Tyrosine phosphorylation and dephosphorylation of proteins is known to play a central role in regulation of cell growth and differentiation. Activation of resting T cells via their antigen-specific receptor (TCR)/CD3 complex is initiated by rapid tyrosine phosphorylation of a number of regulatory proteins, such as phospholipase Cgamma1. Identification of the protein tyrosine kinases (PTKs) involved, their regulators and their substrates are crucial to our understanding of T cell physiology in health and disease. The objective of this proposal is to study the regulation, by reversible tyrosine phosphorylation/dephosphorylation, of the catalytic activity of three src-family PTKs expressed in T cells, i.e., p56lck, p59fyn and p62c-yes, and characterize the two enzymes that mediate these regulatory activities, namely, the cytosolic PTK p50csk and the phosphotyrosine phosphatase CD45. First, the properties and biology of p50csk, which is not well characterized at present, will be studied by analyzing its regulation during T cell activation, substrate specificity, and the details of its interactions and associations with src-family PTKs (by phosphorylation of a negative regulatory tyrosine residue and down- regulation of their catalytic activity) in vitro or in intact T cells. Second, the opposite regulatory event, namely the dephosphorylation of the negative regulatory site by CD45 will be further characterized by analyzing the dephosphorylation of p59fyn and p62c-yes in vitro by isolated CD45 and the in vivo interactions of CD45 with these two src- family PTKs in CD45-/CD45+ pairs of T cell transfectants overexpressing the relevant PTKs. In addition, the potential regulation of CD45 by reversible tyrosine phosphorylation will be assessed. Preliminary experiments indicate that CD45 is phosphorylated on tyrosine in vivo and can be phosphorylated by recombinant p56lck in vitro. The significance of this modification will be evaluated by mapping the (activation- induced?) tyrosine phosphorylation sites of CD45, and determining their potential role by site-directed mutagenesis and functional characterization of the mutated CD45 molecules in transfected T cells.
