The Csk homologous kinase, Chk, binds tyrosine phosphorylated paxillin in human blastic T cells.

In determining the role of Chk in T cell signaling, we have focused on its protein-protein interactions. We detected a tyrosine phosphoprotein that coimmunoprecipitated with Chk from pervanadate stimulated human blastic T cells. Subsequent Western blot analysis identified this tyrosine phosphoprotein as paxillin. Paxillin, a cytoskeletal protein involved in focal adhesions, was first identified as a v-Src substrate in transformed fibroblasts. Interestingly, Chk specifically bound tyrosine phosphorylated paxillin. Consistent with our in vivo data, Chk and paxillin were observed to localize in similar cellular regions prior to and following stimulation. Using GST fusion proteins, we determined that the Chk SH2 domain, not the SH3 domain, bound tyrosine phosphorylated paxillin. Specifically, paxillin bound to the FLVRES motif of the Chk SH2 domain. Using Far Western analysis, we revealed that the Chk SH2 domain directly associates with tyrosine phosphorylated paxillin. Finally, p52(Chk) expression in Csk-deficient mouse embryo fibroblasts decreased total phosphotyrosine levels of paxillin, implying a physiological role for Chk. These studies provide important insight into the role of Chk in tyrosine mediated signaling, as well as T cell physiology.