Recognition of antigen by the T cell antigen receptor complex (TCR) leads to the activation of protein tyrosine kinases (PTKs), the generation of second messengers from the hydrolysis of phosphatidylinositol (PI), and ultimately an appropriate T cell immune response. Recent studies have demonstrated that the transmembrane tyrosine phosphatase (PTP) CD45 is essential for T cell activation. In CD45-negative (CD45-) mutants of various T cell lines, the TCR is uncoupled from PI hydrolysis and PTK activation. In these CD45- T cells, the kinase activity of the src family PTKs lck and fyn is altered, implying that one critical aspect of the role of CD45 in T cell activation is the modulation of lck and fyn activity. Our central hypothesis is that CD45 regulates the activity of lck, fyn, and a third PTK, ZAP-70, and that this regulation is purposefully altered during T cell activation to allow signal transduction to occur. We propose a model in which CD45 normally maintains lck, fyn, and ZAP-70 in a relatively inactive state through the control of phosphorylation of critical regulatory tyrosine residues. The goal of the proposed studies is to explore the validity of this model and, in so doing, elucidate the precise molecular nature of the regulation of lck, fyn, and ZAP-70 by CD45. First, the effect of T cell activation on the catalytic activity and specific phosphorylation of lck, fyn, and ZAP-70 will be characterized using in vitro kinase assays and highly specific phosphopeptide mapping. These studies will be done in normal murine splenic T cells and a variety of T cell lines and clones to allow the selection of a model system that most closely approximates the normal T cell. We will then compare the identified changes in activity and phosphorylation of lck, fyn, and ZAP-70 seen after activation to those seen in situations where CD45 activity is known to be diminished, namely pharmacologic inhibition of CD45 and cD45 mutant T cell lines or clones. The focus will then shift to the effects of TCR-mediated activation on CD45 itself, in terms of its phosphatase activity and phosphorylation. A temporal correlation between the post- activation changes in CD45 and those in lck, fyn, and ZAP-70 can then be evaluated. In this regard, we present new data in this proposal that CD45 activity is decreased after activation through the TCR. Finally, we will determine the effects of the identified CD45-regulated phosphorylations of lck on lck enzymatic activity and the ability of lck to transduce an activation signal after TCR engagement, using a site-directed mutagenesis and transfection approach in an lck-deficient T cell line. Unraveling the molecular mechanisms of the interaction between CD45 and these critical T cell PTKs will aid our understanding of how an immune response is generated and maintained. Furthermore, our results may suggest specific strategies to interrupt aberrant immune responses at a very proximal site, potentially through specific targeting of these PTKs or CD45 itself.
