DIRECT T-CELL ACTIVATION BY CHIMERIC SINGLE-CHAIN FV-SYK PROMOTES SYK-CBL ASSOCIATION AND CBL PHOSPHORYLATION

The protein tyrosine kinase Syk is activated upon engagement of immune recognition receptors. We have focused on the identification of signa ling elements immediately downstream to Syk in the pathway leading to T cell activation. To circumvent T cell receptor (TCR) CD3 activation of Src family kinases, we constructed a signaling molecule with an ext racellular single chain Fv of an anti-TNP antibody, attached via a tra nsmembrane region to Syk (scFv-Syk). In a murine T cell hybridoma, dir ect aggregation of chimeric Syk with antigen culminates in interleukin -2 production and target cell lysis. Initially, it causes an increase in the association between scFv-Syk and the cytosolic protein Cbl and subsequently promotes tyrosine phosphorylation of Cbl. Interestingly, although both Cbl and phospholipase C-gamma (PLC-gamma) are phosphoryl ated in this hybridoma upon TCR CD3 cross-linking, these two events ar e uncoupled in scFv-Syk-transfected cells, in which we were unable to detect antigen-driven PLC-gamma phosphorylation. These results support a model in which Syk can initiate and directly activate the T cell's signaling machinery and position Cbl as a primary tyrosine kinase subs trate in this pathway. Furthermore, for efficient PLC-gamma phosphoryl ation to occur in these cells, the combined actions of different tyros ine kinase families may be required.