On the binding of ATP to the autophosphorylating protein, Ptk, of the bacterium Acinetobacter johnsonii

Obadia

et al. 2002

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.

Section: Fig 2 In Vivo Cross-linking Of Wzc In E Coli Cellsmentioning

confidence: 99%

Structural Organization of the Protein-tyrosine Autokinase Wzc within Escherichia coli Cells

Doublet

Obadia

et al. 2002

“…1). PTKs of Gram-negative bacteria are usually large proteins (ϳ80 kDa) composed of an N-terminal transmembrane domain and a C-terminal cytosolic PTK domain (22) containing the active site Walker motifs A and B (23). When expressed separately, the soluble C-terminal domain of E. coli Wzc still exhibits autophosphorylating activity (24).…”

mentioning

confidence: 99%

Autophosphorylation of the Escherichia coli Protein Kinase Wzc Regulates Tyrosine Phosphorylation of Ugd, a UDP-glucose Dehydrogenase

Obadia

Mijaković

et al. 2003

118

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, UDPglucose 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.

“…These various proteins share several common structural features specific to bacteria, namely the Walker A and B ATPbinding motifs (17), which are not usually found in the counterpart eukaryotic kinases, and a series of tyrosine residues clustered at the C-terminal end of the molecule. For Ptk and Wzc, the Walker A motif has been shown to be effectively employed for autophosphorylation of the protein on tyrosine, suggesting that bacteria utilize for phosphorylation a novel mechanism different from that of eukaryotes (10,17).…”

mentioning

confidence: 99%

“…For Ptk and Wzc, the Walker A motif has been shown to be effectively employed for autophosphorylation of the protein on tyrosine, suggesting that bacteria utilize for phosphorylation a novel mechanism different from that of eukaryotes (10,17). On the other hand, it has been suggested that the tyrosine cluster would be the target sequence for autophosphorylation (10,18), but no accurate characterization of the concerned residues has been made and its function remains unknown.…”

mentioning

confidence: 99%

Tyrosine Phosphorylation of Protein Kinase Wzc fromEscherichia coli K12 Occurs through a Two-step Process

Doublet

Cozzone

2002

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 Nterminal domain, including two transmembrane ␣-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.