ROLES OF LCK, SYK AND ZAP-70 TYROSINE KINASES IN TCR-MEDIATED PHOSPHORYLATION OF THE ADAPTER PROTEIN SHC

The adapter protein Shc has been implicated in mitogenic signaling via growth factor receptors, antigen receptors and cytokine receptors. Re cent studies have suggested that tyrosine phosphorylation of Shc may p lay a key role in T lymphocyte proliferation via interaction of phosph orylated Shc with downstream molecules involved in activation of Ras a nd Myc proteins. However, the sites on Shc that are tyrosine phosphory lated in response to TCR engagement and the ability of different T cel l tyrosine kinases to phosphorylate Shc have not been defined. In this report, we show that during TCR signaling, the tyrosines Y239, Y240 a nd Y317 of Shc are the primary sites of tyrosine phosphorylation. Muta tion of all three tyrosines completely abolished tyrosine phosphorylat ion of Shc following TCR stimulation. Our data also suggest that multi ple T cell tyrosine kinases contribute to tyrosine phosphorylation on Shc. In T cells, CD4/Lck-dependent tyrosine phosphorylation on Shc was markedly diminished when Y317 was mutated, suggesting a preference of Lck for the Y317 site. The syk-family kinases (Syk and ZAP-70) were a ble to phosphorylate the Y239 and Y240 sites, and less efficiently the Y317 site. Moreover, co-expression of Syk or ZAP-70 with Lck resulted in enhanced phosphorylation of Shc on all three sites, suggesting a s ynergy between the syk-family and scr-family kinases. Of the two poten tial Grb2 binding sites (Y239 and Y317), Y239 appears to play a greate r role in recruiting Sos through Grb2. These studies have implications for Ras activation and mitogenic signaling during T cell activation.