The laboratory is concened with identifing the role of the cellular oncogene, cbl, in T lymphocyte activation. While the function of cbl is unknown, it is a major target of tyrosine phosphorylation after the engagement of multiple receptors, including the antigen receptors of T- and B-lymphocytes, Fc-receptors, and receptors for EGF and GM-CSF, suggesting that cbl is involved in the signal transduction pathways triggered by these distinct receptor types. In addition, cbl associates with numerous proteins which are thought to be active in the regulation of receptor mediated activation events. These proteins include adapter molecules (Grb2, nck, p85-PI3K and crk), protein tyrosine kinases (lck, fyn, Zap70 and syk), and other effector molecules, such as PLCg1. In T lymphocytes. we have found that over-expression of cbl significantly reduced the abiltiy of TCR cross-linking to activate both the AP1 (Ras) and the NF-AT (PLC/Ca++) activation pathways. In the case of AP1 activation, cbl appeared to be acting either at the level of Ras activation or upstream of Ras activation. Cbl had no affect on NF-AT activity when cells were stimulated with PMA and ionomycin suggesting that cbl may directly regulate PLCg1 activation. In addition, the association of cbl with the adaptor, Grb2, was found to decrease after T cell activation, whereas the association of the guanidine exchange factor, Sos, with Grb2 increased after TCR cross-linking. Because Grb2 is thought to mediate an activation dependent translocation of Sos, from the cytoplasm to the membrane where it interacts with and activates Ras, we are currently investigating whether cbl may influence Ras activation by regulating the ability of Sos to interact with Grb2. This is being done by analyzing how cbl over-expression affects the association of Sos with Grb2 and by generating and investigating the function of cbl mutants which are no longer able to interact with Grb2. These and additional cbl mutants will also be used to determine the mechanism by which cbl regulates the PLC/Ca++ pathway. Our lab has shown that cbl can directly interact with PLC. Epitope-tagged PLC constructs generated in our lab will be used to analyze the affect cbl has on activation-dependent PLC phosphorylation and translocation. Long range plans are to generate a cbl deficient T cell line to fully elucidate the role of cbl in T cell signaling. Cbl expression in this line can be reconstituted with wild type and mutant forms of cbl to determine the structure/function relationship of different cbl regions.
