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Functional characterization of the low-molecular-mass phosphotyrosine-protein phosphatase of Acinetobacter johnsonii.
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International audience. The ptp gene of Acinetobacter johnsonii was previously reported to encode a low-molecular-mass protein, Ptp, whose amino acid sequence, predicted from the theoretical analysis of the nucleotide sequence of the gene, exhibits a high degree of similarity with those of different eukaryotic and prokaryotic phosphotyrosine-protein phophatases. We have now 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 Ptp can catalyse the dephosphorylation of p-nitrophenyl phosphate and phosphotyrosine, but has no effect on phosphoserine or phosphothreonine. Its activity is blocked by ammonium molybdate and sodium orthovanadate, which are strong inhibitors of phosphotyrosine-protein phosphatases, as well 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 not proteins modified on either serine or threonine. In addition, Ptp has been shown to quantitatively dephosphorylate two exogenous peptides, derived respectively from leech hirudin and human gastrin, previously phosphorylated on tyrosine. Moreover, site-directed mutagenesis experiments performed on Cys11 and Arg16, which are both present in the sequence motif (H/V)C(X5)R(S/T) typical of eukaryotic phosphotyrosine-protein phosphatases, have demonstrated that each amino acid residue is essential for the catalytic activity of Ptp. Taken together, these data provide evidence that Ptp is a member of the phosphotyrosine-protein phosphatase family. Furthermore, in search for the biological function of Ptp, we have found that it can specifically dephosphorylate an endogenous protein kinase, termed Ptk, which is known to autophosphorylate at multiple tyrosine residues in the inner membrane of Acinetobacter johnsonii cells. This represents the first identification of a protein substrate for a bacterial phosphotyrosine-protein phosphatase, and therefore constitutes a possible model for analysing the role of reversible phosphorylation on tyrosine in the regulation of microbial physiology.The ptp gene of Acinetobacter johnsonii was previously reported to encode a low-molecular-mass protein, Ptp, whose amino acid sequence, predicted from the theoretical analysis of the nucleotide sequence of the gene, exhibits a high degree of similarity with those of different eukaryotic and prokaryotic phosphotyrosine-protein phophatases. We have now 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 Ptp can catalyse the dephosphorylation of p-nitrophenyl phosphate and phosphotyrosine, but has no effect on phosphoserine or phosphothreonine. Its activity is blocked by ammonium molybdate and sodium orthovanadate, which are strong inhibitors of phosphotyrosine-protein phosphatases, as well 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 not proteins modified on either serine or threonine. In addition, Ptp has been shown to quantitatively dephosphorylate two exogenous peptides, derived respectively from leech hirudin and human gastrin, previously phosphorylated on tyrosine. Moreover, site-directed mutagenesis experiments performed on Cys11 and Arg16, which are both present in the sequence motif (H/V)C(X5)R(S/T) typical of eukaryotic phosphotyrosine-protein phosphatases, have demonstrated that each amino acid residue is essential for the catalytic activity of Ptp. Taken together, these data provide evidence that Ptp is a member of the phosphotyrosine-protein phosphatase family. Furthermore, in search for the biological function of Ptp, we have found that it can specifically dephosphorylate an endogenous protein kinase, termed Ptk, which is known to autophosphorylate at multiple tyrosine residues in the inner membrane of Acinetobacter johnsonii cells. This represents the first identification of a protein substrate for a bacterial phosphotyrosine-protein phosphatase, and therefore constitutes a possible model for analysing the role of reversible phosphorylation on tyrosine in the regulation of microbial physiology.
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