The broad, long term objectives of this project are to characterize the structure, modes of regulation and physiological function of members of the protein tyrosine phosphatase (PTP) family of enzymes. The coordinated and competing actions of both tyrosine kinases (PTKs) and PTP's are critical for regulation of the signal transduction pathways that underlie fundamental physiological processes such as growth and differentiation. Disturbance of this delicate balance between PTK's and PTP's has been shown to be a cause of a variety of human diseases including cancer, diabetes, and inflammation. Characterization of both PTK's and PTPs will be a prerequisite to understanding the physiological consequences of tyrosine phosphorylation and how this process is abrogated in the pathogenesis of decrease. This proposal describes two basic strategies to characterize the physiological function of PTP1B, TCPTP, PTPH1 and PTP-PEST. First the results os X-ray crystallographic analyses of PTP1B complexed to a peptide substrate have directed the production of forms of the PTP that adopt a catalytically competent conformation but are unable to dephosphorylate the substrate. These mutants will be expressed and used to trap physiological substrates. The second strategy is based on the hypothesis that the noncatalytic segments of the PTPs exert a regulatory function through direct medullation of activity and control of subcellular location, that is mediated by protein; protein interactions. Using a combination of protein purification and molecular biology approaches, including two hybrid screening, these regulatory proteins will be identified and characterized. The sites of interaction with the PTP will be mapped and used to generate specific antagonists of PTP function. The combination of identification of physiological substrates, characterization of regulatory proteins and generation of specific molecular probes should provide important insights into the physiological function of these enzymes. In view of these objectives the specific aims are: 1) To continue our structural analyses of PTP1B and TCPTP, to characterize further their catalytic mechanism and mode of regulation. 2) To utilize the structural information to generate probes with which to examine the physiological function of PTP1B. 3) To identify and characterize potential regulatory proteins that interact with the C-terminal segment of TCPTP. 4) To characterize the regulation and function of the band 4.1-related enzyme, PTPH1. 5) To characterize athe regulation and function of PTP-PEST.
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