The linker phosphorylation site Tyr(292) mediates the negative regulatory effect of Cbl on ZAP-70 in T cells

The protooncogene product Cbl has emerged as a negative regulator of tyrosi ne kinases. We have shown previously that Cbl binds to ZAP-70 through its N -terminal tyrosine kinase binding (TKB) domain. In this study, we demonstra te that overexpression of Cbl in Jurkat T cells decreases the TCR-induced p hosphorylation of ZAP-70 and other cellular phosphoproteins. Coexpression o f Cbl with ZAP-70 in COS cells reproduced the Cbl-induced reduction in the level of phosphorylated ZAP-70, The effect of Cbl was eliminated by the TKB -inactivating G306E mutation in Cbl as well as by a phenylalanine mutation of Tyr(292) within the TKB domain binding site on ZAP-70. Notably, the onco genic Cbl-70Z/3 mutant associated with ZAP-70, but did not reduce the level s of phosphorylated ZAP-70. Overexpression of Cbl, but not Cbl-G306E, in Ju rkat T cells led to a decrease in the TCR-induced NF-AT luciferase reporter activity. Overexpression of the TKB domain itself, but not its G306E mutan t, functioned in a dominant-negative manner and led to an increase in NF-AT reporter activity. Cbl-70Z/3-overexpressing cells exhibited an increase in both basal and TCR-induced NF-AT luciferase reporter activity, and this tr end was reversed by the G306E mutation. Finally, by reconstituting a ZAP-70 -deficient Jurkat T cell line, p116, we demonstrate that wild-type ZAP-70 i s susceptible to the negative regulatory effect of Cbl, whereas the ZAP-70- Y292F mutant is resistant. Together, our results establish that the linker phosphorylation site Tyr(292) mediates the negative regulatory effect of Cb l on ZAP-70 in T cells.