Recognition of foreign antigen by T lymphocytes provokes a series of metabolic alterations leading to cell proliferation and the production of lymphokines. Although the components of the T cell antigen mechanism that links antigen recognition to recognition complex are rather well-defined, the signalling lymphocyte activation remains largely uncharacterized. By analogy with other cell systems, it is widely believed that activation of protein tyrosine kinases figures importantly in the lymphocyte signal transduction cascade. This conjecture has stimulated interest in p56(lck), a lymphocyte-specific membrane-associated protein tyrosine kinase. Previous studies demonstrated that p56(lck) physically associates with components of the T cell antigen recognition complex, particularly the CD4 and CD8 molecules. Physical cross-linking of CD4 activates the intrinsic kinase activity of p56(lck) and stimulates phosphorylation of the CD3 complex, which is essential for T cell antigen receptor function. In addition, stimuli that maximally activate lymphocytes provoke coordinate alterations in lck mRNA and p56(lck) protein expression. This proposal seeks support for a systematic examination of the hypothesis that p56(lck) directly transmits activation signals from the T cell antigen receptor. First, the transcriptional control elements responsible for tissue-specific and developmentally-regulated expression of the lck gene will be defined. These sequences will then be used to direct the expression of constitutively activated forms of p56(lck) in thymocytes and peripheral T lymphocytes, using cell transfection and transgenic mouse systems. In addition, three alternative genetic strategies will be used to reduce the level of functional p56(lck) in normal T cells. The levels of a T cell-specific form of P59(fyn) will be similarly manipulated. In each case, the biochemical consequences of these manipulations will be examined using in vitro activation protocols. Preliminary data encourage the view that these strategies will permit definition of the signalling functions of these protein tyrosine kinases. The long range goal of this application is the molecular description of control mechanisms mediating lymphocyte activation.
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