9631155 Strohl A gene (ptpA) from Streptomyces coelicolor was isolated that encodes a protein having high deduced sequence similarity with mammalian small, acidic phosphotyrosine protein phosphatases (PTPPs). The protein encoded by ptpA was purified and was found to have strong in vitro phosphotyrosine phosphatase activity that is competitively inhibited by dephostatin, a highly specific phosphotyrosine protein phosphatase inhibitor. ptpA was found to be expressed only under minimal medium growth conditions, the same conditions under which growth of S. coelicolor was inhibited by dephostatin, a potent inhibitor of PtpA in vitro. The working hypothesis is that PtpA and(or) other unknown PTPPs dephosphorylate target protein (or proteins), the action of which is (are) required for growth in minimal medium. These and other supportive data strongly suggest that protein tyrosine phosphorylation plays an important, but as yet undefined, role in the regulation of S. coelicolor metabolism. Very little is currently known about the role of protein-tyrosine phosphorylation in bacteria. Thus, the long term goals of this research are to elucidate the function(s) for protein tyrosine phosphorylation in S. coelicolor. The problem will be approached through the discovery of ptpA and the finding that specific PTPP and protein tyrosine kinase inhibitors conditionally inhibit S. coelicolor growth. The specific aims of this project are: (1) To determine precisely under what conditions ptpA is expressed in S. coelicolor; (2) To determine if ptpA mutation or overexpression result in observable, expected phenotypic changes. If not, then the PTPP(s) responsible for dephostatin inhibition of growth in minimal medium will be identified and isolated; (3) To study PtpA activity in vivo and to determine and isolate PtpA substrate(s); and (4) As a lead-in to future studies, mutants of S. coelicolor resistant to PTPP and protein-tyrosine kinase inhibitors will be isolated and used to initiate the isolation of other gen es associated with PTPP-related metabolism. This study will advance the knowledge of a poorly understood biochemical mechanism in bacteria, i.e., protein-tyrosine phosphorylation and dephosphorylation, and its role in regulation of bacterial metabolism. %%% Phosphorylation and dephosphorylation of proteins are involved in the relay of biochemical information from one type of metabolism to another. An unusual protein tyrosine phosphatase, an enzyme that catalyzes the removal of a phosphate group from the tyrosyl residues of other proteins, was discovered in the antibiotic-producing bacterium, Streptomyces coelicolor. Preliminary evidence suggests that this protein phosphatase may be involved in regulation of the central metabolism of this organism and perhaps also morphogenesis and antibiotic production. Thus, the goal of this research is to determine the specific function of this protein phosphatase. This research also will explore additional similar protein phosphatases to determine their potential roles in regulation of metabolism or morphogenesis in this interesting bacterium. ***
