Roles of Lck, Syk and ZAP-70 tyrosine kinases in TCR-mediated phosphorylation of the adapter protein Shc

Abstract

The adapter protein Shc has been implicated in mitogenic signaling via growth factor receptors, antigen receptors and cytokine receptors. Recent studies have suggested that tyrosine phosphorylation of Shc may play a key role in T lymphocyte proliferation via interaction of phosphorylated Shc with downstream molecules involved in activation of Ras and Myc proteins. However, the sites on Shc that are tyrosine phosphorylated in response to TCR engagement and the ability of different T cell tyrosine kinases to phosphorylate Shc have not been defined. In this report, we show that during TCR signaling, the tyrosines Y239, Y240 and Y317 of Shc are the primary sites of tyrosine phosphorylation. Mutation of all three tyrosines completely abolished tyrosine phosphorylation of Shc following TCR stimulation. Our data also suggest that multiple 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 able 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 synergy between the syk-family and scr-family kinases. Of the two potential Grb2 binding sites (Y239 and Y317), Y239 appears to play a greater role in recruiting Sos through Grb2. These studies have implications for Ras activation and mitogenic signaling during T cell activation.

Quantitative Analysis of Phosphotyrosine Signaling Networks Triggered by CD3 and CD28 Costimulation in Jurkat Cells

“…It is interesting to note that phosphorylation of Shc Tyr 317 has been implicated in regulating the specific interaction between Shc and Grap2 (21), which may explain the coregulation of these proteins in our study. Tyr 317 of p52 Shc was reported to be preferred by Lck whereas other sites such as Tyr 239 and Tyr 240 were phosphorylated by Syk and Zap70 in an overexpressed cell line system (22). However, our data showed better correlation between Tyr 317 of Shc and Zap70 as compared with Lck (Figs.…”

Signaling via Shc family adapter proteins

“…During in vitro studies, we observed some subtle, but reproducible di erences in phosphorylation of Y239/ 240 versus Y317 by di erent tyrosine kinases (Walk et al, 1998). It is noteworthy that recently, using speci®c Src-family kinase inhibitors, Blake et al (2000) have observed that the Y239/Y240 sites are targets for Src in vivo, while the Y317 is phosphorylated by another kinase, perhaps the PDGF receptor itself.…”

“…Interestingly, both Y239 and Y317 have the canonical +2 asparagine that is critical for the binding of Grb2 ± SH2 domain. We and others have shown that the SH2 domain of Grb2 can bind to both the Y239 and Y317 (Walk et al, 1998). Velazquez et al (2000) recently reported that Grb2 may bind preferentially to Y317, but Grb2 could bind equally well to the Y239 site when the Y240 was also phosphorylated.…”

Vav1 and Ly-GDI Two Regulators of Rho GTPases, Function Cooperatively as Signal Transducers in T Cell Antigen Receptor-induced Pathways

“…We demonstrate that Ly-GDI associates with the SH2 domain of the adapter protein Shc. Shc is tyrosine-phosphorylated by ZAP-70 upon TCR engagement, and it then forms complexes with Grb2 as well as with other proteins (66). Moreover, Shc was shown to be involved in activation of c-Rel and mitogen-activated protein kinase, and it is required for TCRinduced interleukin-2 production (67).…”