphatase type 7: cloning, expression, and functional analysis.

Huang X, Swingle MR, Honkanen RE
Methods Enzymol. 2000; 315: 579-93

Cantharidin, another natural toxin that inhibits the activity of serine/threonine protein phosphatases types 1 and 2A.

Honkanen RE
FEBS Lett. 1993; 330: 283-6

Cantharidin, a natural toxicant of blister beetles, is a strong inhibitor of protein phosphatases types 1 (PP1) and 2A (PP2A). Like okadaic acid, cantharidin inhibits the activity of the purified catalytic subunit of PP2A (IC50 = 0.16 microM) at a lower concentration than that of PP1 (IC50 = 1.7 microM) and only inhibits the activity of protein phosphatase type 2B (PP2B) at high concentrations. Dose-inhibition studies conducted with whole cell homogenates indicate that cantharidin also inhibits the native forms of these enzymes. Thus, cantharidin, which is economical and readily available, may be useful as an additional probe for studying the functions of serine/threonine protein phosphatases.

Effects of serine/threonine protein phosphatases on ion channels in excitable membranes.

Herzig S, Neumann J
Physiol Rev. 2000; 80: 173-210

This review deals with the influence of serine/threonine-specific protein phosphatases on the function of ion channels in the plasma membrane of excitable tissues. Particular focus is given to developments of the past decade. Most of the electrophysiological experiments have been performed with protein phosphatase inhibitors. Therefore, a synopsis is required incorporating issues from biochemistry, pharmacology, and electrophysiology. First, we summarize the structural and biochemical properties of protein phosphatase (types 1, 2A, 2B, 2C, and 3-7) catalytic subunits and their regulatory subunits. Then the available pharmacological tools (protein inhibitors, nonprotein inhibitors, and activators) are introduced. The use of these inhibitors is discussed based on their biochemical selectivity and a number of methodological caveats. The next section reviews the effects of these tools on various classes of ion channels (i.e., voltage-gated Ca(2+) and Na(+) channels, various K(+) channels, ligand-gated channels, and anion channels). We delineate in which cases a direct interaction between a protein phosphatase and a given channel has been proven and where a more complex regulation is likely involved. Finally, we present ideas for future research and possible pathophysiological implications.

FIN13, a novel growth factor-inducible serine-threonine phosphatase which can inhibit cell cycle progression.

Guthridge MA, Bellosta P, Tavoloni N, Basilico C
Mol Cell Biol. 1997; 17: 5485-98

We have identified a novel type 2C serine-threonine phosphatase, FIN13, whose expression is induced by fibroblast growth factor 4 and serum in late G1 phase. The protein encoded by FIN13 cDNA includes N- and C-terminal domains with significant homologies to type 2C phosphatases, a domain homologous to collagen, and an acidic domain. FIN13 expression predominates in proliferating tissues. Bacterially expressed FIN13 and FIN13 expressed in mammalian cells exhibit serine-threonine phosphatase activity, which requires Mn2+ and is insensitive to inhibition by okadaic acid. FIN13 is localized in the nuclei of transiently transfected cells. Cotransfection of FIN13-expressing plasmids with a plasmid that expresses the neomycin resistance gene inhibits the growth of drug-resistant colonies in NIH 3T3, HeLa and Rat-1 cells. In transiently transfected cells, FIN13 inhibits DNA synthesis and results in the accumulation of cells in G1 and early S phases. Similarly, the induction of expression of FIN13 under the control of a tetracycline-regulated promoter in NIH 3T3 cells leads to growth inhibition, with accumulation of cells in G1 and early S phases. Thus, overexpression and/or unregulated expression of FIN13 inhibits cell cycle progression, indicating that the physiological role of this phosphatase may be that of regulating the orderly progression of cells through the mitotic cycle by dephosphorylating specific substrates which are important for cell proliferation.

The role of protein phosphatase 2A catalytic subunit Calpha in embryogenesis: evidence from sequence analysis and localization studies.

Gotz J, Kues W
Biol Chem. 1999; 380: 1117-20

Protein phosphatase 2A (PP2A) constitutes one of the major families of protein serine/threonine phosphatases found in all eukaryotic cells. PP2A holoenzymes are composed of a catalytic subunit complexed with a structural regulatory subunit of 65 kDa. These core subunits associate with regulatory subunits of various sizes to form different heterotrimers which have been purified and evaluated with regard to substrate specificity. In fully differentiated tissues PP2A expression levels are highest in the brain, however, relatively little is known about expression in the developing embryo. In order to determine the composition of PP2A catalytic subunits in the mouse, cDNAs were cloned and the genomic organization of PP2A Calpha was determined. By a gene targeting approach in the mouse, we have previously shown that the absence of the major catalytic subunit of PP2A, Calpha, resulted in embryonic lethality around embryonic day E6.5. No mesoderm was formed which implied that PP2A plays a crucial role in gastrulation. Here, we extended our studies and analyzed wildtype embryos for Calpha expression at subsequent stages of development. After gastrulation is completed, we find high expression of Calpha restricted to the neural folds, which suggests that PP2A plays an additional pivotal role in neurulation.

Protein phosphatases are pest containing proteins.

Gomes AV, Barnes JA
Biochem Mol Biol Int. 1997; 41: 65-73

Protein phosphatases are required for removing phosphoryl groups in proteins involved in many physiological processes. Investigation of these enzymes for regions rich in proline (P), glutamic acid (E), serine (S) and threonine (T), called PEST regions showed that greater than 85% of the phosphatases investigated contained these regions. These regions are believed to be signals for degradation and could possibly serve as regulators of the intracellular localization and catalytic activity via limited proteolysis or as conditional signals for rapid degradation of these proteins by the ATP/ubiquitin-dependent and/or the ATP non-ubiquitin dependent proteolytic pathway. Many of these phosphatases were also found to contain a pentapeptide sequence biochemically related to the KFERQ motif which targets proteins for lysosomal degradation which suggest that several pathways may exist for the degradation of protein phosphatases.

Three-dimensional structure of the catalytic subunit of protein serine/threonine phosphatase-1.

Goldberg J, Huang HB, Kwon YG, Greengard P, Nairn AC, Kuriyan J
Nature. 1995; 376: 745-53

The crystal structure of mammalian protein phosphatase-1, complexed with the toxin microcystin and determined at 2.1 A resolution, reveals that it is a metalloenzyme unrelated in architecture to the tyrosine phosphatases. Two metal ions are positioned by a central beta-alpha-beta-alpha-beta scaffold at the active site, from which emanate three surface grooves that are potential binding sites for substrates and inhibitors. The carboxy terminus is positioned at the end of one of the grooves such that regulatory sequences following the domain might modulate function. The fold of the catalytic domain is expected to be closely preserved in protein phosphatases 2A and 2B (calcineurin).

Inhibition of protein serine/threonine phosphatases by fumonisin B1, a mycotoxin.

Fukuda H, Shima H, Vesonder RF, Tokuda H, Nishino H, Katoh S, Tamura S, Sugimura T, Nagao M
Biochem Biophys Res Commun. 1996; 220: 160-5

Fumonisin B1 (FB1), a mycotoxin produced by the fungus Fusarium moniliforme, which is a common contaminant of corn, is suspected to be a cause of human esophageal cancer. FB1 is hepatotoxic and hepatocarcinogenic in rats, and although the mechanisms involved have not been clarified, the latter is associated with a weak initiating activity. The effects of FB1 on the activity of protein serine/threonine phosphatases (PPs) (PP1, PP2A, PP2B, PP2C and PP5/T/K/H) were investigated in the present study. Inhibition of dephosphorylation was noted for all five PPs with IC50 values of 80 microM-3000 microM. Among the five PPs examined, PP5 was most sensitive with an IC50 of 80 microM. This concentration is comparable to that estimated to be reached in the rat body by feeding FB1 to obtain hepatic tumors. Inhibition of PP5 could thus play important roles in the toxicity and carcinogenic action of FB1.

Importance of the C28-C38 hydrophobic domain of okadaic acid for potent inhibition of protein serine-threonine phosphatases 1 and 2A.

Frydrychowski VA, Urbanek RA, Dounay AB, Forsyth CJ
Bioorg Med Chem Lett. 2001; 11: 647-9

Okadaic acid is a potent inhibitor of select serine/threonine protein phosphatases. The importance of the C28-C38 hydrophobic domain of okadaic acid for inhibition of PP1 and PP2A was investigated. The hydrophobic domain is required but not sufficient for potent inhibition, and it also contributes to differential inhibition between PP1 and PP2A.

Kinetic analysis of human serine/threonine protein phosphatase 2Calpha.

Fjeld CC, Denu JM
J Biol Chem. 1999; 274: 20336-43

The PPM family of Ser/Thr protein phosphatases have recently been shown to down-regulate the stress response pathways in eukaryotes. Within the stress pathway, key signaling kinases, which are activated by protein phosphorylation, have been proposed as the in vivo substrates of PP2C, the prototypical member of the PPM family. Although it is known that these phosphatases require metal cations for activity, the molecular details of these important reactions have not been established. Therefore, here we report a detailed biochemical study to elucidate the kinetic and chemical mechanism of PP2Calpha. Steady-state kinetic and product inhibition studies revealed that PP2Calpha employs an ordered sequential mechanism, where the metal cations bind before phosphorylated substrate, and phosphate is the last product to be released. The metal-dependent activity of PP2C (as reflected in kcat and kcat/Km), indicated that Fe2+ was 1000-fold better than Mg2+. The pH rate profiles revealed two ionizations critical for catalytic activity. An enzyme ionization with a pKa value of 7 must be unprotonated for catalysis, and an enzyme ionization with a pKa of 9 must be protonated for substrate binding. Bronsted analysis of substrate leaving group pKa indicated that phosphomonoester hydrolysis is rate-limiting at pH 7. 0, but not at pH 8.5 where a common step independent of the nature of the substrate and alcohol product limits turnover (kcat). Rapid reaction kinetics between phosphomonoester and PP2C yielded exponential "bursts" of product formation, consistent with phosphate release being the slow catalytic step at pH 8.5. Dephosphorylation of synthetic phosphopeptides corresponding to several protein kinases revealed that PP2C displays a strong preference for diphosphorylated peptides in which the phosphorylated residues are in close proximity.

Identification of a type 6 protein ser/thr phosphatase regulated by interleukin-2 stimulation.

Filali M, Li S, Kim HW, Wadzinski B, Kamoun M
J Cell Biochem. 1999; 73: 153-63

We have identified a 36 kD phosphoprotein that forms a complex with spliceosomal small nuclear ribonucleoproteins in lymphocyte extracts. This 36 kD protein is differentially phosphorylated in transformed human lymphoid cell lines and is regulated by IL-2 in peripheral blood T cells. We purified the 36 kD protein from human lymphocytes by employing a combination of immuno-affinity chromatography and preparative two-dimensional gel electrophoresis. Internal amino acid sequence analysis of the purified protein yielded two peptides that had perfect matches with sequences in the human protein serine/threonine phosphatase 6 (PP6). Using degenerate primers corresponding to the peptides, we obtained from a human T lymphocyte cDNA library a DNA fragment whose sequence is homologous to an EST cDNA clone (R05547). The predicted amino acid sequence of this clone showed over 98% sequence identity to human PP6. The identification of an IL-2 regulated type 6 protein serine/threonine phosphatase in lymphocytes was further substantiated by immunoblotting with anti-peptide antibodies. These findings suggest that PP6 is a component of a signaling pathway regulating cell cycle progression in response to IL-2 receptor stimulation.

Structure-based functional motif identifies a potential disulfide oxidoreductase active site in the serine/threonine protein phosphatase-1 subfamily.

Fetrow JS, Siew N, Skolnick J
FASEB J. 1999; 13: 1866-74

In previous work, 3-dimensional descriptors of protein function ('fuzzy functional forms') were used to identify disulfide oxidoreductase active sites in high-resolution protein structures. During this analysis, a potential disulfide oxidoreductase active site in the serine/threonine protein phosphatase-1 (PP1) crystal structure was discovered. In PP1, the potential redox active site is located in close proximity to the phosphatase active site. This result is interesting in view of literature suggesting that serine/threonine phosphatases could be subject to redox control mechanisms within the cell; however, the actual source of this control is unknown. Additional analysis presented here shows that the putative oxidoreductase active site is highly conserved in the serine/threonine phosphatase-1 subfamily, but not in the serine/threonine phosphatase-2A or -2B subfamilies. These results demonstrate the significant advantages of using structure-based motifs for protein functional site identification. First, a putative disulfide oxidoreductase active site has been identified in serine-threonine phosphatases using a descriptor built from the glutaredoxin/thioredoxin family, proteins that have no apparent evolutionary relationship whatsoever to the PP1 proteins. Second, the proximity of the putative disulfide oxidoreductase active site to the phosphatase active site provides evidence toward a regulatory control mechanism. No sequence-based method could provide either piece of information.

Serine/threonine protein phosphatases in the control of cell function.

Depaoli-Roach AA, Park IK, Cerovsky V, Csortos C, Durbin SD, Kuntz MJ, Sitikov A, Tang PM, Verin A, Zolnierowicz S
Adv Enzyme Regul. 1994; 34: 199-224

Reversible protein phosphorylation is a fundamental mechanism by which many biological functions are regulated. Achievement of such control requires the coordinated action of the interconverting enzymes, the protein kinases and protein phosphatases. By comparison with protein kinases, a limited number of protein phosphatase catalytic subunits are present in the cell, which raises the question of how such a small number of dephosphorylating enzymes can counterbalance the action of the more numerous protein kinases. In mammalian cells, four major classes of Ser/Thr-specific phosphatase catalytic subunits have been identified, comprising two distinct gene families. The high degree of homology among members of the same family, PP1, PP2A and PP2B, and the high degree of evolutionary conservation between organisms as divergent as mammals and yeast, implies that these enzymes are involved in fundamental cell functions. Type 1 enzymes appear to acquire specificity by association with targeting regulatory subunits which direct the enzymes to specific cellular compartments, confer substrate specificity and control enzyme activity. In spite of the progress made in determining the structure of the PP2A subunits, very little is known about the control of this activity and about substrate selection. Recent studies have unravelled a significant number of regulatory subunits. The potential existence of five distinct B or B-related polypeptides, some of which are present in multiple isoforms, two A and two C subunit isoforms, raises the possibility that a combinatorial association could generate a large number of specific PP2A forms with different substrate specificity and/or cellular localization. Moreover, biochemical, biological and genetic studies all concur in suggesting that the regulatory subunits may play an important role in determining the properties of the Ser/Thr protein phosphatases and hence their physiological functions.

Molecular mechanisms underlying inhibition of protein phosphatases by marine toxins.

Dawson JF, Holmes CF
Front Biosci. 1999; 4: 64658-64658

The protein serine/threonine phosphatases constitute a unique class of enzymes that are critical regulatory enzymes as they must counteract the activities of thousands of protein kinases in human cells. Uncontrolled inhibition of phosphatase activity by toxic inhibitors can lead to widespread catastrophic effects. Over the past decade, a number of natural product toxins have been identified which specifically and potently inhibit protein phosphatase-1 and -2A. Among these are the cyanobacteria-derived cyclic heptapeptide microcystin-LR and the polyether fatty acid okadaic acid from dinoflagellate sources. The molecular mechanism of the potent inhibition of protein phosphatase-1 by these toxins is becoming clear through insights gathered from diverse sources. These include: 1. Comparison of structural variants of the toxins, 2. Delineating the structural differences between protein phosphatase-1 and -2A accounting for their differing sensitivity to okadaic acid, 3. Determination of the crystal structure of protein phosphatase-1 with microcystin-LR bound and, most recently, 4. Mutagenesis of protein phosphatase-1. Taken together, these data point to a common binding site on protein phosphatase-1 for okadaic acid and microcystin-LR. However, the details of these data suggest that each toxin binds to the common site in a subtly different way, relying on common structural interactions to different degrees. Finally, the insights derived from protein phosphatase-1 may help explain different sensitivities of other protein serine/threonine phosphatases to toxin inhibition due to the high degree of structural conservation among many members of this enzyme family.

Identification of sds21 in fission yeast in an inhibitor-resistant high molecular mass protein phosphatase-1 complex.

Dawson JF, Holmes CF
Biochem Cell Biol. 1999; 77: 551-8

While characterizing the type-1 protein phosphatases sds21 and dis2 in fission yeast (Schizosaccharomyces pombe) a novel high molecular mass protein was identified with serine/threonine phosphatase activity (referred to as PP-R) that was resistant to a panel of characteristic inhibitors of protein phosphatases. Purification of the native sds21 catalytic isoform of protein phosphatase-1 (PP-1) from an S. pombe knockout strain lacking dis2 (deltadis2) resulted predominantly in identification of PP-R. To test the hypothesis that the catalytic activity of PP-R comprised sds21, a parallel purification was performed of PP-1 activity from an S. pombe knockout strain lacking sds21 (deltasds21). Both deltasds21 and deltadis2 strains exhibited similar protein phosphatase activity profiles as determined by DEAE-sepharose, Mono-Q and Superdex gel filtration chromatography. However, the peak of protein phosphatase activity from deltasds21 S. pombe that co-migrated with PP-R from deltadis2 S. pombe exhibited the sensitivity to a panel of inhibitors that was characteristic of a type-1 protein phosphatase. These data suggest that the catalytic subunit of PP-R comprises sds21 and that the resistance to inhibitors may originate from structural differences between dis2 and sds21 isoforms. A key structural feature present in sds21, but lacking in dis2, is a classical phosphorylation consensus sequence surrounding serine-145 of sds21. The previous hypothesis was that PP-1 activity among several lower eukaryotes may be regulated directly by cAMP-dependent protein kinase (PKA) phosphorylation. However, this study demonstrated that recombinant sds21 is not a target for PKA in vitro. The constrained configuration of the putative PKA site on the PP-1 holoenzyme may restrict its ability to be targeted by PKA.

Importance of the beta12-beta13 loop in protein phosphatase-1 catalytic subunit for inhibition by toxins and mammalian protein inhibitors.

Connor JH, Kleeman T, Barik S, Honkanen RE, Shenolikar S
J Biol Chem. 1999; 274: 22366-72

Type-1 protein serine/threonine phosphatases (PP1) are uniquely inhibited by the mammalian proteins, inhibitor-1 (I-1), inhibitor-2 (I-2), and nuclear inhibitor of PP1 (NIPP-1). In addition, several natural compounds inhibit both PP1 and the type-2 phosphatase, PP2A. Deletion of C-terminal sequences that included the beta12-beta13 loop attenuated the inhibition of the resulting PP1alpha catalytic core by I-1, I-2, NIPP-1, and several toxins, including tautomycin, microcystin-LR, calyculin A, and okadaic acid. Substitution of C-terminal sequences from the PP2A catalytic subunit produced a chimeric enzyme, CRHM2, that was inhibited by toxins with dose-response characteristics of PP1 and not PP2A. However, CRHM2 was insensitive to the PP1-specific inhibitors, I-1, I-2, and NIPP-1. The anticancer compound, fostriecin, differed from other phosphatase inhibitors in that it inhibited wild-type PP1alpha, the PP1alpha catalytic core, and CRHM2 with identical IC(50). Binding of wild-type and mutant phosphatases to immobilized microcystin-LR, NIPP-1, and I-2 established that the beta12-beta13 loop was essential for the association of PP1 with toxins and the protein inhibitors. These studies point to the importance of the beta12-beta13 loop structure and conformation for the control of PP1 functions by toxins and endogenous proteins.

Cellular mechanisms regulating protein phosphatase-1. A key functional interaction between inhibitor-2 and the type 1 protein phosphatase catalytic subunit.

Connor JH, Frederick D, Huang Hb, Yang J, Helps NR, Cohen PT, Nairn AC, DePaoli-Roach A, Tatchell K, Shenolikar S
J Biol Chem. 2000; 275: 18670-5

Inhibitor-1 (I-1) and inhibitor-2 (I-2) selectively inhibit type 1 protein serine/threonine phosphatases (PP1). To define the molecular basis for PP1 inhibition by I-1 and I-2 charged-to-alanine substitutions in the Saccharomyces cerevisiae, PP1 catalytic subunit (GLC7), were analyzed. Two PP1 mutants, E53A/E55A and K165A/E166A/K167A, showed reduced sensitivity to I-2 when compared with wild-type PP1. Both mutants were effectively inhibited by I-1. Two-hybrid analysis and coprecipitation or pull-down assays established that wild-type and mutant PP1 catalytic subunits bound I-2 in an identical manner and suggested a role for the mutated amino acids in enzyme inhibition. Inhibition of wild-type and mutant PP1 enzymes by full-length I-2(1-204), I-2(1-114), and I-2(36-204) indicated that the mutant enzymes were impaired in their interaction with the N-terminal 35 amino acids of I-2. Site-directed mutagenesis of amino acids near the N terminus of I-2 and competition for PP1 binding by a synthetic peptide encompassing an I-2 N-terminal sequence suggested that a PP1 domain composed of amino acids Glu-53, Glu-55, Asp-165, Glu-166, and Lys-167 interacts with the N terminus of I-2. This defined a novel regulatory interaction between I-2 and PP1 that determines I-2 potency and perhaps selectivity as a PP1 inhibitor.

Inhibitors of serine/threonine phosphoprotein phosphatases alter circadian properties in Gonyaulax polyedra.

Comolli J, Taylor W, Rehman J, Hastings JW
Plant Physiol. 1996; 111: 285-91

Protein serine/threonine phosphatases were implicated in the regulation of circadian rhythmicity in the marine dinoflagellate Gonyaulax polyedra based on the effects of three inhibitors specific for protein phosphatases 1 and 2A (okadaic acid, calyculin A, and cantharidin). Chronic exposure to okadaic acid resulted in a significant period lengthening, as measured by the bioluminescent glow rhythm, whereas cantharidin and calyculin A caused large phase delays but no persistent effect on period. Short pulses of the phosphatase inhibitors resulted in phase delays that were greatest near subjective dawn. Unlike 6-dimethylaminopurine, a protein kinase inhibitor, okadaic acid, calyculin A, and cantharidin did not block light-induced phase shifts. The inhibitors tested also increased radiolabeled phosphate incorporation into Gonyaulax proteins in vivo and blocked protein phosphatase 1 and 2A activities in Gonyaulax extracts. This study indicates that protein dephosphorylation catalyzed by protein serine/threonine phosphatases is necessary for proper functioning of the circadian system.

Protein serine/threonine phosphatases; an expanding family.

Cohen PT, Brewis ND, Hughes V, Mann DJ
FEBS Lett. 1990; 268: 355-9

Five protein serine/threonine phosphatases (PP) have been identified by cloning cDNA from mammalian and Drosophila libraries. These novel enzymes, which have not yet been detected by the techniques of protein chemistry and enzymology, are termed PPV, PP2Bw, PPX, PPY and PPZ. The complete amino acid sequences of PPX, PPY and PPZ and an almost complete sequence of PPV are presented. In the catalytic domain PPV and PPX are more similar to PP2A (57-69% identity) than PP1 (45-49% identity), while PPY and PPZ are more similar to PP1 (66-68% identity) than PP2A (44% identity). The cDNA for PP2Bw encodes a novel Ca2+/calmodulin-dependent protein phosphatase only 62% identical to PP2B in the catalytic domain. Approaches for determining the cellular functions of these protein phosphatases are discussed.

Important roles for novel protein phosphatases dephosphorylating serine and threonine residues.

Cohen PT
Biochem Soc Trans. 1993; 21: 884-8

Classification of protein-serine/threonine phosphatases: identification and quantitation in cell extracts.

Cohen P
Methods Enzymol. 1991; 201: 389-98

A novel human protein serine/threonine phosphatase, which possesses four tetratricopeptide repeat motifs and localizes to the nucleus.

Chen MX, McPartlin AE, Brown L, Chen YH, Barker HM, Cohen PT
EMBO J. 1994; 13: 4278-90

A novel human protein serine/threonine phosphatase, PP5, and a structurally related phosphatase in Saccharomyces cerevisiae, PPT1, have been identified from their cDNA and gene respectively. Their predicted molecular mass is 58 kDa and they comprise a C-terminal phosphatase catalytic domain and an N-terminal domain, which has four repeats of 34 amino acids, three of which are tandemly arranged. The phosphatase domain possesses all the invariant motifs of the PP1/PP2A/PP2B gene family, but is not closely related to any other known member (< or = 40% identity). Thus PP5 and PPT1 comprise a new subfamily. The repeats in the N-terminal domain are similar to the tetratricopeptide repeat (TPR) motifs which have been found in several proteins that are required for mitosis, transcription and RNA splicing. Bacterially expressed PP5 is able to dephosphorylate serine residues in proteins and is more sensitive than PP1 to the tumour promoter okadaic acid. A 2.3 kb mRNA encoding PP5 is present in all human tissues examined. Investigation of the intracellular distribution of PP5 by immunofluorescence, using two different antibodies raised against the TPR and phosphatase domains, localizes PP5 predominantly to the nucleus. This suggests that, like other nuclear TPR-containing proteins, it may play a role in the regulation of RNA biogenesis and/or mitosis.

Dephosphorylation of human cyclin-dependent kinases by protein phosphatase type 2C alpha and beta 2 isoforms.

Cheng A, Kaldis P, Solomon MJ
J Biol Chem. 2000; 275: 34744-9

We previously reported that the activating phosphorylation on cyclin-dependent kinases in yeast (Cdc28p) and in humans (Cdk2) is removed by type 2C protein phosphatases. In this study, we characterize this PP2C-like activity in HeLa cell extract and determine that it is due to PP2C beta 2, a novel PP2C beta isoform, and to PP2C alpha. PP2C alpha and PP2C beta 2 co-purified with Mg(2+)-dependent Cdk2/Cdk6 phosphatase activity in DEAE-Sepharose, Superdex-200, and Mono Q chromatographies. Moreover, purified recombinant PP2C alpha and PP2C beta 2 proteins efficiently dephosphorylated monomeric Cdk2/Cdk6 in vitro. The dephosphorylation of Cdk2 and Cdk6 by PP2C isoforms was inhibited by the binding of cyclins. We found that the PP2C-like activity in HeLa cell extract, partially purified HeLa PP2C alpha and PP2C beta 2 isoforms, and the recombinant PP2Cs exhibited a comparable substrate preference for a phosphothreonine containing substrate, consistent with the conservation of threonine residues at the site of activating phosphorylation in CDKs.

[Protein phosphatases: structure and function]

Bulanova EG, Budagian VM
Mol Biol (Mosk). 1994; 28: 991-1001

The process of protein and enzyme systems phosphorylation is necessary for cell growth, differentiation and preparation for division and mitosis. The conformation changes of protein as a result of phosphorylation lead to increased enzyme activity and enhanced affinity to substrates. A large group of enzymes--protein kinases--is responsible for phosphorylation process in cell, which are divided into tyrosine- and serine-threonine-kinases depending on their ability to phosphorylate appropriate amino acid residues. In this review has been considered the functional importance and structure of protein phosphatases--enzymes, which are functional antagonists of protein kinases.

Possible protein phosphatase inhibition by bis(hydroxyethyl)sulfide, a hydrolysis product of mustard gas.

Brimfield AA
Toxicol Lett. 1995; 78: 43-8

Recently, the natural vesicant cantharidin was shown to bind exclusively to and inhibit protein phosphatase 2A (PP2A) in mouse tissue extracts (Li and Casida (1992) Proc. Natl. Acad. Sci. USA 89, 11867-11870). To explore the generality of this effect in vesicant action, we measured the protein serine/threonine phosphatase activity in mouse liver cytosol (in the form of the okadaic acid inhibitable increment of p-nitrophenyl phosphate (p-NPP) phosphatase activity) in the presence of aqueous sulfur mustard or its hydrolysis product, bis(hydroxyethyl)sulfide (TDG). Sulfur mustard inhibited p-NPP hydrolysis. However, inhibition correlated with the time elapsed between thawing and the addition of mustard to the enzyme preparation, not with concentration. TDG exhibited a direct, concentration-related inhibition of p-NPP hydrolysis between 30 and 300 microM. We conclude that sulfur mustard also has an inhibitory effect on protein serine/threonine phosphatases. However, the inhibition is an effect of its non-alkykating hydrolysis product TDG, not of sulfur mustard itself.

PPX, a novel protein serine/threonine phosphatase localized to centrosomes.

Brewis ND, Street AJ, Prescott AR, Cohen PT
EMBO J. 1993; 12: 987-96

The amino acid sequence of a novel mammalian protein phosphatase, termed PPX (and designated PPP4 in the human genome nomenclature), has been deduced from the cDNA and shown to be 65% identical to PP2A alpha and PP2A beta and 45% identical to PPI isoforms, the predicted molecular mass being 35 kDa. PPX was expressed in the baculovirus system. Its substrate specificity and sensitivity to the inhibitors, okadaic acid and microcystin, were similar (but not identical) to the catalytic subunit of PP2A. However, PPX did not bind the 65 kDa regulatory subunit of PP2A. The intracellular localization of PPX was investigated by immunofluorescence using two different antibodies raised against bacterially expressed PPX and a PPX-specific peptide. These showed that although PPX was distributed throughout the cytoplasm and the nucleus, intense staining occurred at centrosomes. The centrosomal staining was apparent in interphase and at all stages of mitosis, except telophase. In contrast, antibodies directed against bacterially expressed PP2A were not specifically localized to centrosomes. The human autoantibody #5051, which stains the pericentriolar material, colocalizes with PPX antibodies, suggesting that PPX may play a role in microtubule nucleation.

Flicking the switches: phosphorylation of serine/threonine protein phosphatases.

Brautigan DL
Semin Cancer Biol. 1995; 6: 211-7

Signal transduction involves protein phosphorylation and dephosphorylation. To produce both substantial and transient changes requires coordinated and reciprocal regulation of kinases and phosphatases. One mechanism to accomplish this is phosphorylation, and there are now reports of phosphorylation of all the major types of protein Ser/Thr phosphatases. Phosphorylation of type-1 and type-2A phosphatases occurs within characteristic C-terminal sequences and results in the loss of phosphatase activity. The phosphatases catalyse intramolecular dephosphorylation, with a restoration of activity. This property probably accounts for the apparent constitutive activity of phosphatases in cell and tissue extracts. Phosphorylation of phosphatases is a way to flick the activity off and on in cells during the growth cycle and in response to stimuli.

Protein phosphatases.

Brautigan DL
Recent Prog Horm Res. 1994; 49: 197-214

Characterization of targeting domains by sequence analysis: glycogen-binding domains in protein phosphatases.

Bork P, Dandekar T, Eisenhaber F, Huynen M
J Mol Med. 1998; 76: 77-9

Higher eucaryotic cdc25 proteins are structurally related to phosphoseryl/threonyl protein phosphatases.

Belle R, Ollivier E, Guerrucci MA
Biol Cell. 1992; 75: 139-43

cdc25 proteins are universally involved in the control of cell division. Using an original method of sequence analysis, cdc25 proteins from different sources were compared to protein phosphatases. Protein phosphatases could clearly be characterized as two distinct protein families, the phospho-seryl/threonyl phosphatases, and the phospho-tyrosyl phosphatases. None of the cdc25 proteins analyzed fitted with the phospho-tyrosyl phosphatases, indicating that if they indeed possess this biochemical activity, they form a distinct phsophatase protein group. Unexpectedly, higher eucaryotic cdc25 proteins (from human and fly) were found to be structurally related to phospho-seryl/threonyl phosphatases. These results fit well with expected function of the proteins, associated solely in higher eucaryotes, to dephosphorylation of threonine in the cell cycle control protein cdc2.

Molecular cloning of a protein serine/threonine phosphatase containing a putative regulatory tetratricopeptide repeat domain.

Becker W, Kentrup H, Klumpp S, Schultz JE, Joost HG
J Biol Chem. 1994; 269: 22586-92

Two novel protein serine/threonine phosphatases were cloned from a rat fat cell library with probes generated by a polymerase chain reaction-based cloning approach. One of these cDNAs encoded a protein presumably representing the rat homologue of PPV from Drosophila (75% identity of amino acids). The other novel cDNA encoded a protein phosphatase of 499 amino acids and was designated PPT. Its catalytic domain contains motifs typical for protein phosphatases but is only distantly related with PP1, PP2A, and PP2B (38-42% identical amino acids). When expressed in Escherichia coli, the catalytic domain of PPT exhibited protein phosphatase activity (dephosphorylation of phosphorylase a) that was inhibitable by okadaic acid. As a unique feature among other members of this gene family, PPT has an amino-terminal extension of 200 amino acids harboring three tandemly arranged tetratricopeptide repeat (TPR) motifs. This domain has previously been found in other proteins involved in the regulation of RNA synthesis or mitosis. mRNA of PPT was predominantly found in brain and, in lower levels, in testis, but was nearly undetectable in spleen, lung, skeletal muscle, kidney, and liver. It is suggested that the TPR domain of PPT may be involved in the regulation of the function of this novel protein phosphatase.

Conservation analysis and structure prediction of the protein serine/threonine phosphatases. Sequence similarity with diadenosine tetraphosphatase from Escherichia coli suggests homology to the protein phosphatases.

Barton GJ, Cohen PT, Barford D
Eur J Biochem. 1994; 220: 225-37

A multiple sequence alignment of 44 serine/threonine-specific protein phosphatases has been performed. This reveals the position of a common conserved catalytic core, the location of invariant residues, insertions and deletions. The multiple alignment has been used to guide and improve a consensus secondary-structure prediction for the common catalytic core. The location of insertions and deletions has aided in defining the positions of surface loops and turns. The prediction suggests that the core protein phosphatase structure comprises two domains: the first has a single, beta sheet flanked by alpha helices, while the second is predominantly alpha helical. Knowledge of the core secondary structures provides a guide for the design of site-directed-mutagenesis experiments that will not disrupt the native phosphatase fold. A sequence similarity between eukaryotic serine/threonine protein phosphatases and the Escherichia coli diadenosine tetraphosphatase has been identified. This extends over the N-terminal 100 residues of bacteriophage phosphatases and E. coli diadenosine tetraphosphatase. Residues which are invariant amongst these classes are likely to be important in catalysis and protein folding. These include Arg92, Asn138, Asp59, Asp88, Gly58, Gly62, Gly87, Gly93, Gly137, His61, His139 and Val90 and fall into three clusters with the consensus sequences GD(IVTL)HG, GD(LYF)V(DA)RG and GNH, where brackets surround alternative amino acids. The first two consensus sequences are predicted to fall in the beta-alpha and beta-beta loops of a beta-alpha-beta-beta secondary-structure motif. This places the predicted phosphate-binding site at the N-terminus of the alpha helix, where phosphate binding may be stabilised by the alpha-helix dipole.

Expression and biochemical properties of a protein serine/threonine phosphatase encoded by bacteriophage lambda.

Barik S
Proc Natl Acad Sci U S A. 1993; 90: 10633-7

The predicted amino acid sequence encoded by the open reading frame 221 (orf221) of bacteriophage lambda exhibited a high degree of similarity to the catalytic subunits of a variety of protein serine/threonine phosphatases belonging to PP1, PP2A, and PP2B groups. Cloning and expression of the orf221 gene in Escherichia coli provided direct evidence that the gene codes for a protein serine/threonine phosphatase. The single-subunit recombinant enzyme was purified in soluble form and shown to possess a unique repertoire of biochemical properties--e.g., an absolute requirement for Mn2+, resistance to okadaic acid, inhibitors 1 and 2, and ability to dephosphorylate casein, adenovirus E1A proteins, and the alpha subunit of phosphorylase kinase. No phosphotyrosine phosphatase activity was observed. Mutational and biochemical analyses identified the conserved residues 73-77 and Cys138 to be important for activity. The name PP-lambda is proposed for this unusual prokaryotic enzyme.

The structure and mechanism of protein phosphatases: insights into catalysis and regulation.

Barford D, Das AK, Egloff MP
Annu Rev Biophys Biomol Struct. 1998; 27: 133-64

Eukaryotic protein phosphatases are structurally and functionally diverse enzymes that are represented by three distinct gene families. Two of these, the PPP and PPM families, dephosphorylate phosphoserine and phosphothreonine residues, whereas the protein tyrosine phosphatases (PTPs) dephosphorylate phosphotyrosine amino acids. A subfamily of the PTPs, the dual-specificity phosphatases, dephosphorylate all three phosphoamino acids. Within each family, the catalytic domains are highly conserved, with functional diversity endowed by regulatory domains and subunits. The protein Ser/Thr phosphatases are metalloenzymes and dephosphorylate their substrates in a single reaction step using a metal-activated nucleophilic water molecule. In contrast, the PTPs catalyze dephosphorylation by use of a cysteinyl-phosphate enzyme intermediate. The crystal structures of a number of protein phosphatases have been determined, enabling us to understand their catalytic mechanisms and the basis for substrate recognition and to begin to provide insights into molecular mechanisms of protein phosphatase regulation.

Molecular mechanisms of the protein serine/threonine phosphatases.

Barford D
Trends Biochem Sci. 1996; 21: 407-12

The dephosphorylation of proteins on their serine, threonine and tyrosine residues is catalysed by three families of protein phosphatases that regulate numerous intracellular processes. Diversity of structure within a family is generated by targeting and regulatory subunits and domains. Structural studies of these enzymes have revealed that although the two families of protein Ser/Thr phosphatases are unrelated in sequence, the architecture of their catalytic domains is remarkably similar and distinct from the protein tyrosine phosphatases. Insights into the molecular mechanisms of catalysis and regulation of these enzymes have been obtained.

Colworth Medal Lecture. Structural studies of reversible protein phosphorylation and protein phosphatases.

Barford D
Biochem Soc Trans. 1999; 27: 751-66

Autoregulation of protein phosphatase type 2A expression.

Baharians Z, Schonthal AH
J Biol Chem. 1998; 273: 19019-24

Protein phosphatases are involved in many cellular processes. One of the most abundant of these enzymes, the serine/threonine-specific protein phosphatase type 2A (PP2A), is present in most eukaryotic cells and serves a variety of functions. However, the detailed study of its regulation and function has been hampered by the difficulty of manipulating its expression level in cell culture. By using a new mammalian expression vector to forcibly overexpress PP2A in the mouse fibroblast cell line NIH3T3, we now show that the catalytic subunit of PP2A is subject to a potent autoregulatory mechanism that adjusts PP2A protein to constant levels. This control is exerted at the translational level and does not involve regulation of transcription or RNA processing. Thus, our results demonstrate tight control of PP2A expression, and provide an explanation for the difficulty of increasing PP2A expression experimentally.

Cloning of a novel testis specific protein serine/threonine phosphatase, PPN 58A, from Drosophila melanogaster.

Armstrong CG, Dombradi V, Mann DJ, Cohen PT
Biochim Biophys Acta. 1998; 1399: 234-8

A gene encoding a novel member of the PPP family of protein serine/threonine phosphatases, termed PPN 58A, was cloned from Drosophila melanogaster. The deduced amino acid sequence of PPN 58A exhibits 59-62% identity to D. melanogaster PP1 isoforms, 51% identity to D. melanogaster PPY 55A and < or = 40% identity to other members of the PPP family. The single copy gene PPN 58A maps to chromosome 2 locus 58A. Analysis of PPN 58A mRNA reveals that, like PPY 55A, PPN 58A is a testis specific enzyme.

The search for the biological function of novel yeast Ser/Thr phosphatases.

Arino J, Posas F, Clotet J
Methods Mol Biol. 1998; 93: 305-13

Interactions between a minimal protein serine/threonine phosphatase and its phosphopeptide substrate sequence.

Ansai T, Dupuy LC, Barik S
J Biol Chem. 1996; 271: 24401-7

The protein phosphatase encoded by coliphage lambda (PPlambda) was found to be the equivalent of the minimal catalytic core of serine/threonine protein phosphatases (PP) by biochemical and mutational criteria. Bacterially expressed truncated versions of PP1 and PP5 phosphatases, representing the catalytic cores homologous to PPlambda, exhibited potent phosphatase activity. Unlike full-length PP1, but like PPlambda, the recombinant cores could use casein, p-nitrophenyl phosphate, and a wide variety of peptides as substrates and were resistant to okadaic acid, microcystin-LR, and trypsin. Mutations of His173, Asp208, or Arg221 had little effect on the activity of the PP1 core protein, indicating its closer identity with PPlambda than with full-length PP1. Terminal deletions of a few amino acids of the cores destroyed their activity, supporting their minimal nature. Analysis of PPlambda mutants suggested an influence of the substrate on metal ion binding. The minimal length of a phosphopeptide substrate of PPlambda appeared to be a phosphorylated serine/threonine flanked by 1 or 2 amino acid residues on either side, the N-terminal ones being more effective.

Non-classical protein Ser/Thr phosphatases: what are they for?

Andreeva AV, Kutuzov MA
Biochemistry (Mosc). 1999; 64: 228-87

Protein serine/threonine phosphatases are involved in regulation of diverse cellular functions. This review is devoted to a novel group of protein Ser/Thr phosphatases, rdgC/PP5, which has been recently discovered in animals, fungi, and plants. Their structure, location, and possible functions are discussed.

RdgC/PP5-related phosphatases: novel components in signal transduction.

Andreeva AV, Kutuzov MA
Cell Signal. 1999; 11: 555-62

A great variety of cellular functions are regulated by protein serine/threonine phosphatases (PP). This review summarises the current knowledge of the structural features, patterns of expression and involvement in signal transduction pathways of protein serine/threonine phosphatases related to PP5 and RdgC. Designated now as PP5/RdgC subfamily by P. T. W. Cohen in her 1997 study published in Trends in Biochemical Sciences, (Vol. 22, pp. 245-251), this heterogeneous group comprises phosphatases PP5/PPT, containing regulatory domains with tetratricopeptide repeats, RdgC/PPEF, which possess Ca2+-binding EF hand-type sites, and, recently discovered in plants, PP7. PP5 is ubiquitously expressed and appears to be a multifunctional phosphatase involved in a number of different signalling pathways. In contrast, expression of RdgC/PPEF phosphatases and PP7 is confined primarily to specialised sensory cells in animals and plants, respectively, which may be indicative of their more specialised roles in sensory signal transduction.

PP7, a plant phosphatase representing a novel evolutionary branch of eukaryotic protein Ser/Thr phosphatases.

Andreeva AV, Evans DE, Hawes CR, Bennett N, Kutuzov MA
Biochem Mol Biol Int. 1998; 44: 703-15

We describe a novel protein Ser/Thr phosphatase from Arabidopsis thaliana, PP7, which is only 27-32% identical in amino acid sequence to the known phosphatases and is the most divergent member of the PPP (PP1/2A/2B) family for today. Some structural features suggest more close relationship of PP7 to the PP5/rdgC subfamily. PP7 contains all of the residues essential for the phosphatase activity and possesses three major insertions in its presumable C-terminal subdomain, which suggest its unique regulation and/or optimisation of its structure for interaction with specific substrates or regulators. A phosphatase structurally related to PP7 is expressed in rice. PP7 conservation between mono- and dicotyledonous plants may point to its essential role in the plant cell.

Regulation of protein phosphatase-1.

Aggen JB, Nairn AC, Chamberlin R
Chem Biol. 2000; 7: 1323-1323

Reversible protein phosphorylation is a major regulatory mechanism of intracellular signal transduction. Protein phosphatase 1 (PP1) is one of four major types of serine-threonine phosphatases mediating signaling pathways, but the means by which its activity is modulated has only recently begun to come into focus.

Dephosphorylation of the focal adhesion protein VASP in vitro and in intact human platelets.

Abel K, Mieskes G, Walter U
FEBS Lett. 1995; 370: 184-8

The focal adhesion protein VASP, a possible link between signal transduction pathways and the microfilament system, is phosphorylated by both cAMP- and cGMP-dependent protein kinases in vitro and in intact cells. Here, the analysis of VASP dephosphorylation by the serine/threonine protein phosphatases (PP) PP1, PP2A, PP2B and PP2C in vitro is reported. The phosphatases differed in their selectivity with respect to the dephosphorylation of individual VASP phosphorylation sites. Incubation of human platelets with okadaic acid, a potent inhibitor of PP1 and PP2A, caused the accumulation of phosphorylated VASP indicating that the phosphorylation status of VASP in intact cells is regulated to a major extent by serine/threonine protein phosphatases. Furthermore, the accumulation of phosphorylated cAMP-dependent protein kinase substrate(s) appears to account for inhibitory effects of okadaic acid on platelet function.