The long-term objectives of this proposal are to define the mechanisms by which the T cell receptor (TcR) is coupled to activation of serine/threonine and tyrosine kinases, and the mechanisms by which activation of these kinases results in gene induction. Antigen-unresponsive mutants of a murine T cell clone (D5, Ar-5), which are uncoupled from PI turnover and subsequent steps of activation, will be used in these studies. The T cell receptors of these cells are functional in binding ligand, and accessory molecules including CD4 and CD45 are expressed at normal levels. Hence the mutation(s) responsible for impaired PI turnover must reside either in one or more PI-specific phospholipases C (PLC), or in mechanisms coupling the phospholipases C to the T cell receptor.
The Specific aims of this proposal are to define: 1. Coupling of he T cell receptor to PI turnover: The PLC isozymes that are present in murine T cells will be identified. Isozymes which are modified or translocated in activated T cells, and thus represent potential candidates for coupling to the T cell receptor, will be tested for whether their function or coupling is impaired in the mutant cells. H. Involvement of the Fyn tyrosine kinase in TcR-PLC coupling: The mutants uncoupled from PI turnover also show a marked decrease in T cell receptor-associated Fyn tyrosine kinase activity. The structure of the Fyn protein in parent and mutant cells will be compared. The hypothesis that Fyn is implicated in TcR-PLC coupling will be tested by determining: whether Fyn regulates or associates with a phospholipase C in resting and activated T cells, whether deleting Fyn from wild-type cells by antisense or targeted mutagenesis results in loss of PI turnover, and conversely whether introducing wild-type Fyn into mutants which express reduced levels of functional Fyn restores PI turnover. III. Involvement of the CD45 tyrosine phosphatase in TcR-PLC coupling: T cells which lack the cell-surface tyrosine phosphatase CD45 appear very similar in their activation phenotype to the mutants derived in this laboratory, in that they are uncoupled from PI turnover and subsequent steps of activation. CD45 is thought to be required for maintaining the Lck tyrosine kinase in a dephosphorylated and active state. The hypotheses that CD45 is also required for Fyn activity, and that a low level of functional Fyn (and/or Lck) activity is a primary reason for the uncoupled phenotype of CD45- cells, will be tested. IV. Relation between kinase activation and induction of DNA-binding proteins: Using nuclear extracts from the D5 T cell clone, inducible proteins binding to the NFAT, AP-1, and NFkappaB sites in the 5' regions of the IL2 gene, and to the NFkappaB site of the IL2Ralpha gene, have been identified. The cells will be subjected to pharmacological manipulations which independently inhibit protein kinase C, calcium-dependent kinases, and tyrosine kinases, and tested for induction of DNA-binding proteins. These experiments will establish whether different kinases are responsible for the induction of different DNA-binding proteins.
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