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1.
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.  相似文献   

2.
Reversible phosphorylation is the key mechanism regulating several cellular events in prokaryotes and eukaryotes. In prokaryotes, signal transduction is perceived to occur primarily via the two-component signaling system involving histidine kinases and cognate response regulators. Although an alternative regulatory pathway controlled by the eukaryote-type serine/threonine kinase (Streptococcus pyogenes serine/threonine kinase; SP-STK) has been shown to modulate bacterial growth, division, adherence, invasion, and virulence in group A Streptococcus (GAS; S. pyogenes), the precise role of the co-transcribing serine/threonine phosphatase (SP-STP) has remained enigmatic. In this context, this is the first report describing the construction and characterization of non-polar SP-STP mutants in two different strains of Type M1 GAS. The STP knock-out mutants displayed increased bacterial chain lengths in conjunction with thickened cell walls, significantly reduced capsule and hemolysin production, and restoration of the phenotypes postcomplementation. The present study also reveals important contribution of cognately regulated-reversible phosphorylation by SP-STK/SP-STP on two major response regulators of two-component systems, WalRK and CovRS. We also demonstrate a distinct role of SP-STP in terms of expression of surface proteins and SpeB in a strain-specific manner. Further, the attenuation of virulence in the absence of STP and its restoration only in the complemented strains that were generated by the use of a low copy plasmid and not by a high copy one emphasize not only the essential role of STP in virulence but also highlight the tightly regulated SP-STP/SP-STK-mediated cognate functions. SP-STP thus is an important regulator of GAS virulence and plays a critical role in GAS pathogenesis.  相似文献   

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Kinetic analysis of human serine/threonine protein phosphatase 2Calpha.   总被引:1,自引:0,他引:1  
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. Br?nsted 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.  相似文献   

5.
Protein phosphorylation is essential for the regulation of cell growth, division, and differentiation in both prokaryotes and eukaryotes. Signal transduction in prokaryotes was previously thought to occur primarily by histidine kinases, involved in two-component signaling pathways. Lately, bacterial homologues of eukaryotic-type serine/threonine kinases and phosphatases have been found to be necessary for cellular functions such as growth, differentiation, pathogenicity, and secondary metabolism. The Gram-positive bacteria Streptococcus agalactiae (group B streptococci, GBS) is an important human pathogen. We have identified and characterized a eukaryotic-type serine/threonine protein kinase (Stk1) and its cognate phosphatase (Stp1) in GBS. Biochemical assays revealed that Stk1 has kinase activity and localizes to the membrane and that Stp1 is a soluble protein with manganese-dependent phosphatase activity on Stk1. Mutations in these genes exhibited pleiotropic effects on growth, virulence, and cell segregation of GBS. Complementation of these mutations restored the wild type phenotype linking these genes to the regulation of various cellular processes in GBS. In vitro phosphorylation of cell extracts from wild type and mutant strains revealed that Stk1 is essential for phosphorylation of six GBS proteins. We have identified the predominant endogenous substrate of both Stk1 and Stp1 as a manganese-dependent inorganic pyrophosphatase (PpaC) by liquid chromatography/tandem mass spectrometry. These results suggest that these eukaryotic-type enzymes regulate pyrophosphatase activity and other cellular functions of S. agalactiae.  相似文献   

6.
Xenopus MAP kinase activator, a 45 kDa protein, has been shown to function as a direct upstream factor sufficient for full activation and both tyrosine and serine/threonine phosphorylation of inactive MAP kinase. We have now shown by using an anti-MAP kinase activator antiserum that MAP kinase activator is ubiquitous in tissues and is regulated post-translationally. Activation of MAP kinase activator is correlated precisely with its threonine phosphorylation during the oocyte maturation process. It is a key question whether MAP kinase activator is a kinase or not. We have shown that Xenopus MAP kinase activator purified from mature oocytes is capable of undergoing autophosphorylation on serine, threonine and tyrosine residues. Dephosphorylation of purified activator by protein phosphatase 2A treatment inactivates its autophosphorylation activity as well as its activator activity. Thus, Xenopus MAP kinase activator is a protein kinase with specificity for both serine/threonine and tyrosine. Partial protein sequencing of purified activator indicates that it contains a sequence homologous to kinase subdomains VI and VII of two yeast protein kinases, STE7 and byrl.  相似文献   

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9.
The interplay between serine/threonine and tyrosine phosphorylation was studied in human neutrophils. The direct effects of calyculin and okadaic acid, potent inhibitors of PP1 and PP2A serine/threonine phosphatases, on the patterns of neutrophil phosphorylation, and their effects on the responses of neutrophils to CD32 cross-linking were monitored. After a 2-min incubation with 10-6 M calyculin, a transient tyrosine phosphorylation of a subset of proteins, among which Cbl and Syk, was observed. After a longer incubation (>5 min) with calyculin, concomitant with an accumulation of serine and threonine phosphorylation, neutrophil responses to CD32 cross-linking were selectively altered. Tyrosine phosphorylation of Cbl in response to CD32 cross-linking was inhibited by calyculin, and this inhibition was linked with a slower electrophoretic mobility of Cbl as a consequence of its phosphorylation on serine/threonine residues. However, tyrosine phosphorylation of Syk and of the receptor itself were not affected. Furthermore, the mobilization of intracellular calcium stimulated by CD32 cross-linking was totally abrogated by calyculin. Finally, the stimulation of superoxide production observed in response to CD32 cross-linking was enhanced in calyculin-treated cells. These results suggest that serine/threonine phosphorylation events regulate the signaling pathways activated by CD32 cross-linking in neutrophils and identify a novel mechanism of modulation of the functional responsiveness of human neutrophils to CD32 cross-linking.  相似文献   

10.
We have cloned and characterized a novel mammalian serine/threonine protein kinase WNK1 (with no lysine (K)) from a rat brain cDNA library. WNK1 has 2126 amino acids and can be detected as a protein of approximately 230 kDa in various cell lines and rat tissues. WNK1 contains a small N-terminal domain followed by the kinase domain and a long C-terminal tail. The WNK1 kinase domain has the greatest similarity to the MEKK protein kinase family. However, overexpression of WNK1 in HEK293 cells exerts no detectable effect on the activity of known, co-transfected mitogen-activated protein kinases, suggesting that it belongs to a distinct pathway. WNK1 phosphorylates the exogenous substrate myelin basic protein as well as itself mostly on serine residues, confirming that it is a serine/threonine protein kinase. The demonstration of activity was striking because WNK1, and its homologs in other organisms lack the invariant catalytic lysine in subdomain II of protein kinases that is crucial for binding to ATP. A model of WNK1 using the structure of cAMP-dependent protein kinase suggests that lysine 233 in kinase subdomain I may provide this function. Mutation of this lysine residue to methionine eliminates WNK1 activity, consistent with the conclusion that it is required for catalysis. This distinct organization of catalytic residues indicates that WNK1 belongs to a novel family of serine/threonine protein kinases.  相似文献   

11.
Searching the genome sequence of Streptococcus pneumoniae revealed the presence of a single Ser/Thr protein kinase gene stkP linked to protein phosphatase phpP. Biochemical studies performed with recombinant StkP suggest that this protein is a functional eukaryotic-type Ser/Thr protein kinase. In vitro kinase assays and Western blots of S. pneumoniae subcellular fractions revealed that StkP is a membrane protein. PhpP is a soluble protein with manganese-dependent phosphatase activity in vitro against a synthetic substrate RRA(pT)VA. Mutations in the invariant aspartate residues implicated in the metal binding completely abolished PhpP activity. Autophosphorylated form of StkP was shown to be a substrate for PhpP. These results suggest that StkP and PhpP could operate as a functional pair in vivo. Analysis of phosphoproteome maps of both wild-type and stkP null mutant strains labeled in vivo and subsequent phosphoprotein identification by peptide mass fingerprinting revealed two possible substrates for StkP. The evidence is presented that StkP can phosphorylate in vitro phosphoglucosamine mutase GlmM which catalyzes the first step in the biosynthetic pathway leading to the formation of UDP-N-acetylglucosamine, an essential common precursor to cell envelope components.  相似文献   

12.
Here we isolated tautomycetin, TC, and examined its phosphatase inhibitory activity. Recently we have reported that the left-hand moiety of tautomycin, TM, and the right one containing the spiroketal are essentially required for inhibition of protein phosphatase, PP, and induction of apoptosis, respectively. TC is structurally almost identical to TM except that TC is lacking the spiroketal, which has the potential apoptosis-inducing activity. TC specifically inhibited PP1 activity, IC50 values for purified PP1 and PP2A enzymes being 1.6 and 62 nM, respectively, whereas the IC50 values of TM were 0.21 and 0.94 nM, respectively. These results demonstrate that TC is the most specific PP1 inhibitor out of over 40 species of natural phosphatase inhibitors reported, strongly suggesting that TC is a novel powerful tool to elucidate the physiological roles of PP1 in various biological events.  相似文献   

13.
14.
The catalytic subunit of protein serine/threonine phosphatase 4 (PP4C) has greater than 65% amino acid identity to the catalytic subunit of protein phosphatase 2A (PP2AC). Despite this high homology, PP4 does not appear to associate with known PP2A regulatory subunits. As a first step toward characterization of PP4 holoenzymes and identification of putative PP4 regulatory subunits, PP4 was purified from bovine testis soluble extracts. PP4 existed in two complexes of approximately 270-300 and 400-450 kDa as determined by gel filtration chromatography. The smaller PP4 complex was purified by sequential phenyl-Sepharose, Source 15Q, DEAE2, and Superdex 200 gel filtration chromatographies. The final product contained two major proteins: the PP4 catalytic subunit plus a protein that migrated as a doublet of 120-125 kDa on SDS-polyacrylamide gel electrophoresis. The associated protein, termed PP4R1, and PP4C also bound to microcystin-Sepharose. Mass spectrometry analysis of the purified complex revealed two major peaks, at 35 (PP4C) and 105 kDa (PP4R1). Amino acid sequence information of several peptides derived from the 105 kDa protein was utilized to isolate a human cDNA clone. Analysis of the predicted amino acid sequence revealed 13 nonidentical repeats similar to repeats found in the A subunit of PP2A (PP2AA). The PP4R1 cDNA clone engineered with an N-terminal Myc tag was expressed in COS M6 cells and PP4C co-immunoprecipitated with Myc-tagged PP4R1. These data indicate that one form of PP4 is similar to the core complex of PP2A in that it consists of a catalytic subunit and a "PP2AA-like" structural subunit.  相似文献   

15.
Calcineurin was demonstrated to regulate the phosphorylation of threonine (T)-172 of CDK4. We further investigated how calcineurin can regulate this essential post-translational modification on CDK4. In this study, we demonstrate that calcineurin can associate predominantly with the cytoplasmic form of CDK4 in the absence of cyclin D. The inhibition of calcineurin phosphatase activity resulted in the specific increase of the phosphorylation and activity levels of CDK4 within the mitotic fraction. The association of calcineurin with CDK4 peaked during the mitotic phase of the cell cycle and coincided with reduction of CDK4 phosphorylation. Using structural mutants to CDK4, we localized the interaction site of calcineurin within the amino terminal residues of CDK4 that are important for both cyclin D and p16INK4a binding. Our data suggest that calcineurin may regulate the kinase activity of CDK4 in a cell cycle-dependent manner and may be an important component of the negative regulation of CDK4.  相似文献   

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17.
S Lin  W Chen    S S Broyles 《Journal of virology》1992,66(5):2717-2723
The nucleotide sequence of the vaccinia virus open reading frame B1 predicts a polypeptide with significant sequence similarity to the catalytic domain of known protein kinases. To determine whether the B1R polypeptide is a protein kinase, we have expressed it in bacteria as a fusion with glutathione S-transferase. Affinity-purified preparations of the fusion protein were found to undergo autophosphorylation and also phosphorylated the exogenous substrates casein and histone H1. Mutation of lysine 41 to glutamine within the conserved kinase catalytic domain II abrogated protein kinase activity on all three protein substrates, supporting the notion that the protein kinase activity is inherent to the B1R polypeptide. Casein and histone H1 were phosphorylated on serine and threonine residues. The B1R fusion protein was phosphorylated on a threonine residue(s) by an apparently intramolecular mechanism. The autophosphorylation reaction resulted in phosphorylation of the glutathione S-transferase portion of the fusion and not the protein kinase domain. The protein kinase activity of B1R was specific for ATP as the phosphate donor; GTP was not utilized to a detectable extent. Immunoblotting experiments with anti-B1R antiserum showed that the protein kinase is located in the virion particle. Chromatography of virion extracts resulted in separation of the B1R protein kinase from the bulk of the total protein kinase activity, indicating that multiple protein kinases are present in the virion particle and that B1R is distinct from the previously described vaccinia virus-associated protein kinase.  相似文献   

18.
Protein phosphatase 2C (PP2C) is a Mn2+- or Mg2+-dependent protein Ser/Thr phosphatase that is essential for regulating cellular stress responses in eukaryotes. The crystal structure of human PP2C reveals a novel protein fold with a catalytic domain composed of a central beta-sandwich that binds two manganese ions, which is surrounded by alpha-helices. Mn2+-bound water molecules at the binuclear metal centre coordinate the phosphate group of the substrate and provide a nucleophile and general acid in the dephosphorylation reaction. Our model presents a framework for understanding not only the classical Mn2+/Mg2+-dependent protein phosphatases but also the sequence-related domains of mitochondrial pyruvate dehydrogenase phosphatase, the Bacillus subtilus phosphatase SpoIIE and a 300-residue domain within yeast adenyl cyclase. The protein architecture and deduced catalytic mechanism are strikingly similar to the PP1, PP2A, PP2B family of protein Ser/Thr phosphatases, with which PP2C shares no sequence similarity, suggestive of convergent evolution of protein Ser/Thr phosphatases.  相似文献   

19.
The binding of Ag- and Cd-substituted plastocyanin to reduced photosystem 1 of spinach has been studied through the rotational correlation time of plastocyanin measured by the technique of perturbed angular correlation of gamma-rays (PAC). Ag and Cd are used as models for native Cu(I) and Cu(II), respectively. A dissociation constant of 5 microM was found for Ag-plastocyanin, whereas the dissociation constant was at least 24 times higher for Cd-plastocyanin. PAC was further used to characterize the structure of the metal site of Cd- and Ag-plastocyanin. The Cd spectra are characteristic of a planar configuration of one cysteine and two histidines. However, the spectra show an unusual peak broadening and a high degree of internal motion, interpreted as motion of one of the histidines within the plane. (111)Ag decays to (111)Cd, followed by the emission of two gamma-rays used for the PAC experiment. The (111)Ag PAC spectra indicate that one of the coordinating histidines has a different position in the Ag protein than in the Cd protein but that the decay of Ag to Cd causes a relaxation of the position of this histidine to the position in the Cd protein within 20 ns. Binding of Ag-plastocyanin to photosystem I stabilized the Ag metal site structure so that no relaxation was observed on a time scale of 100 ns. This stabilization of the Ag structure upon binding indicates that the metal site structure is involved in regulating how the dissociation constant for plastocyanin depends on the charge of the metal ion.  相似文献   

20.
Autophagy is a cellular degradation process that is up-regulated upon starvation. Nutrition-dependent regulation of mTOR (mammalian target of rapamycin) is a major determinant of autophagy. RTK (receptor tyrosine kinase) signalling and AMPK (AMP-activated protein kinase) converge upon mTOR to suppress or activate autophagy. Nutrition-dependent regulation of autophagy is mediated via mTOR phosphorylation of the serine/threonine kinase ULK1 (unc51-like kinase 1). In the present study, we also describe ULK1 as an mTOR-independent convergence point for AMPK and RTK signalling. We initially identified ULK1 as a 14-3-3-binding protein and this interaction was enhanced by treatment with AMPK agonists. AMPK interacted with ULK1 and phosphorylated ULK1 at Ser(555) in vitro. Mutation of this residue to alanine abrogated 14-3-3 binding to ULK1, and in vivo phosphorylation of ULK1 was blocked by a dominant-negative AMPK mutant. We next identified a high-stringency Akt site in ULK1 at Ser(774) and showed that phosphorylation at this site was increased by insulin. Finally, we found that the kinase-activation loop of ULK1 contains a consensus phosphorylation site at Thr(180) that is required for ULK1 autophosphorylation activity. Collectively, our results suggest that ULK1 may act as a major node for regulation by multiple kinases including AMPK and Akt that play both stimulatory and inhibitory roles in regulating autophagy.  相似文献   

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