A major phosphotyrosyl-protein phosphatase from bovine heart is associated with a low-molecular-weight acid phosphatase

The phosphotyrosyl [Tyr(P)]-immunoglobulin G (IgG) phosphatase activity in the extracts of bovine heart, bovine brain, human kidney, and rabbit liver can be separated by DEAE-cellulose at neutral pH into two fractions. The unbound fraction exhibits a higher activity at acidic than neutral pH while the reverse is true for the bound fraction. Of all tissues examined, the Tyr(P)-IgG phosphatase activity in the unbound fraction measured at pH 5.0 is higher than that in the bound fraction measured at pH 7.2. The acid Tyr(P)-IgG phosphatase activity has been extensively purified from bovine heart. It copurified with an acid phosphatase activity (p-nitrophenyl phosphate (PNPP) as a substrate) throughout the purification procedure. These two activities coelute from various ion-exchange and gel filtration chromatographies and comigrate on polyacrylamide gel electrophoresis, indicating that they reside on the same protein molecule. The phosphatase has a Mr = 15,000 by gel filtration and exhibits an optimum between pH 5.0 and 6.0 when either Tyr(P)-IgG-casein or PNPP is the substrate. It is highly specific for Tyr(P)-protein with little activities toward phosphoseryl [Ser(P)]- or phosphothreonyl [Thr(P)]-protein. The enzyme activities toward Tyr(P)-casein and PNPP are strongly inhibited by microM molybdate and vanadate but insensitive to inhibition by L(+)-tartrate, NaF, or Zn2+. The molecular and catalytic properties of the acid Tyr(P)-protein phosphatase purified from bovine heart are very similar to those of the low-molecular-weight acid phosphatases of Mr = 14,000 previously identified and purified from the cytosolic fraction of human liver, placenta, and other animal tissues.     

A synthetic beta-casein phosphopeptide and analogues as model substrates for casein kinase-1, a ubiquitous, phosphate directed protein kinase

The phosphopeptide Ser (P)-Ser(P)-Ser-(P)-Glu-Glu-Ser22-Ile-Thr, reproducing the 17-24 segment of beta-casein A2 including the seryl residue (Ser-22) which is targeted by casein kinase-1 was synthesized and used as model substrate for this enzyme. Its phosphorylation efficiency is actually higher than that of intact beta-casein (similar Vmax and 14 microM Km). Conversely the fully dephosphorylated peptide SSSEESIT is not affected by CK-1 to any detectable extent and its glutamyl derivative EEEEESIT displays a more than 50-fold higher Km and a 5-fold lower Vmax as compared to the parent phosphopeptide. The relevance of the individual phosphoseryl residues has been assessed by comparing the phosphorylation efficiencies of the phosphopeptides EESpEESIT, ESpEEESIT and SpEEEESIT: while the first is a substrate almost as good as the tris Ser (P)-peptide (Km = 62 microM), and the third one is almost as poor as EEEEESIT (Km = 1.55 mM), ESpEEESIT displays a intermediate efficiency (Km = 277 microM). These data in conjunction with the finding that the phosphopentapeptide Ser(P)-Ser(P)-Ser(P)-Ser-Ser(P), but neither Ser(P)-Ser(P)-Ser-Ser(P) nor Ser-Ser(P)-Ser(P)-Glu-Glu and Ser-Ala-Ala-Ser(P)-Ser(P), is readily phosphorylated by CK-1, support the concept that CK-1 is a phosphate directed protein kinase recognizing the Ser(P)-X-X-Ser-X and, less efficiently, the Ser(P)-X-X-X-Ser-X motifs.     

Phosphotyrosine Substrate Sequence Motifs for Dual Specificity Phosphatases

Protein tyrosine phosphatases dephosphorylate tyrosine residues of proteins, whereas, dual specificity phosphatases (DUSPs) are a subgroup of protein tyrosine phosphatases that dephosphorylate not only Tyr(P) residue, but also the Ser(P) and Thr(P) residues of proteins. The DUSPs are linked to the regulation of many cellular functions and signaling pathways. Though many cellular targets of DUSPs are known, the relationship between catalytic activity and substrate specificity is poorly defined. We investigated the interactions of peptide substrates with select DUSPs of four types: MAP kinases (DUSP1 and DUSP7), atypical (DUSP3, DUSP14, DUSP22 and DUSP27), viral (variola VH1), and Cdc25 (A-C). Phosphatase recognition sites were experimentally determined by measuring dephosphorylation of 6,218 microarrayed Tyr(P) peptides representing confirmed and theoretical phosphorylation motifs from the cellular proteome. A broad continuum of dephosphorylation was observed across the microarrayed peptide substrates for all phosphatases, suggesting a complex relationship between substrate sequence recognition and optimal activity. Further analysis of peptide dephosphorylation by hierarchical clustering indicated that DUSPs could be organized by substrate sequence motifs, and peptide-specificities by phylogenetic relationships among the catalytic domains. The most highly dephosphorylated peptides represented proteins from 29 cell-signaling pathways, greatly expanding the list of potential targets of DUSPs. These newly identified DUSP substrates will be important for examining structure-activity relationships with physiologically relevant targets.     

Regulation of stress-activated protein kinase signaling pathways by protein phosphatases.

Stress-activated protein kinase (SAPK) signaling plays essential roles in eliciting adequate cellular responses to stresses and proinflammatory cytokines. SAPK pathways are composed of three successive protein kinase reactions. The phosphorylation of SAPK signaling components on Ser/Thr or Thr/Tyr residues suggests the involvement of various protein phosphatases in the negative regulation of these systems. Accumulating evidence indicates that three families of protein phosphatases, namely the Ser/Thr phosphatases, the Tyr phosphatases and the dual specificity Ser/Thr/Tyr phosphatases regulate these pathways, each mediating a distinct function. Differences in substrate specificities and regulatory mechanisms for these phosphatases form the molecular basis for the complex regulation of SAPK signaling. Here we describe the properties of the protein phosphatases responsible for the regulation of SAPK signaling pathways.     

Role of serine 214 and tyrosine 171, components of the S2 subsite of alpha-lytic protease, in catalysis.

The function of a hydrogen bond network, comprised of the hydroxyl groups of Tyr 171 and Ser 214, in the hydrophobic S2 subsite of alpha-lytic protease, was investigated by mutagenesis and the kinetics of a substrate analog series. To study the catalytic role of the Tyr 171 and Ser 214 hydroxyl groups, Tyr 171 was converted to phenylalanine (Y171F) and Ser 214 to alanine (S214A). The double mutant (Y171F: S214A) also was generated. The single S214A and double Y171F:S214A mutations cause differential effects on catalysis and proenzyme processing. For S214A, kcat/Km is (4.9 x 10(3))-fold lower than that of wild type and proenzyme processing is blocked. For the double mutant (Y171F:S214A), kcat/Km is 82-fold lower than that of wild type and proenzyme processing occurs. In Y171F, kcat/Km is 34-fold lower than that of wild type, and the proenzyme is processed. The data indicate that Ser 214, although conserved among serine proteases and hydrogen bonded to the catalytic triad [Brayer, G. D., Delbaere, L. T. J., & James, M. N. G. (1979) J. Mol. Biol. 131, 743], is not essential for catalytic function in alpha-lytic protease. A substrate series (in which peptide length is varied) established that the mutations (Y171F and Y171F:S214A) do not alter enzyme-substrate interactions in subsites other than S2. The pH dependence of kcat/Km for Y171F and Y171F:S214A has changed less than 0.5 unit from that of wild type; this suggests the catalytic triad is unperturbed. In wild type, hydrophobic interactions at S2 increase kcat/Km by up to (1.2 x 10(3))-fold with no effect on Km.(ABSTRACT TRUNCATED AT 250 WORDS)     

Disruption of distal interactions of Arg 262 and of substrate binding to Ser 52 affect catalysis of sheep liver cytosolic serine hydroxymethyltransferase

The crystal structure of human liver cytosolic recombinant serine hydroxymethyltransferase (hcSHMT) suggested that Ser53 and Arg 263 could participate in the reaction catalyzed by SHMT. The mutation of Arg262 (corresponding to Arg263 in hcSHMT) to "A" in sheep liver cytosolic SHMT (scSHMT) resulted in a 5-fold increase in Km for L-Ser and a 5-fold decrease in kcat compared to scSHMT. Further, in R262A SHMT-glycine complex, the peak at 343 nm (geminal diamine) was more pronounced, compared to wild-type enzyme. Stopped-flow studies showed that the rate constant for the formation of glycine-geminal diamine for R262A SHMT was also decreased. The rate of reaction, concentration of spectral intermediates, fluorescence excitation maximum of glycine geminal diamine and interaction with methoxyamine were altered in R262A SHMT. Although Arg263 in hcSHMT is located outside the PLP binding pocket, it positions Tyr73 for interaction with PLP, by forked H-bonding with the carbonyl groups of main chain residues, Asn71 and Lys72 of the other subunit of the tight dimer. Mutation of Arg262 to Ala and the consequent alteration in orientation of PLP leads to decreased catalytic efficiency. Ser53 (in hcSHMT) is in hydrogen bonding distance to one of the carboxylate oxygens of the amino acid substrate, which also interacts with Tyr83 and Arg402. Replacement of Ser53 with Cys (using 'O' software program) in the structure of hcSHMT resulted in disruption of these interactions, whereas replacement with Ala (S53A) only weakened the substrate interactions. There was a 10-fold increase in Km and 20-fold decrease in catalytic activity efficiency for S52C SHMT, whereas S52A SHMT retained 20% of the activity without change in Km for serine. These results suggest that S52 affects substrate binding and catalysis.     

Regulation of protein kinase C delta by phorbol ester, endothelin-1, and platelet-derived growth factor in cardiac myocytes

Protein kinase C (PKC) delta is regulated allosterically by phosphatidylserine and diacylglycerol (which promote its translocation to the membrane) and by phosphorylation of Ser/Thr and Tyr residues. Although phosphorylation on Thr-505/Ser-643/Ser-662 may simply "prime" PKCdelta for activation, it could be regulatory. We examined the regulation of PKCdelta in cardiac myocytes by endothelin-1 (Gq protein-coupled receptor agonist) and platelet-derived growth factor (receptor tyrosine kinase agonist) in comparison with phorbol 12-myristate 13-acetate (PMA). All increased phosphorylation of PKCdelta(Thr-505/Ser-643) and of Tyr residues, although to differing extents. De novo phosphorylation occurred mainly after translocation of PKCdelta to the particulate fraction, and phosphorylations of Thr-505/Ser-643 versus Tyr residues were essentially independent events. Following chromatographic separation of the PKCdelta subspecies, activities were correlated with immunoreactivity profiles of total and phosphorylated forms. In unstimulated cells, approximately 25% of PKCdelta lacked phosphorylation of Thr-505/Ser-643 and displayed minimal activity (assayed in the presence of phosphatidylserine/PMA following chromatography). Endothelin-1 or PMA (10 min) promoted Thr-505/Ser-643 phosphorylation of this pool, and this was associated with an increase in total recoverable PKCdelta activity. Meanwhile, in cells exposed to endothelin-1 or PMA, the overall pool of PKCdelta translocated rapidly (30 s) to the particulate fraction and was phosphorylated on Tyr residues. This was associated with an increase in lipid-independent activity (i.e. the phosphatidylserine/PMA requirement disappeared). For endothelin-1, Tyr phosphorylation of PKCdelta and the increase in phosphatidylserine/PMA-independent activity persisted after PKCdelta retrotranslocated to the soluble fraction. We concluded that, with this physiological agonist, PKCdelta becomes activated in the particulate fraction but retains activity following its retrotranslocation, presumably to phosphorylate substrates elsewhere.     

Molecular determinants of substrate recognition in hematopoietic protein-tyrosine phosphatase

The extracellular signal-regulated protein kinase 2 (ERK2) plays a central role in cellular proliferation and differentiation. Full activation of ERK2 requires dual phosphorylation of Thr183 and Tyr185 in the activation loop. Tyr185 dephosphorylation by the hematopoietic protein-tyrosine phosphatase (HePTP) represents an important mechanism for down-regulating ERK2 activity. The bisphosphorylated ERK2 is a highly efficient substrate for HePTP with a kcat/Km of 2.6 x 10(6) m(-1) s(-1). In contrast, the kcat/Km values for the HePTP-catalyzed hydrolysis of Tyr(P) peptides are 3 orders of magnitude lower. To gain insight into the molecular basis for HePTP substrate specificity, we analyzed the effects of altering structural features unique to HePTP on the HePTP-catalyzed hydrolysis of p-nitrophenyl phosphate, Tyr(P) peptides, and its physiological substrate ERK2. Our results suggest that substrate specificity is conferred upon HePTP by both negative and positive selections. To avoid nonspecific tyrosine dephosphorylation, HePTP employs Thr106 in the substrate recognition loop as a key negative determinant to restrain its protein-tyrosine phosphatase activity. The extremely high efficiency and fidelity of ERK2 dephosphorylation by HePTP is achieved by a bipartite protein-protein interaction mechanism, in which docking interactions between the kinase interaction motif in HePTP and the common docking site in ERK2 promote the HePTP-catalyzed ERK2 dephosphorylation (approximately 20-fold increase in kcat/Km) by increasing the local substrate concentration, and second site interactions between the HePTP catalytic site and the ERK2 substrate-binding region enhance catalysis (approximately 20-fold increase in kcat/Km) by organizing the catalytic residues with respect to Tyr(P)185 for optimal phosphoryl transfer.     

Schizosaccharomyces pombe carboxyl-terminal domain (CTD) phosphatase Fcp1: distributive mechanism, minimal CTD substrate, and active site mapping

Schizosaccharomyces pombe Fcp1 is an essential protein serine phosphatase that preferentially dephosphorylates Ser(2) of the RNA polymerase II C-terminal domain (CTD) heptad repeat Y(1)S(2)P(3)T(4)S(5)P(6)S(7). Here we show that: (i) Fcp1 acts distributively during the hydrolysis of substrates containing tandem Ser(2)-PO(4) heptads; (ii) the minimal optimal CTD substrate for Fcp1 is a single heptad of phasing S(5)P(6)S(7)Y(1)S(2)P(3)T(4); and (iii) single alanine mutations of flanking residues Tyr(1) or Pro(3) result in 6-fold decrements in CTD phosphatase activity. Fcp1 belongs to the DXDX(T/V) family of phosphotransferases that act via an acyl-phosphoenzyme intermediate. An alanine scan of 11 conserved positions of S. pombe Fcp1 identifies Thr(174), Tyr(237), Thr(243), and Tyr(249) as important for phosphatase activity. Structure-activity relationships at these positions were determined by introducing conservative substitutions. Our results, together with previous mutational studies, highlight a constellation of 11 amino acids that are conserved in all Fcp1 orthologs and likely comprise the active site.     

Synthetic peptides as model substrates for the study of the specificity of the polycation-stimulated protein phosphatases

The substrate specificity of the different forms of the polycation-stimulated (PCS, type 2A) protein phosphatases and of the active catalytic subunit of the ATP, Mg-dependent (type 1) phosphatase (AMDC) was investigated, using synthetic peptides phosphorylated by either cyclic-AMP-dependent protein kinase or by casein kinase-2. The PCS phosphatases are very efficient toward the Thr(P) peptides RRAT(P)VA and RRREEET(P)EEE when compared with the Ser(P) analogues RRAS(P)VA and RRREEES(P)EEEAA. Despite their distinct sequence, both Thr(P) peptides are excellent substrates for the PCSM and PCSH1 phosphatases, being dephosphorylated faster than phosphorylase a. The slow dephosphorylation of RRAS(P)VA by the PCS phosphatases could be increased substantially by the insertion of N-terminal (Arg) basic residues. In contrast with the latter, the AMDC phosphatase shows very poor activity toward all the phosphopeptides tested, without preference for either Ser(P) or Thr(P) peptides. However, N-terminal basic residues also favor the dephosphorylation of otherwise almost inert substrates by the AMDC phosphatase. Hence, while the dephosphorylation of Thr(P) substrates by the PCS phosphatases is highly favored by the nature of the phosphorylated amino acid, phosphatase activity toward Ser(P)-containing peptides may require specific determinants in the primary structure of the phosphorylation site.     

FYVE-DSP1, a dual-specificity protein phosphatase containing an FYVE domain

Dual-specificity protein phosphatases (DSPs) dephosphorylate proteins at Ser/Thr and Tyr. FYVE domain is a double zinc finger motif which specifically binds phosphatidylinositol(3)-phosphate. Here, we report a novel dual specificity phosphatase that contains a FYVE domain at the C-terminus. We designate the protein FYVE-DSP1. Molecular cloning yielded three isoforms of the enzyme presumably derived from alternate RNA splicing. Sequence alignment revealed that the catalytic phosphatase domain of FYVE-DSP1 closely resembled that of myotubularin, while its FYVE domain has all the conserved amino acid residues found in other proteins of the same family. Recombinant FYVE-DSP1 is partitioned in both cytosolic and membrane fractions. It dephosphorylates proteins phosphorylated on Ser, Thr, and Tyr residues and low molecular weight phosphatase substrate para-nitrophenylphosphate. It shows typical characteristics of other DSPs and protein tyrosine phosphatases (PTPs). These include inhibition by sodium vanadate and pervanadate, pH dependency, and inactivation by mutation of the key cysteinyl residue at the phosphatase signature motif. Finally, PCR analyses demonstrated that FYVE-DSP1 is widely distributed in human tissues but different spliced forms expressed differently.     

Phosphorylation of insulin receptor substrate-1 (IRS-1) by protein kinase B positively regulates IRS-1 function.

Incubation of cells with insulin leads to a transient rise in Tyr phosphorylation of insulin receptor substrate (IRS) proteins, accompanied by elevation in their Ser(P)/Thr(P) content and their dissociation from the insulin receptor (IR). Wortmannin, a phosphatidylinositol 3-kinase inhibitor, selectively prevented the increase in Ser(P)/Thr(P) content of IRS-1, its dissociation from IR, and the decrease in its Tyr(P) content following 60 min of insulin treatment. Four conserved phosphorylation sites within the phosphotyrosine binding/SAIN domains of IRS-1 and IRS-2 served as in vitro substrates for protein kinase B (PKB), a Ser/Thr kinase downstream of phosphatidylinositol 3-kinase. Furthermore, PKB and IRS-1 formed stable complexes in vivo, and overexpression of PKB enhanced Ser phosphorylation of IRS-1. Overexpression of PKB did not affect the acute Tyr phosphorylation of IRS-1; however, it significantly attenuated its rate of Tyr dephosphorylation following 60 min of treatment with insulin. Accordingly, overexpression of IRS-1(4A), lacking the four potential PKB phosphorylation sites, markedly enhanced the rate of Tyr dephosphorylation of IRS-1, while inclusion of vanadate reversed this effect. These results implicate a wortmannin-sensitive Ser/Thr kinase, different from PKB, as the kinase that phosphorylates IRS-1 and acts as the feedback control regulator that turns off insulin signals by inducting the dissociation of IRS proteins from IR. In contrast, insulin-stimulated PKB-mediated phosphorylation of Ser residues within the phosphotyrosine binding/SAIN domain of IRS-1 protects IRS-1 from the rapid action of protein-tyrosine phosphatases and enables it to maintain its Tyr-phosphorylated active conformation. These findings implicate PKB as a positive regulator of IRS-1 functions.     

Catalytic mechanism of scytalone dehydratase: site-directed mutagenisis, kinetic isotope effects, and alternate substrates.

On the basis of the X-ray crystal structure of scytalone dehydratase complexed with an active center inhibitor [Lundqvist, T., Rice, J., Hodge, C. N., Basarab, G. S., Pierce, J. and Lindqvist, Y. (1994) Structure (London) 2, 937-944], eight active-site residues were mutated to examine their roles in the catalytic mechanism. All but one residue (Lys73, a potential base in an anti elimination mechanism) were found to be important to catalysis or substrate binding. Steady-state kinetic parameters for the mutants support the native roles for the residues (Asn131, Asp31, His85, His110, Ser129, Tyr30, and Tyr50) within a syn elimination mechanism. Relative substrate specificities for the two physiological substrates, scytalone and veremelone, versus a Ser129 mutant help assign the orientation of the substrates within the active site. His85Asn was the most damaging mutation to catalysis consistent with its native roles as a general base and a general acid in a syn elimination. The additive effect of Tyr30Phe and Tyr50Phe mutations in the double mutant is consistent with their roles in protonating the substrate's carbonyl through a water molecule. Studies on a synthetic substrate, which has an anomeric carbon atom which can better stabilize a carbocation than the physiological substrate (vermelone), suggest that His110Asn prefers this substrate over vermelone in order to balance the mutation-imposed weakness in promoting the elimination of hydroxide from substrates. All mutant enzymes bound a potent active-site inhibitor in near 1:1 stoichiometry, thereby supporting their active-site integrity. An X-ray crystal structure of the Tyr50Phe mutant indicated that both active-site waters were retained, likely accounting for its residual catalytic activity. Steady-state kinetic parameters with deuterated scytalone gave kinetic isotope effects of 2.7 on kcat and 4.2 on kcat/Km, suggesting that steps after dehydration partially limit kcat. Pre-steady-state measurements of a single-enzyme turnover with scytalone gave a rate that was 6-fold larger than kcat. kcat/Km with scytalone has a pKa of 7.9 similar to the pKa value for the ionization of the substrate's C6 phenolic hydroxyl, whereas kcat was unaffected by pH, indicating that the anionic form of scytalone does not bind well to enzyme. With an alternate substrate having a pKa above 11, kcat/Km had a pKa of 9.3 likely due to the ionization of Tyr50. The non-enzyme-catalyzed rate of dehydration of scytalone was nearly a billion-fold slower than the enzyme-catalyzed rate at pH 7.0 and 25 degrees C. The non-enzyme-catalyzed rate of dehydration of scytalone had a deuterium kinetic isotope effect of 1.2 at pH 7.0 and 25 degrees C, and scytalone incorporated deuterium from D2O in the C2 position about 70-fold more rapidly than the dehydration rate. Thus, scytalone dehydrates through an E1cb mechanism off the enzyme.     

Polycation-dependent, Ca2+-antagonized phosphorylation of calmodulin by casein kinase-2 and a spleen tyrosine protein kinase

Ten distinct protein kinases have been tested for their ability to phosphorylate calmodulin. Only casein kinase-2 and a spleen tyrosine protein kinase (TPK-III) proved effective, their phosphorylation efficiency being dramatically enhanced by histones and other polybasic peptides while being depressed by 50 microM Ca2+. Phosphorylation by CK-2 takes place with a Km of 12 microM calmodulin, leading to the incorporation of more than 1.5 mol P/mol substrate. Ser81 and Thr79 are among the residues affected. On the other hand, the two tyrosyl residues of calmodulin are both phosphorylated by TPK-III, Tyr99 being preferred over Tyr138.     

Site-directed mutagenesis of human aldolase isozymes: the role of Cys-72 and Cys-338 residues of aldolase A and of the carboxy-terminal Tyr residues of aldolases A and B

In order to elucidate the role of particular amino acid residues in the catalytic activity and conformational stability of human aldolases A and B [EC 4.1.2.13], the cDNAs encoding these isoenzyme were modified using oligonucleotide-directed, site-specific mutagenesis. The Cys-72 and/or Cys-338 of aldolase A were replaced by Ala and the COOH-terminal Tyr of aldolases A and B was replaced by Ser. The three mutant aldolases A thus prepared, A-C72A, A-C338A, and A-C72,338A, were indistinguishable from the wild-type enzyme with respect to general catalytic properties, while the replacement of Tyr-363 by Ser in aldolase A (A-Y363S) resulted in decreases of the Vmax of the fructose-1, 6-bisphosphate (FDP) cleavage reaction, activity ratio of FDP/fructose-1-phosphate (F1P), and the Km values for FDP and F1P. The wild-type and all the mutant aldolase A proteins exhibited similar thermal stabilities. In contrast, the mutant aldolase A proteins were more stable than the wild-type enzyme against tryptic and alpha-chymotryptic digestions. Based upon these results it is concluded that the strictly conserved Tyr-363 of human aldolase A is required for the catalytic function with FDP as the substrate, while neither Cys-72 nor Cys-338 directly takes part in the catalytic function although the two Cys residues may be involved in maintaining the correct spatial conformation of aldolase A. Replacement of Tyr-363 by Ser in human aldolase B lowered the Km value for FDP appreciably and also diminished the stability against elevated temperatures and tryptic digestion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Src kinase regulation by phosphorylation and dephosphorylation

Src and Src-family protein-tyrosine kinases are regulatory proteins that play key roles in cell differentiation, motility, proliferation, and survival. The initially described phosphorylation sites of Src include an activating phosphotyrosine 416 that results from autophosphorylation, and an inhibiting phosphotyrosine 527 that results from phosphorylation by C-terminal Src kinase (Csk) and Csk homologous kinase. Dephosphorylation of phosphotyrosine 527 increases Src kinase activity. Candidate phosphotyrosine 527 phosphatases include cytoplasmic PTP1B, Shp1 and Shp2, and transmembrane enzymes include CD45, PTPalpha, PTPepsilon, and PTPlambda. Dephosphorylation of phosphotyrosine 416 decreases Src kinase activity. Thus far PTP-BL, the mouse homologue of human PTP-BAS, has been shown to dephosphorylate phosphotyrosine 416 in a regulatory fashion. The platelet-derived growth factor receptor protein-tyrosine kinase mediates the phosphorylation of Src Tyr138; this phosphorylation has no direct effect on Src kinase activity. The platelet-derived growth factor receptor and the ErbB2/HER2 growth factor receptor protein-tyrosine kinases mediate the phosphorylation of Src Tyr213 and activation of Src kinase activity. Src kinase is also a substrate for protein-serine/threonine kinases including protein kinase C (Ser12), protein kinase A (Ser17), and CDK1/cdc2 (Thr34, Thr46, and Ser72). Of the three protein-serine/threonine kinases, only phosphorylation by CDK1/cdc2 has been demonstrated to increase Src kinase activity. Although considerable information on the phosphoprotein phosphatases that catalyze the hydrolysis of Src phosphotyrosine 527 is at hand, the nature of the phosphatases that mediate the hydrolysis of phosphotyrosine 138 and 213, and phosphoserine and phosphothreonine residues has not been determined.     

Protein phosphorylation on Ser, Thr and Tyr residues in cyanobacteria

Cyanobacteria belong to an extremely diverse group of gram-negative prokaryotes. They are all able to perform oxygen-evolving photosynthesis, but differ in morphology, ecological habitats, and physiology. This diversity is also reflected in the complexity of regulatory proteins involved in protein phosphorylation on Ser, Thr and Tyr residues. For those strains whose genomes are completely sequenced, for example, the number of genes identified so far that encode Ser/Thr and Tyr kinases range from none to 52. Genetic, molecular as well as functional genomic analyses demonstrate that Ser/Thr and Tyr kinases and phosphatases are involved in the regulation of a variety of activities according to changes in growth conditions or cell metabolism, such as cell motility, carbon and nitrogen metabolism, photosynthesis and stress response. The major challenge in the near future is to integrate these components into signaling pathways and identify their targets. Some of the Ser/Thr and Tyr kinases and phosphatases are expected to interact with classical two-component signaling pathways.     

Polarized subcellular distribution of the 1-, 4- and 5-phosphatase activities that metabolize inositol 1,4,5-trisphosphate in intestinal epithelial cells.

In intestinal epithelial cells, Ins(1,4,5)P3 is metabolized both by an intracellular 5-phosphatase and by less specific extracellular phosphatases [Rubiera, Velasco, Michell, Lazo & Shears (1988) Biochem. J. 255, 131-137]. A total of 91% of intracellular Ins(1,4,5)P3 5-phosphatase was particulate, and was preferentially associated with plasma membranes rather than with other subcellular organelles. A soluble Ins(1,4,5)P3 3-kinase activity was also characterized, further supporting the idea that inositol phosphates are important in enterocyte function. We have studied the distribution of Ins(1,4,5)P3 phosphatase activities in basolateral and brush-border domains of the plasma membrane. Compared with homogenates, the extracellular phosphatases were 13-17-fold enriched in brush-border membranes, but only 2-fold enriched in basolateral membranes. The 1- and 4-phosphates of Ins(1,4,5)P3 were hydrolysed at equal rates by the extracellular phosphatases; these enzymes are proposed to have digestive functions. The intracellular particulate 5-phosphatase was 2-fold enriched in brush-border membranes and 13-fold enriched in basolateral membranes, at the same pole of the cell where Ins(1,4,5)P3 is believed to be generated. This is opposite to the polarized distribution of particulate 5-phosphatase in hepatocytes [Shears, Evans, Kirk & Michell (1988) Biochem. J. 256, 363-369]; these differences in subcellular distribution may be important in determining cell-specific metabolism of Ins(1,4,5)P3.     

Purification and characterization of a protein-phosphotyrosine phosphatase from rat spleen which dephosphorylates and inactivates a tyrosine-specific protein kinase

A particulate form of protein-phosphotyrosine phosphatase was solubilized and purified over 2,000-fold from the particulate fraction of rat spleen. Phosphorylated poly(Glu, Tyr), a random copolymer of glutamic acid and tyrosine, was used as substrate for measuring protein-phosphotyrosine phosphatase activity. Nonionic detergents like Triton X-100 increased the protein-phosphotyrosine phosphatase activity of the particulate fraction (but not of the soluble fraction) by 4-8-fold. Chromatography of the Triton extract of the particulate fraction on DEAE-Sephacel gave three peaks of protein-phosphotyrosine phosphatase activity. The major peak of activity was further purified on Bio-Gel HTP, Sephadex G-75, and phosphocellulose columns. On polyacrylamide gel electrophoresis in the presence of Na-dodecyl-SO4 the purified enzyme showed a major protein band of Mr 36,000 which comigrated with enzyme activity on the phosphocellulose column. The apparent Vmax and Km for phosphorylated poly(Glu,Tyr) were 6,150 nmol min-1 mg-1 and 1.6 microM, respectively. This enzyme was strongly inhibited by microM concentrations of orthovanadate and zinc acetate. Fluoride (50 mM) inhibited this enzyme only by 30-40%. Divalent metal ions Ca2+, Mg2+, and Mn2+ were inhibitory at 1-10 mM concentration. EDTA had no effect on the activity of the purified enzyme. This phosphatase could dephosphorylate and inactivate the phosphorylated form of a tyrosine-specific protein kinase (TK-I) previously purified from rat spleen. Dephosphorylation and inactivation of TK-I by purified phosphatase were inhibited by orthovanadate. After dephosphorylation and inactivation by phosphatase, TK-I could be rephosphorylated and reactivated on incubation with ATP. These results suggest that this protein-phosphotyrosine phosphatase may be involved in the regulation of the kinase activity of TK-I.     

Determination of the Substrate Specificity of Protein-tyrosine Phosphatase TULA-2 and Identification of Syk as a TULA-2 Substrate

TULA-1 (UBASH3A/STS-2) and TULA-2 (p70/STS-1) represent a novel class of protein-tyrosine phosphatases. Previous studies suggest that TULA-2 is sequence-selective toward phosphotyrosyl (Tyr(P)) peptides. In this work the substrate specificity of TULA-1 and -2 was systematically evaluated by screening a combinatorial Tyr(P) peptide library. Although TULA-1 showed no detectable activity toward any of the Tyr(P) peptides in the library, TULA-2 recognizes two distinct classes of Tyr(P) substrates. On the N-terminal side of Tyr(P), the class I substrates contain a proline at the Tyr(P)−1 position, a hydrophilic residue at the Tyr(P)−2 position, and aromatic hydrophobic residues at positions Tyr(P)−3 and beyond. The class II substrates typically contain two or more acidic residues, especially at Tyr(P)−1 to Tyr(P)−3 positions, and aromatic hydrophobic residues at other positions. At the C-terminal side of Tyr(P), TULA-2 generally prefers acidic and aromatic residues. The library screening results were confirmed by kinetic analysis of representative peptides selected from the library as well as Tyr(P) peptides derived from various Tyr(P) proteins. TULA-2 is highly active toward peptides corresponding to the Tyr(P)-323 and Tyr(P)-352 sites of Syk, and the Tyr(P)-397 site of focal adhesion kinase and has lower activity toward other Tyr(P) sites in these proteins. In glycoprotein VI-stimulated platelets, knock-out of the TULA-2 gene significantly increased the phosphorylation level of Syk at Tyr-323 and Tyr-352 sites and to a lesser degree at the Tyr-525/526 sites. These results suggest that Syk is a bona fide TULA-2 substrate in platelets.