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.