It is now well established that a the phosphorylation of proteins on tyrosyl residues is an essential element in the control of both normal and neoplastic cell growth. This is a reversible process representing a balance between the activity of kinases and phosphates. Considerable research effort has focused on the kinases while the phosphatases have been relatively neglected. Thus, the broad, long term objectives of this research program are to characterize the structure properties and mode of regulation of the protein tyrosine phosphatases (PTPase), of which very little is known at present. This will provide a necessary and complementary perspective from which to achieve an understanding of the role of protein tyrosine phosphorylation in the control of cell function. In reference to the health-relatedness of the project it is anticipated that information pertaining to the structure and regulation of the PTPases will provide indicators to direct the search for potential biochemical causes of pathological conditions such as diabetes and transformation, and suggest means by which they may be countered.
Specific Aim 1 deals with the role of the C terminal segment of a low Mr T Cell-PTPase, in modulating its activity; it also addresses the identification and characterization of proteins that blind to, and appear to regulate the activity of the enzyme following expression in BHK cells. Synthetic peptides derived from sequences in the C terminal segment will be tested as inhibitors of a truncated PTPase expressed in Sf9 cells. Co-immunoprecipitation studies and affinity chromatography over columns constructed using the C terminal segment of the PTPase will be used to identify binding proteins. The phosphorylation of PTPase by PTKs in vivo will be investigated, and coprecipitation with the PTKs will be sought as a means to look for phosphorylation on activity. The role of phosphorylation of seryl residues in the TC-PTPase by p34cdc2 in modulating activity in vitro and in vivo will also be studied.
In specific Aim 3, the identity and mode of regulation of the PTPase that dephosphorylates p34cdc2 and commits the cell to mitosis will be ascertained. Protein biochemical approaches will be utilized with particular attention paid to the fundamental step towards understanding the control of the cell cycle. Finally, in specific Aim 4, three new PTPase cDNAs that have been identified in HeLa cells by PCR will be sequenced. The proteins will be expressed and characterized at the enzymatic level using site directed mutagenesis to elucidate the significance of some of the structural features.
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