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.     

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.     

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.     

Periplasmic protein-protein contacts in the inner membrane protein Wzc form a tetrameric complex required for the assembly of Escherichia coli group 1 capsules

The K antigenic capsular polysaccharide forms a structural layer, the capsule, on the surfaces of Escherichia coli cells. The capsule provides an important protective covering that helps protect encapsulated bacteria from host immune defenses. The assembly and translocation of the capsule requires proteins in the inner and outer membranes. The inner membrane protein Wzc is a tyrosine autokinase that plays an essential role in what is believed to be a coordinated biosynthesis and secretion process. Mutants lacking Wzc can form K antigen oligosaccharides but are unable to polymerize high molecular weight capsular polymers. Wzc homologs have been identified in exopolymer biosynthesis systems in many different Gram-negative and -positive bacteria. Using single particle averaging on cryo-negatively stained samples, we have produced the first three-dimensional structure of this type of membrane protein in its phosphorylated state at approximately 14 A resolution. Perfluoro-octanoate-PAGE analysis of detergent-solubilized oligomeric Wzc and symmetry analysis of the transmission electron microscopy data clearly demonstrated that Wzc forms a tetrameric complex with C4 rotational symmetry. Viewed from the top of the complex, the oligomer is square with a diameter of approximately 100 A and can be divided into four separate densities. From the side, Wzc is approximately 110 A high and has a distinctive appearance similar to an extracted molar tooth. The upper "crown" region is approximately 55 A high and forms a continuous ring of density. Four unconnected "roots" ( approximately 65 A high) emerge from the underside of the crown. We propose that the crown is formed by protein-protein contacts from the four Wzc periplasmic domains, while each root represents an individual cytoplasmic tyrosine autokinase domain.     

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.     

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.     

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.     

Relationship between exopolysaccharide production and protein-tyrosine phosphorylation in gram-negative bacteria

The phosphorylation of proteins at tyrosine residues is known to play a key role in the control of numerous fundamental processes in animal systems. In contrast, the biological significance of protein-tyrosine phosphorylation in bacteria, which has only been recognised recently, is still unclear. Here, we have analysed the role in Escherichia coli cells of an autophosphorylating protein-tyrosine kinase, Wzc, and a phosphotyrosine-protein phosphatase, Wzb, by performing knock-out experiments on the corresponding genes, wzc and wzb, and looking at the metabolic consequences induced. The results demonstrate that the phosphorylation of Wzc, as regulated by Wzb, is directly connected with the production of a particular capsular polysaccharide, colanic acid. Thus, when Wzc is phosphorylated on tyrosine, no colanic acid is synthesised by bacteria, but when dephosphorylated by Wzb, colanic acid is produced. This process is rather specific to the pair of proteins Wzc/Wzb. Indeed, a much lesser effect, if any, on colanic acid synthesis is observed when knock-out experiments are performed on another pair of genes, etk and etp, which also encode respectively a protein-tyrosine kinase, Etk, and a phosphotyrosine-protein phosphatase, Etp, in E. coli. In addition, the analysis of the phosphorylation reaction at the molecular level reveals differences between Gram-negative and Gram-positive bacteria, namely in the number of protein components required for this reaction to occur.     

Translocation of group 1 capsular polysaccharide in Escherichia coli serotype K30. Structural and functional analysis of the outer membrane lipoprotein Wza

The late steps in assembly of capsular polysaccharides (CPS) and their translocation to the bacterial cell surface are not well understood. The Wza protein was shown previously to be required for the formation of the prototype group 1 capsule structure on the surface of Escherichia coli serotype K30 (Drummelsmith, J., and Whitfield, C. (2000) EMBO J. 19, 57-66). Wza is a conserved outer membrane lipoprotein that forms multimers adopting a ringlike structure, and collective evidence suggests a role for these structures in the export of capsular polymer across the outer membrane. Wza was purified in the native form and with a C-terminal hexahistidine tag. WzaHis6 was acylated and functional in capsule assembly, although its efficiency was slightly reduced in comparison to the native Wza protein. Ordered two-dimensional crystals of WzaHis6 were obtained after reconstitution of purified multimers into lipids. Electron microscopy of negatively stained crystals and Fourier filtering revealed ringlike multimers with an average outer diameter of 8.84 nm and an average central cavity diameter of 2.28 nm. Single particle analysis yielded projection structures at an estimated resolution of 3 nm, favoring a structure for the WzaHis6 containing eight identical subunits. A derivative of Wza (Wza*) in which the original signal sequence was replaced with that from OmpF showed that the native acylated N terminus of Wza is critical for formation of normal multimeric structures and for their competence for CPS assembly, but not for targeting Wza to the outer membrane. In the presence of Wza*, CPS accumulated in the periplasm but was not detected on the cell surface. Chemical cross-linking of intact cells suggested formation of a transmembrane complex minimally containing Wza and the inner membrane tyrosine autokinase Wzc.     

Mutational analysis of the carboxy-terminal (YGX)4 repeat domain of CpsD,an autophosphorylating tyrosine kinase required for capsule biosynthesis in Streptococcus pneumoniae

In Streptococcus pneumoniae, CpsB, CpsC, and CpsD are essential for encapsulation, and mutants containing deletions of cpsB, cpsC, or cpsD exhibit rough colony morphologies. CpsD is an autophosphorylating protein-tyrosine kinase, CpsC is required for CpsD tyrosine phosphorylation, and CpsB is a phosphotyrosine-protein phosphatase. We have previously shown that autophosphorylation of CpsD at tyrosine attenuates its activity and consequently reduces the level of encapsulation and negatively regulates CPS production. In this study, we further investigated the role of the carboxy-terminal (YGX)(4) repeat domain of CpsD in encapsulation. A CpsD truncation mutant in which the entire (YGX)(4) repeat domain was removed was indistinguishable from a strain in which the entire cpsD gene had been deleted, indicating that the carboxy-terminal (YGX)(4) tail is required for CpsD activity in capsular polysaccharide production. Double mutants having a single tyrosine residue at position 2, 3, or 4 in the (YGX)(4) repeat domain and lacking CpsB exhibited a rough colony morphology, indicating that in the absence of an active protein-tyrosine phosphatase, phosphorylation of just one of the tyrosine residues in the (YGX)(4) repeat was sufficient to inactivate CpsD. When various mutants in which CpsD had either one or combinations of two or three tyrosine residues in the (YGX)(4) repeat domain were examined, only those with three tyrosine residues in the (YGX)(4) repeat domain were indistinguishable from the wild-type strain. The mutants with either one or two tyrosine residues exhibited mucoid colony morphologies. Further analysis of the mucoid strains indicated that the mucoid phenotype was not due to overproduction of capsular polysaccharide, as these strains actually produced less capsular polysaccharide than the wild-type strain. Thus, the tyrosine residues in the (YGX)(4) repeat domain are essential for normal functioning of CpsD.     

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.     

Analysis of deletions of the carboxyl terminus of the epidermal growth factor receptor reveals self-phosphorylation at tyrosine 992 and enhanced in vivo tyrosine phosphorylation of cell substrates.

The human epidermal growth factor receptor (EGFR) contains a large C' terminus distal to the protein tyrosine kinase domain that is conserved among members of its extended gene family. To investigate the C' terminus, a series of mutant EGFR cDNAs encoding progressive C'-terminal deletions were prepared and expressed in null recipient B82L cells. In vivo self-phosphorylation was retained in receptors truncated to residues 1052 and 1022 which lack the three identified sites of tyrosine self-phosphorylation. Receptors truncated to residue 991 did not undergo in vivo self-phosphorylation. Purified 1022 truncated receptor was self-phosphorylated to the extent of 1 mol of phosphate/mol of receptor protein. The deduced additional site of tyrosine self-phosphorylation at residue 992 was confirmed by tryptic phosphopeptide mapping and protein sequencing. EGFRs deleted to give C'-terminal residues 1052, 1022, 991, and 973 exhibited enhanced EGF-stimulated tyrosine phosphorylation of cell substrates in vivo, whereas deletion at residue 944 abolished all detectable EGF-stimulated protein tyrosine phosphorylation. These results indicate that ligand-induced self-phosphorylation is limited to the C' terminus of the EGFR and suggest that this region of the holoreceptor has an inhibitory function.     

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.     

Identification of structural and molecular determinants of the tyrosine‐kinase Wzc and implications in capsular polysaccharide export

Capsular polysaccharides are well‐established virulence factors of pathogenic bacteria. Their biosynthesis and export are regulated within the transmembrane polysaccharide assembly machinery by the autophosphorylation of atypical tyrosine‐kinases, named BY‐kinases. However, the accurate functioning of these tyrosine‐kinases remains unknown. Here, we report the crystal structure of the non‐phosphorylated cytoplasmic domain of the tyrosine‐kinase Wzc from Escherichia coli in complex with ADP showing that it forms a ring‐shaped octamer. Mutational analysis demonstrates that a conserved EX₂RX₂R motif involved in subunit interactions is essential for polysaccharide export. We also elucidate the role of a putative internal regulatory tyrosine and we show that BY‐kinases from proteobacteria autophosphorylate on their C‐terminal tyrosine cluster via a single‐step intermolecular mechanism. This structure‐function analysis also allows us to demonstrate that two different parts of a conserved basic region called the RK‐cluster are essential for polysaccharide export and for kinase activity respectively. Based on these data, we revisit the dichotomy made between BY‐kinases from proteobacteria and firmicutes and we propose a unique process of oligomerization and phosphorylation. We also reassess the function of BY‐kinases in the capsular polysaccharide assembly machinery.     

Expression and characterization of wild type, truncated, and mutant forms of the intracellular region of the receptor-like protein tyrosine phosphatase HPTP beta.

Human HPTP beta is unique among mammalian receptor-like protein tyrosine phosphatases in that it has only a single catalytic domain. The intracellular region of HPTP beta was expressed in bacteria, purified, and characterized. It exhibits high activity toward all substrates tested and is potently inhibited by zinc. Vanadate and polyanions also inhibited activity. The juxta-membrane segment of HPTP beta (residues 1622-1639) potentially functions as a negative regulatory sequence since its deletion can increase HPTP beta activity 5-fold. This segment contains up to two sites for protein kinase C phosphorylation, although in vitro phosphorylation by this kinase did not affect HPTP beta activity. The boundaries of the catalytic domain were delineated by truncation analyses. Successive deletion of N-terminal sequence prior to residue 1684 had little effect on substrate affinity and at most reduced activity about 6-fold. Further removal of residues 1684-1686 resulted in a marked 50-500-fold drop in activity, and loss of N-terminal sequence prior to residue 1690 abolished activity. Based on these analyses a highly conserved motif was identified in all mammalian tyrosine phosphatases (E/q) (F/y)XX(L/i), corresponding to positions 1684-1688 of HPTP beta. Mutation of residue 1684 or 1685 generally gave rise to proteins with marked temperature sensitivity. These mutant HPTP beta were active but had reduced activity compared to the wild type enzyme. In conjunction, these results suggest that this region represents the N-terminal border of the catalytic domain and is essential for correct phosphatase folding although not directly involved in catalysis. Parallel truncation studies have defined residues 1930-1939/40 as the C-terminal border of the catalytic domain.     

v-Src induces tyrosine phosphorylation of focal adhesion kinase independently of tyrosine 397 and formation of a complex with Src

The non-receptor tyrosine kinase FAK plays a key role at sites of cellular adhesion. It is subject to regulatory tyrosine phosphorylation in response to a variety of stimuli, including integrin engagement after attachment to extracellular matrix, oncogene activation, and growth factor stimulation. Here we use an antibody that specifically recognizes the phosphorylated form of the putative FAK autophosphorylation site, Tyr(397). We demonstrate that FAK phosphorylation induced by integrins during focal adhesion assembly differs from that induced by activation of a temperature-sensitive v-Src, which is associated with focal adhesion turnover and transformation. Specifically, although v-Src induces tyrosine phosphorylation of FAK, there is no detectable phosphorylation of Tyr(397). Moreover, activation of v-Src results in a net decrease in fibronectin-stimulated phosphorylation of Tyr(397), suggesting possible antagonism between v-Src and integrin-induced phosphorylation. Our mutational analysis further indicates that the binding of v-Src to Tyr(397) of FAK in its phosphorylated form, which is normally mediated, at least in part, by the SH2 domain of Src, is not essential for v-Src-induced cell transformation. We conclude that different stimuli can induce phosphorylation of FAK on distinct tyrosine residues, linking specific phosphorylation events to ensuing biological responses.     

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.     

Tyrosine phosphorylation of the well packed ephrinB cytoplasmic beta-hairpin for reverse signaling. Structural consequences and binding properties

Tyrosine phosphorylation of the 22-residue cytoplasmic region of ephrinB induces its binding to the SH2 domain of Grb4, thus initiating reverse signaling pathways controlling cytoskeleton assembly and remodeling. Recently, the region corresponding to this 22-residue motif was demonstrated to adopt a well packed beta-hairpin structure with a high conformational stability in the unphosphorylated cytoplasmic subdomain. However, because the binding to Grb4 is phosphorylation-dependent and the hairpin contains three conserved tyrosine residues that may be phosphorylated, the key events remain unknown as to how tyrosine phosphorylation affects the structure of this well packed beta-hairpin and which phosphorylation site is relevant to SH2 domain binding. By characterizing the structural and binding properties of six 22-residue SH2 domain-binding motifs with different phosphorylated sites, the present study reveals that, as shown by circular dichroism and NMR, the unphosphorylated 22-residue motif adopts a well formed beta-hairpin structure in isolation from the ephrinB cytoplasmic subdomain. However, this beta-hairpin is radically abolished by tyrosine phosphorylation, regardless of the relative location and number of Tyr residues. Unexpectedly, the peptides with either Tyr304 or Tyr316 phosphorylated show high affinity binding to SH2 domain, whereas the peptide with Tyr311 phosphorylated has no detectable binding. This implies that ephrinB with Tyr311 phosphorylated might have a currently unidentified binding partner distinct from the Grb4 protein, because Tyr311 is known to be phosphorylated in vivo. Based on the results above, it is thus proposed that the disruption of the tight side-chain packing by tyrosine phosphorylation in the well structured region of a signaling protein may represent a general activation mechanism by which a cryptic binding site is disclosed for new protein-protein interactions.     

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.     

Distinct signal transduction through the tyrosine-containing domains of the granulocyte colony-stimulating factor receptor.

The receptor for granulocyte colony-stimulating factor (G-CSFR) is a hemopoietic growth factor receptor, which mediates proliferation and differentiation signals. The cytoplasmic region of G-CSFR carries four tyrosine residues in its C-terminal half. We constructed mutant receptors in which each tyrosine residue of G-CSFR was mutated to phenylalanine. Two mutant receptors (Tyr703 and Tyr728) neither transduced the growth-inhibitory signal nor induced the neutrophil-specific myeloperoxidase (MPO) gene. The Tyr703 mutant did not induce morphological changes in cells, whereas transformants expressing the Tyr728 mutant adhered to plates with a macrophage-like morphology upon G-CSF stimulation. Mutation of the most distal tyrosine residue (Tyr763) abolished the ability of G-CSFR to stimulate the tyrosine phosphorylation of a cellular protein with an M(r) of 54 kDa. These results indicated that the regions around the three tyrosine residues of G-CSFR play essential and distinct roles in signal transduction.