The laboratory is concened with identifying 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 T cell receptor (TCR)), suggesting that Cbl is involved in the signal transduction pathways triggered by distinct receptor types. In addition, Cbl associates with numerous proteins which are thought to be active in the regulation of receptor mediated activation events. In T cells 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 appears to be acting upstream of Ras itself, possibly by regulating the association of Grb2 with Sos (or other guanidine exchange factors). Although Cbl over- expression led to a significant decrease in NF-AT activation after TCR cross-linking, it had no affect on NF-AT activity when cells were stimulated with PMA and ionomycin or an activated form of the Ca++-sensitive phosphatase, calcineurin, suggesting that Cbl may also regulate PLCg1 activation. Recent studies indicate that cells that over-express Cbl have a significant decrease in the generation of phosphoinositol phosphates (IP3) after TCR-cross-linking suggesting PLCg1 activity is indeed reduced. Activation induced tyrosine phosphorylation of PLCg1 in also reduced in Cbl over-expressing cells. This further suggests that Cbl participates in the regulation of PLCg1 activation during receptor-mediated T cell activation. Because Cbl can regulate both the RAS and PLCg1/ Ca++ activation pathways we feel these data suggest that Cbl acts as a """"""""complex"""""""" adapter molecule. In this role, Cbl may act by pre-assembling an ordered complex of effector and adapter molecules prior to TCR activation, and facilitating efficient signal transduction after receptor engagement. In assembling such a complex in unactivated cells, Cbl may also egulate the interaction of specific effectors with this complex. Cbl over-expression negatively affects activation by disrupting the pyhsiologic stoichiometry of the activation complexes. We are currently generating Cbl truncation and deletion mutants which will be used to address which regions of Cbl are required for the regulation of each pathway and to identify the Cbl-associated molecules which are critical for both TCR-mediated RAS and PLCg1 activation.
