Perturbed regulation of ZAP-70 and sustained tyrosine phosphorylation of LAT and SLP-76 in c-Cbl-deficient thymocytes

Recent studies indicate that c-Cbl and its oncogenic variants can modulate the activity of protein tyrosine kinases, This finding is supported by stud ies showing that c-Cbl interacts directly with a negative regulatory tyrosi ne in ZAP-70, and that the levels of tyrosine-phosphorylated ZAP-70 and num erous other proteins are increased in TCR-stimulated thymocytes from c-Cbl- deficient mice. Here, we demonstrate that this enhanced phosphorylation of ZAP-70 add that of two substrates, LAT and SLP-76, is not due to altered pr otein levels but is the consequence of two separate events. First, we find increased expression of tyrosine-phosphorylated TCR zeta chain in c-Cbl-def icient thymocytes, which results in a higher level of zeta-chain-associated ZAP-70 that is initially accessible for activation. Thus, more ZAP-70 is a ctivated and more of its substrates (LAT and SLP-76) become tyrosine-phosph orylated after TCR stimulation. However, an additional mechanism of ZAP-70 regulation is evident at a later time poststimulation At this time, ZAP-70 from both normal and c-Cbl(-/-) thymocytes becomes hyperphosphorylated; how ever, only in normal thymocytes does this correlate with ZAP-70 down-regula tion and a diminished ability to phosphorylate LAT and SLP-76, In contrast, c-Cbl-deficient thymocytes display altered phosphorylation kinetics, for w hich LAT phosphorylation is increased and SLP-76 phosphorylation is sustain ed. Thus, the ability to down-regulate the phosphorylation of two ZAP-70 su bstrates is impaired in c-Cbl(-/-) thymocytes, These findings provide evide nce that c-Cbl is involved in the negative regulation of the phosphorylatio n of LAT and SLP-76 by ZAP-70.