FUNCTIONAL-CHARACTERIZATION OF THE LOW-MOLECULAR-MASS PHOSPHOTYROSINE-PROTEIN PHOSPHATASE OF ACINETOBACTER-JOHNSONII

The ptp gene of Acinetobacter johnsonii was previously reported to enc ode a low-molecular-mass protein, Ptp, whose amino acid sequence, pred icted from the theoretical analysis of the nucleotide sequence of the gene, exhibits a high degree of similarity with those of different euk aryotic and prokaryotic phosphotyrosine-protein phophatases. We have n ow overexpressed the ptp gene in Escherichia coli cells, purified the Ptp protein to homogeneity by a single-step chromatographic procedure, and analysed its functional properties. We have shown that Pt-p can c atalyse the dephosphorylation of p-nitrophenyl phosphate and phosphoty rosine, but has no effect on phosphoserine or phosphothreonine. Its ac tivity is blocked by ammonium molybdate and sodium orthovanadate, whic h are strong inhibitors of phosphotyrosine-protein phosphatases, as we ll as by N-ethylmaleimide and iodoacetic acid. Such specificity of Ptp for phosphotyrosine has been confirmed by the observation that it can dephosphorylate endogenous proteins phosphorylated on tyrosine, but n ot proteins modified on either serine or:threonine. In addition, Ptp h as been shown to quantitatively dephosphorylate two exogenous peptides , derived respectively from leech hirudin and human gastrin, previousl y phosphorylated on tyrosine. Moreover, site-directed mutagenesis expe riments performed on Cys11 and Arg16, which are both present in the se quence motif (H/V)C(X-5)R(S/T) typical of eukaryotic phosphotryosine-p rotein phosphatases, have demonstrated that each amino acid residue is essential for the catalytic activity of Ptp. Taken together, these da ta provide evidence that Ptp is a member of the phosphotyrosine-protei n phosphatase family. Furthermore, in search for the biological functi on of Ptp, we have found that it can specifically dephosphorylate an e ndogenous protein kinase, termed Ptk, which is known to autophosphoryl ate at multiple tyrosine residues in the inner membrane of Acinetobact er johnsonii cells. This represents the first identification of a prot ein substrate for a bacterial phosphotyrosine-protein phosphatase, and therefore constitutes a possible model for analysing the role of reve rsible phosphorylation on tyrosine in the regulation of microbial phys iology. (C) 1998 Academic Press Limited.