Grgurevich S, Mikhael A, McVicar D W
Laboratory of Experimental Immunology, Division of Basic Sciences, National Cancer Institute, NCI-FCRDC, Frederick, Maryland, 21702-1201, USA.
Biochem Biophys Res Commun. 1999 Mar 24;256(3):668-75. doi: 10.1006/bbrc.1999.0398.
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.
在确定Chk在T细胞信号传导中的作用时，我们着重研究了其蛋白质-蛋白质相互作用。我们从过钒酸盐刺激的人母细胞性T细胞中检测到一种与Chk共免疫沉淀的酪氨酸磷酸化蛋白。随后的蛋白质印迹分析确定这种酪氨酸磷酸化蛋白为桩蛋白。桩蛋白是一种参与粘着斑的细胞骨架蛋白，最初在转化的成纤维细胞中被鉴定为v-Src底物。有趣的是，Chk特异性结合酪氨酸磷酸化的桩蛋白。与我们的体内数据一致，在刺激前后，Chk和桩蛋白都定位于相似的细胞区域。使用谷胱甘肽S-转移酶（GST）融合蛋白，我们确定Chk的SH2结构域而非SH3结构域结合酪氨酸磷酸化的桩蛋白。具体而言，桩蛋白与Chk SH2结构域的FLVRES基序结合。通过Far Western分析，我们发现Chk的SH2结构域直接与酪氨酸磷酸化的桩蛋白相关联。最后，在Csk缺陷型小鼠胚胎成纤维细胞中p52(Chk)的表达降低了桩蛋白总的磷酸酪氨酸水平，这暗示了Chk的生理作用。这些研究为Chk在酪氨酸介导的信号传导以及T细胞生理学中的作用提供了重要的见解。
