The oncogenic 70Z Cbl mutation blocks the phosphotyrosine binding domain-dependent negative regulation of ZAP-70 by c-Cbl in Jurkat T cells

T-cell receptor (TCR) engagement results in the activation of Src family (L ck and Fyn) and ZAP-70 protein tyrosine kinases, leading to tyrosine phosph orylation of multiple cellular substrates including the complex adapter pro tein c-Cbl. Moreover, Cbl is tyrosine phosphorylated upon engagement of gro wth factor receptors, cytokine receptors, and immunoreceptors and functions as a negative regulator of tyrosine kinase signalling pathways. Cbl associ ates via its phosphotyrosine binding (PTB) domain to the ZAP-70 pY292 negat ive regulatory phosphotyrosine. me recently demonstrated that the oncogenic Cbl mutant, 70Z Cbl, requires its PTB domain to upregulate NFAT in unstimu lated Jurkat T cells, Here, we demonstrate that kinase-dead but not wild-ty pe forms of Fyn, Lck, and ZAP-70 block 70Z Cbl-mediated NFAT activation. Mo reover, 70Z Cbl does not upregulate NFAT in the ZAP-70-deficient P116 Jurka t T-cell line. The requirement for Fyn, Lck, and ZAP-70 is not due to tyros ine phosphorylation of 70Z Cbl, as mutation of all tyrosines in, or deletio n of, the C-terminal region of 70Z Cbl (amino acids 655 to 906) blocks 70Z Cbl tyrosine phosphorylation but enhances 70Z Cbl-mediated NFAT activation. Further, 70Z Cbl does not cooperate with ZAP-70 Y292F to upregulate NFAT, indicating that 70Z Cbl and ZAP-70 do not activate parallel signalling path ways, Finally, the upregulation of NFAT observed upon ZAP-70 overexpression is blocked by Cbl in a PTB domain-dependent manner. We conclude that oncog enic 70Z Cbl acts as a dominant negative to block the PTB domain-dependent negative regulatory role of endogenous Cbl on ZAP-70, leading to constituti ve ZAP-70 signalling and activation of transcription factors.