Autophosphorylation of the Escherichia coli protein kinase Wzc regulates tyrosine phosphorylation of Ugd,a UDP-glucose dehydrogenase

Autophosphorylation of protein-tyrosine kinases (PTKs) involved in exopolysaccharide and capsular polysaccharide biosynthesis and transport has been observed in a number of Gram-negative and Gram-positive bacteria. However, besides their own phosphorylation, little is known about other substrates targeted by these protein-modifying enzymes. Here, we present evidence that the protein-tyrosine kinase Wzc of Escherichia coli is able to phosphorylate an endogenous enzyme, UDP-glucose dehydrogenase (Ugd), which participates in the synthesis of the exopolysaccharide colanic acid. The process of phosphorylation of Ugd by Wzc was shown to be stimulated by previous autophosphorylation of Wzc on tyrosine 569. The phosphorylation of Ugd was demonstrated to actually occur on tyrosine and result in a significant increase of its dehydrogenase activity. In addition, the phosphotyrosine-protein phosphatase Wzb, which is known to effectively dephosphorylate Wzc, exhibited only a low effect, if any, on the dephosphorylation of Ugd. These data were related to the recent observation that two other UDP-glucose dehydrogenases have been also shown to be phosphorylated by a PTK in the Gram-positive bacterium Bacillus subtilis. Comparative analysis of the activities of PTKs from Gram-negative and Gram-positive bacteria showed that they are regulated by different mechanisms that involve, respectively, either the autophosphorylation of kinases or their interaction with a membrane protein activator.     

Cells of Escherichia coli Contain a Protein-Tyrosine Kinase, Wzc, and a Phosphotyrosine-Protein Phosphatase, Wzb

Two proteins of Escherichia coli, termed Wzc and Wzb, were analyzed for their capacity to participate in the reversible phosphorylation of proteins on tyrosine. First, Wzc was overproduced from its specific gene and purified to homogeneity by affinity chromatography. Upon incubation in the presence of radioactive ATP, it was found to effectively autophosphorylate. Two-dimensional analysis of its phosphoamino acid content revealed that it was modified exclusively at tyrosine. Second, Wzb was also overproduced from the corresponding gene and purified to homogeneity by affinity chromatography. It was shown to contain a phosphatase activity capable of cleaving the synthetic substrate p-nitrophenyl phosphate into p-nitrophenol and free phosphate. In addition, it was assayed on individual phosphorylated amino acids and appeared to dephosphorylate specifically phosphotyrosine, with no effect on phosphoserine or phosphothreonine. Such specificity for phosphotyrosine was confirmed by the observation that Wzb was able to dephosphorylate previously autophosphorylated Wzc. Together, these data demonstrate, for the first time, that E. coli cells contain both a protein-tyrosine kinase and a phosphotyrosine-protein phosphatase. They also provide evidence that this phosphatase can utilize the kinase as an endogenous substrate, which suggests the occurrence of a regulatory mechanism connected with reversible protein phosphorylation on tyrosine. From comparative analysis of amino acid sequences, Wzc was found to be similar to a number of proteins present in other bacterial species which are all involved in the synthesis or export of exopolysaccharides. Since these polymers are considered important virulence factors, we suggest that reversible protein phosphorylation on tyrosine may be part of the cascade of reactions that determine the pathogenicity of bacteria.     

functional analysis of conserved gene products involved in assembly of Escherichia coli capsules and exopolysaccharides: evidence for molecular recognition between Wza and Wzc for colanic acid biosynthesis

Group 1 capsular polysaccharides (CPSs) of Escherichia coli and some loosely cell-associated exopolysaccharides (EPSs), such as colanic acid, are assembled by a Wzy-dependent polymerization system. In this biosynthesis pathway, Wza, Wzb, and Wzc homologues are required for surface expression of wild-type CPS or EPS. Multimeric complexes of Wza in the outer membrane are believed to provide a channel for polymer export; Wzc is an inner membrane tyrosine autokinase and Wzb is its cognate phosphatase. This study was performed to determine whether the Wza, Wzb, and Wzc proteins for colanic acid expression in E. coli K-12 could function in the E. coli K30 prototype group 1 capsule system. When expressed together, colanic acid Wza, Wzb, and Wzc could complement a wza-wzb-wzc defect in E. coli K30, suggesting conservation in their collective function in Wzy-dependent CPS and EPS systems. Expressed individually, colanic acid Wza and Wzb could also function in K30 CPS expression. In contrast, the structural requirements for Wzc function were more stringent because colanic acid Wzc could restore translocation of K30 CPS to the cell surface only when expressed with its cognate Wza protein. Chimeric colanic acid-K30 Wzc proteins were constructed to further study this interaction. These proteins could restore K30 biosynthesis but were unable to couple synthesis to export. The chimeric protein comprising the periplasmic domain of colanic acid Wzc was functional for effective K30 CPS surface expression only when coexpressed with colanic acid Wza. These data highlight the importance of Wza-Wzc interactions in group 1 CPS assembly.     

The solution structure of Escherichia coli Wzb reveals a novel substrate recognition mechanism of prokaryotic low molecular weight protein-tyrosine phosphatases

Low molecular weight protein-tyrosine phosphatases (LMW-PTPs) are small enzymes that ubiquitously exist in various organisms and play important roles in many biological processes. In Escherichia coli, the LMW-PTP Wzb dephosphorylates the autokinase Wzc, and the Wzc/Wzb pair regulates colanic acid production. However, the substrate recognition mechanism of Wzb is still poorly understood thus far. To elucidate the molecular basis of the catalytic mechanism, we have determined the solution structure of Wzb at high resolution by NMR spectroscopy. The Wzb structure highly resembles that of the typical LMW-PTP fold, suggesting that Wzb may adopt a similar catalytic mechanism with other LMW-PTPs. Nevertheless, in comparison with eukaryotic LMW-PTPs, the absence of an aromatic amino acid at the bottom of the active site significantly alters the molecular surface and implicates Wzb may adopt a novel substrate recognition mechanism. Furthermore, a structure-based multiple sequence alignment suggests that a class of the prokaryotic LMW-PTPs may share a similar substrate recognition mechanism with Wzb. The current studies provide the structural basis for rational drug design against the pathogenic bacteria.     

Phosphorylation of Wzc, a tyrosine autokinase, is essential for assembly of group 1 capsular polysaccharides in Escherichia coli

Wzc proteins are tyrosine autokinases. They are found in some important bacterial pathogens of humans and livestock as well as plant-associated bacteria, and are often encoded within gene clusters determining synthesis and assembly of capsular and extracellular polysaccharides. Autophosphorylation of Wzc(cps) is essential for assembly of the serotype K30 group 1 capsule in Escherichia coli O9a:K30, although a genetically unlinked Wzc(cps)-homologue (Etk) can also participate with low efficiency. While autophosphorylation of Wzc(cps) is required for assembly of high molecular weight K30 capsular polysaccharide, it is not essential for either the synthesis of the K30 repeat units or for activity of the K30 polymerase enzyme. Paradoxically, the cognate phosphotyrosine protein phosphatase for Wzc(cps), Wzb(cps), is also required for capsule expression. The tyrosine-rich domain at the C terminus of Wzc(cps) was identified as the site of phosphorylation and autophosphorylation of Wzc requires a functional Walker A motif. Intermolecular transphosphorylation of Wzc(cps) was detected in strains expressing a combination of mutant Wzc(cps) derivatives. The N- and C-terminal domains of Wzc(cps) were expressed independently to mimic the situation found naturally in Gram-positive bacteria. In this format, both domains were required for phosphorylation of the Wzc(cps) C terminus, and for capsule assembly. Regulation by a post-translational phosphorylation event represents a new dimension in the assembly of bacterial cell-surface polysaccharides.     

Involvement of a protein tyrosine kinase in production of the polymeric bioemulsifier emulsan from the oil-degrading strain Acinetobacter lwoffii RAG-1

The genes associated with the biosynthesis of the polymeric bioemulsifier emulsan, produced by the oil-degrading Acinetobacter lwoffii RAG-1 are clustered within a 27-kbp region termed the wee cluster. This report demonstrates the involvement of two genes of the wee cluster of RAG-1, wzb and wzc, in emulsan biosynthesis. The two gene products, Wzc and Wzb were overexpressed and purified. Wzc exhibited ATP-dependent autophosphorylating protein tyrosine kinase activity. Wzb was found to be a protein tyrosine phosphatase capable of dephosphorylating the phosphorylated Wzc. Using the synthetic substrate p-nitrophenyl phosphate (PNPP) Wzb exhibited a V(max) of 12 micromol of PNPP min(-1) mg(-1) and a K(m) of 8 mM PNPP at 30 degrees C. The emulsifying activity of mutants lacking either wzb or wzc was 16 and 15% of RAG-1 activity, respectively, suggesting a role for the two enzymes in emulsan production. Phosphorylation of Wzc was found to occur within a cluster of five tyrosine residues at the C terminus. Colonies from a mutant in which these five tyrosine residues were replaced by five phenylalanine residues along with those of a second mutant, which also lacked Wzb, exhibited a highly viscous colony consistency. Emulsan activity of these mutants was 25 and 24% of that of RAG-1, respectively. Neither of these mutants contained cell-associated emulsan. However, they did produce an extracellular high-molecular-mass galactosamine-containing polysaccharide. A model is proposed in which subunit polymerization, translocation and release of emulsan are all associated and coregulated by tyrosine phosphorylation.     

Crystal Structures of Wzb of Escherichia coli and CpsB of Streptococcus pneumoniae, Representatives of Two Families of Tyrosine Phosphatases that Regulate Capsule Assembly

Many Gram-positive and Gram-negative bacteria utilize polysaccharide surface layers called capsules to evade the immune system; consequently, the synthesis and export of the capsule are a potential therapeutic target. In Escherichia coli K-30, the integral membrane tyrosine autokinase Wzc and the cognate phosphatase Wzb have been shown to be key for both synthesis and assembly of capsular polysaccharides. In the Gram-positive bacterium Streptococcus pneumoniae, the CpsCD complex is analogous to Wzc and the phosphatase CpsB is the corresponding cognate phosphatase. The phosphatases are known to dephosphorylate their corresponding autokinases, yet despite their functional equivalence, they share no sequence homology. We present the structure of Wzb in complex with phosphate and high-resolution structures of apo-CpsB and a phosphate-complexed CpsB. We show that both proteins are active toward Wzc and thereby demonstrate that CpsB is not specific for CpsCD. CpsB is a novel enzyme and represents the first solved structure of a tyrosine phosphatase from a Gram-positive bacterium. Wzb and CpsB have completely different structures, suggesting that they must operate by very different mechanisms. Although the mechanism of Wzb can be inferred from previous studies, CpsB appears to have a tyrosine phosphatase mechanism not observed before. We propose a chemical mechanism for CpsB based on site-directed mutagenesis and structural data.     

Regulatory Interactions between a Bacterial Tyrosine Kinase and Its Cognate Phosphatase

The cyclic process of autophosphorylation of the C-terminal tyrosine cluster (YC) of a bacterial tyrosine kinase and its subsequent dephosphorylation following interactions with a counteracting tyrosine phosphatase regulates diverse physiological processes, including the biosynthesis and export of polysaccharides responsible for the formation of biofilms or virulence-determining capsules. We provide here the first detailed insight into this hitherto uncharacterized regulatory interaction at residue-specific resolution using Escherichia coli Wzc, a canonical bacterial tyrosine kinase, and its opposing tyrosine phosphatase, Wzb. The phosphatase Wzb utilizes a surface distal to the catalytic elements of the kinase, Wzc, to dock onto its catalytic domain (Wzc_CD). Wzc_CD binds in a largely YC-independent fashion near the Wzb catalytic site, inducing allosteric changes therein. YC dephosphorylation is proximity-mediated and reliant on the elevated concentration of phosphorylated YC near the Wzb active site resulting from Wzc_CD docking. Wzb principally recognizes the phosphate of its phosphotyrosine substrate and further stabilizes the tyrosine moiety through ring stacking interactions with a conserved active site tyrosine.     

Impact of phosphorylation of specific residues in the tyrosine autokinase,Wzc, on its activity in assembly of group 1 capsules in Escherichia coli

Wzc(CPS) is a tyrosine autokinase essential for the assembly of a high-molecular-weight (HMW) group 1 capsular polysaccharide (CPS) in Escherichia coli. Homologues of Wzc participate in the formation of CPS and exopolysaccharides in a variety of gram-positive and gram-negative bacteria. Phosphorylation of tyrosine residues in the Wzc(CPS) C terminus is essential for HMW CPS assembly. Overexpression of Wzb(CPS) (phosphatase) in a wild-type background caused a 3.7-fold decrease in the amount of cell-associated K30 CPS produced, confirming the importance of Wzc(CPS) phosphorylation for capsule assembly. In this study, the tyrosine-rich region was dissected in an attempt to identify residues critical for Wzc(CPS) phosphorylation and/or capsule expression. Site-directed mutagenesis demonstrated that no single tyrosine residue in this region is sufficient for detectable phosphorylation of Wzc(CPS) in vivo or for HMW CPS expression. Furthermore, no single tyrosine residue is essential for phosphorylation or capsule assembly, since removal of any one tyrosine residue has no detectable effect. Altering combinations of tyrosine residues (from two to five) led to Wzc(CPS) derivatives that were still competent for phosphorylation but that could not support assembly of HMW CPS, showing that phosphorylation of Wzc per se is not an accurate measure of its ability to function in capsule assembly. One interpretation of these data is that the overall level of phosphorylation in this region, rather than the precise combination of residues accessible to phosphorylation, is important for the activity of Wzc(CPS). Tyrosine 569, a residue shown to modulate the in vitro phosphorylation of Wzc(CA) from E. coli K-12, was also mutated. The derivative with this mutation still functioned in capsule assembly. Quantitation of K30(CPS) from this mutant revealed no difference in the amount of polymer produced. Finally, dithiobis(succinimidylpropionate) cross-linking was used to confirm that Wzc(CPS) forms complexes in vivo, independent of the phosphorylation state of the protein.     

Tyrosine phosphorylation of protein kinase Wzc from Escherichia coli K12 occurs through a two-step process.

In bacteria, several proteins have been shown to autophosphorylate on tyrosine residues, but little is known on the molecular mechanism of this modification. To get more information on this matter, we have analyzed in detail the phosphorylation of a particular autokinase, protein Wzc, from Escherichia coli K12. The analysis of the hydropathic profile of this protein indicates that it is composed of two main domains: an N-terminal domain, including two transmembrane alpha-helices, and a C-terminal cytoplasmic domain. The C-terminal domain alone can undergo autophosphorylation and thus appears to harbor the protein-tyrosine kinase activity. By contrast, the N-terminal domain is not phosphorylated when incubated either alone or in the presence of the C-domain, and does not influence the extent of phosphorylation of the C-domain. The C-domain contains six different sites of phosphorylation. Among these, five are located at the C-terminal end of the molecule in the form of a tyrosine cluster (Tyr(708), Tyr(710), Tyr(711), Tyr(713), and Tyr(715)), and one site is located upstream, at Tyr(569). The Tyr(569) residue can autophosphorylate through an intramolecular process, whereas the tyrosine cluster cannot. The phosphorylation of Tyr(569) results in an increased protein kinase activity of Wzc, which can, in turn, phosphorylate the five terminal tyrosines through an intermolecular process. It is concluded that protein Wzc autophosphorylates by using a cooperative two-step mechanism that involves both intra- and interphosphorylation. This mechanism may be of biological significance in the signal transduction mediated by Wzc.     

Structural organization of the protein-tyrosine autokinase Wzc within Escherichia coli cells

Protein Wzc from Escherichia coli is a member of a newly defined family of protein-tyrosine autokinases that are essential for surface polysaccharide production in both Gram-negative and Gram-positive bacteria. Although the catalytic mechanism of the autophosphorylation of Wzc was recently described, the in vivo structural organization of this protein remained unclear. Here, we have determined the membrane topology of Wzc by performing translational fusions of lacZ and phoA reporter genes to the wzc gene. It has been shown that Wzc consists of two main structural domains: an N-terminal domain, bordered by two transmembrane helices, which is located in the periplasm of cells, and a C-terminal domain, harboring all phosphorylation sites of the protein, which is located in the cytoplasm. In addition, it has been demonstrated for the first time that Wzc can oligomerize in vivo to form essentially trimers and hexamers. Cross-linking experiments performed on strains expressing various domains of Wzc have shown that the cytoplasmic C-terminal domain is sufficient to generate oligomerization of Wzc. Mutant proteins, modified in either the ATP-binding site or the different phosphorylation sites, i.e. rendered unable to undergo autophosphorylation, have appeared to oligomerize into high molecular mass species identical to those formed by the wild-type protein. It was concluded that phosphorylation of Wzc is not essential to its oligomerization. These data, connected with the phosphorylation mechanism of Wzc, may be of biological significance in the regulatory role played by this kinase in polysaccharide synthesis.     

Influence of tyrosine-kinase Wzc activity on colanic acid production in Escherichia coli K12 cells

Bacterial tyrosine-kinases have been demonstrated to participate in the regulation of capsule polysaccharides (CPS) and exopolysaccharides (EPS) production and export. However, discrepant data have been reported on the molecular mechanism responsible for this regulation depending on the bacterial species analyzed. Special attention was previously paid to the tyrosine-kinase Wzc_ca of Escherichia coli K-12, which is involved in the production of the exopolysaccharide, colanic acid, and autophosphorylates by using a cooperative two-step process. In this work, we took advantage of these observations to investigate in further detail the effect of Wzc_ca phosphorylation on the colanic acid production. First, it is shown that expression of the phosphorylated form of Wzc prevents production of colanic acid whereas expression of the non-phosphorylated form allows biosynthesis of this exopolysaccharide. However, we provide evidence that, in the latter case, the size distribution of the colanic acid polymer is less scattered than in the case of the wild-type strain expressing both phosphorylated and non-phosphorylated forms of Wzc. It is then demonstrated that colanic acid production is not merely regulated by an on/off mechanism and that, instead, both phosphorylated and non-phosphorylated forms of Wzc are required to promote colanic acid synthesis. Moreover, a series of data suggests that besides the involvement of phosphorylated and non-phosphorylated forms of Wzc in the production of colanic acid, two particular regions of this kinase play as such an important role in the synthesis of this exopolysaccharide: a proline-rich domain located in the N-terminal part of Wzc_ca, and a tyrosine cluster present in the C-terminal portion of the enzyme. Furthermore, considering that polysaccharides are known to facilitate bacterial resistance to certain environmental stresses, it is shown that the resistance of E. coli to desiccation is directly connected with the phosphorylation state of Wzc_ca.     

Tyrosine phosphorylation of CpsD negatively regulates capsular polysaccharide biosynthesis in streptococcus pneumoniae

In Streptococcus pneumoniae, the first four genes of the capsule locus (cpsA to cpsD) are common to most serotypes. By analysis of various in-frame deletion and site-directed mutants, the function of their gene products in capsular polysaccharide (CPS) biosynthesis was investigated. We found that while CpsB, C and D are essential for encapsulation, CpsA is not. CpsC and CpsD have similarity to the amino-terminal and carboxy-terminal regions, respectively, of the autophosphorylating protein-tyrosine kinase Wzc from Escherichia coli. Alignment of CpsD with Wzc and other related proteins identified conserved Walker A and B sequence motifs and a tyrosine rich domain close to the carboxy-terminus. We have shown that CpsD is also an autophosphorylating protein-tyrosine kinase and that point mutations in cpsD affecting either the ATP-binding domain (Walker A motif) or the carboxy-terminal [YGX]4 repeat domain eliminated tyrosine phosphorylation of CpsD. We describe, for the first time, the phenotypic impact of these two mutations on polysaccharide production and show that they affect CPS production differently. Whereas a mutation in the Walker A motif resulted in loss of encapsulation, mutation of the tyrosines in the [YGX]4 repeat domain resulted in an apparent increase in encapsulation and a mucoid phenotype. These data suggest that autophosphorylation of CpsD at tyrosine attenuates its activity and reduces the level of encapsulation. Additionally, we demonstrated that CpsC is required for CpsD tyrosine phosphorylation and that CpsB influences dephosphorylation of CpsD. These results are consistent with CpsD tyrosine phosphorylation acting to negatively regulate CPS production. This has implications for the function of CpsC/CpsD homologues in both Gram-positive and Gram-negative bacteria and provides a mechanism to explain regulation of CPS production during pathogenesis.     

H(2)O(2)-induced tyrosine phosphorylation of protein kinase cdelta by a mechanism independent of inhibition of protein-tyrosine phosphatase in CHO and COS-7 cells

It has been proposed that H(2)O(2) increases tyrosine phosphorylation of cellular proteins by inhibiting protein-tyrosine phosphatase through oxidation of the cysteine residue of the enzyme essential for its catalytic activity. Tyrosine phosphorylation of the delta isoform of protein kinase C (PKC) was induced by H(2)O(2) in CHO and COS-7 cells. H(2)O(2) also induced activation of mitogen-activated protein kinase. Vanadate and molybdate, which inhibit protein-tyrosine phosphatase by binding to its active site, did not induce tyrosine phosphorylation of PKCdelta, but enhanced H(2)O(2)-induced tyrosine phosphorylation of PKCdelta in the cell. The oxoanions, however, generated the active form of mitogen-activated protein kinase. Another protein-tyrosine phosphatase inhibitor, phenylarsine oxide, which bridges the thiol residues of the enzyme, induced tyrosine phosphorylation of PKCdelta, and the reaction was enhanced by vanadate. These results suggest that inhibition of protein-tyrosine phosphatase is insufficient for induction of tyrosine phosphorylation of PKCdelta in the cells, and that presumably activation of protein-tyrosine kinase may be essential for tyrosine phosphorylation of the PKC isoform.     

How tyrosine phosphorylation affects the UDP-glucose dehydrogenase activity of Bacillus subtilis YwqF

The UDP-glucose dehydrogenase activity of Bacillus subtilis YwqF is regulated by reversible phosphorylation on a tyrosine residue. This reaction, which is catalyzed by the protein-tyrosine kinase YwqD, activates the enzyme, while dephosphorylation of phosphotyrosine-YwqF by the phosphotyrosine-protein phosphatase YwqE reduces its enzyme activity. Our kinetic data indicate that the phosphorylated and unphosphorylated forms of YwqF differ in binding the substrates. The UDP-glucose dehydrogenase reaction catalyzed by YwqF is inhibited by one of its substrates, UDP-glucose, and the extent of this inhibition seems to be reduced upon YwqF phosphorylation. We propose that this effect could at least partly account for the observed activation of YwqF induced by tyrosine phosphorylation. Potential physiological implications of this finding are discussed.     

Isolation and characterization of a protein-tyrosine kinase and a phosphotyrosine-protein phosphatase from Klebsiella pneumoniae

Two proteins of Klebsiella pneumoniae, termed Yor5 and Yco6, were analyzed for their capacity to participate in the reversible phosphorylation of proteins on tyrosine. First, protein Yco6 was overproduced from its specific gene and purified to homogeneity by affinity chromatography. Upon incubation in the presence of radioactive adenosine triphosphate, it was found to effectively autophosphorylate. Two-dimensional analysis of its phosphoamino acid content revealed that it was modified exclusively at tyrosine. Second, protein Yor5 was also overproduced from the corresponding gene and purified to homogeneity by affinity chromatography. It was shown to contain a phosphatase activity capable of cleaving the synthetic substrate p-nitrophenyl phosphate into p-nitrophenol and free phosphate. In addition, it was assayed on individual phosphorylated amino acids and appeared to dephosphorylate specifically phosphotyrosine, with no effect on phosphoserine or phosphothreonine. Such specificity for phosphotyrosine was confirmed by the observation that Yor5 was able to dephosphorylate protein Yco6 previously autophosphorylated. Together, these data demonstrate that similarly to other bacterial species including Acinetobacter johnsonii and Escherichia coli, the cells of K. pneumoniae contain both a protein-tyrosine kinase and a phosphotyrosine-protein phosphatase. They also provide evidence that this phosphatase can utilize the kinase as an endogenous substrate, which suggests the occurrence of a regulatory mechanism connected with reversible protein phosphorylation on tyrosine. Since Yco6 and Yor5 are both involved in the synthesis of capsular polysaccharide and since capsules are essential to the virulence of K. pneumoniae, we suggest that reversible protein phosphorylation on tyrosine may be part of the cascade of reactions that determine the pathogenicity of bacteria.     

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

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

Budding and fission yeast casein kinase I isoforms have dual-specificity protein kinase activity.

We have examined the activity and substrate specificity of the Saccharomyces cerevisiae Hrr25p and the Schizosaccharomyces pombe Hhp1, Hhp2, and Cki1 protein kinase isoforms. These four gene products are isotypes of casein kinase I (CKI), and the sequence of these protein kinases predicts that they are protein serine/threonine kinases. However, each of these four protein kinases, when expressed in Escherichia coli in an active form, was recognized by anti-phosphotyrosine antibodies. Phosphoamino acid analysis of 32P-labeled proteins showed phosphorylation on serine, threonine, and tyrosine residues. The E. coli produced forms of Hhp1, Hhp2, and Cki1 were autophosphorylated on tyrosine, and both Hhp1 and Hhp2 were capable of phosphorylating the tyrosine-protein kinase synthetic peptide substrate polymer poly-E4Y1. Immune complex protein kinases assays from S. pombe cells showed that Hhp1-containing precipitates were associated with a protein-tyrosine kinase activity, and the Hhp1 present in these immunoprecipitates was phosphorylated on tyrosine residues. Although dephosphorylation of Hhp1 and Hhp2 by Ser/Thr phosphatase had little effect on the specific activity, tyrosine dephosphorylation of Hhp1 and Hhp2 caused a 1.8-to 3.1-fold increase in the Km for poly-E4Y1 and casein. These data demonstrate that four different CKI isoforms from two different yeasts are capable of protein-tyrosine kinase activity and encode dual-specificity protein kinases.     

Protein phosphorylation on tyrosine in bacteria

Protein phosphorylation on tyrosine has been demonstrated to occur in a wide array of bacterial species and appears to be ubiquitous among prokaryotes. This covalent modification is catalyzed by autophosphorylating ATP-dependent protein-tyrosine kinases that exhibit structural and functional features similar, but not identical, to those of their eukaryotic counterparts. The reversibility of the reaction is effected by two main classes of protein-tyrosine phosphatases: one includes conventional eukaryotic-like phosphatases and dual-specific phosphatases, and the other comprises acidic phosphatases of low molecular weight. Less frequently, a third class concerns enzymes of the polymerase-histidinol phosphatase type. In terms of genomic organization, the genes encoding a protein-tyrosine phosphatase and a protein-tyrosine kinase in a bacterial species are most often located next to each other on the chromosome. In addition, these genes are generally part of large operons that direct the coordinate synthesis of proteins involved in the production or regulation of exopolysaccharides and capsular polysaccharides. Recent data provide evidence that there exists a direct relationship between the reversible phosphorylation of proteins on tyrosine and the production of these polysaccharidic polymers, which are also known to be important virulence factors. Therefore, a new concept has emerged suggesting the existence of a biological link between protein-tyrosine phosphorylation and bacterial pathogenicity.     

Tyrosine-phosphorylated caveolin is a physiological substrate of the low M(r) protein-tyrosine phosphatase

Low M(r) phosphotyrosine-protein phosphatase is involved in the regulation of several tyrosine kinase growth factor receptors. The best characterized action of this enzyme is on the signaling pathways activated by platelet-derived growth factor, where it plays multiple roles. In this study we identify tyrosine-phosphorylated caveolin as a new potential substrate for low M(r) phosphotyrosine-protein phosphatase. Caveolin is tyrosine-phosphorylated in vivo by Src kinases, recruits into caveolae, and hence regulates the activities of several proteins involved in cellular signaling cascades. Our results demonstrate that caveolin and low M(r) phosphotyrosine-protein phosphatase coimmunoprecipitate from cell lysates, and that a fraction of the enzyme localizes in caveolae. Furthermore, in a cell line sensitive to insulin, the overexpression of the C12S dominant negative mutant of low M(r) phosphotyrosine-protein phosphatase (a form lacking activity but able to bind substrates) causes the enhancement of tyrosine-phosphorylated caveolin. Insulin stimulation of these cells induces a strong increase of caveolin phosphorylation. The localization of low M(r) phosphotyrosine-protein phosphatase in caveolae, the in vivo interaction between this enzyme and caveolin, and the capacity of this enzyme to rapidly dephosphorylate phosphocaveolin, all indicate that tyrosine-phosphorylated caveolin is a relevant substrate for this phosphatase.