Sawasdikosol S, Chang J H, Pratt J C, Wolf G, Shoelson S E, Burakoff S J
Division of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA.
J Immunol. 1996 Jul 1;157(1):110-6.
Crk is a Src homology 2 (SH2)/Src homology 3 (SH3)-containing adapter protein that has been implicated in intracellular signaling in fibroblasts and PC12 pheochromocytoma cells. Crk has been shown to bind to a tyrosine-phosphorylated protein of 116 kDa after TCR-mediated T cell activation. Here we demonstrate that the Crk-associated p116 phosphoprotein is not the Crk-associated substrate (Cas) but, rather, is a protein product of the c-cbl proto-oncogene. Whereas Cas was not tyrosine-phosphorylated after T cell activation, Cbl became highly phosphorylated. Crk immunoprecipitates from activated T cell lysates contain tyrosine-phosphorylated Cbl. This association is mediated by the SH2 domain of Crk, as evidenced by the interaction between Cbl and the fusion protein product of a glutathione S-transferase (GST) expression construct encoding the Crk-SH2 domain in vitro. Furthermore, phosphopeptide-binding studies revealed that the GST-Crk SH2 domain binds to a tyrosine-phosphorylated peptide corresponding to amino acids 770-781 of Cbl with high affinity. Cbl is a protein tyrosine kinase (PTK) substrate that becomes phosphorylated after engagement of numerous cell surface receptors including the TCR. Data revealed by genetic studies in the nematode, Caenorhabditis elegans, implicates a Cbl-like molecule, Sli-1, as a negative regulator of the Let-23-signaling pathway. Because the signal from the Let-23 pathway affects the activation status of the Let-60 (Ras homologue in C. elegans) pathway, the activation-dependent association between Crk and Cbl may represent another TCR-generated signal leading to Ras-related pathways.
Crk是一种含有Src同源2（SH2）/Src同源3（SH3）的衔接蛋白，已被证明参与成纤维细胞和PC12嗜铬细胞瘤细胞的细胞内信号传导。研究表明，TCR介导的T细胞活化后，Crk可与116 kDa的酪氨酸磷酸化蛋白结合。在此我们证明，与Crk相关的p116磷蛋白并非Crk相关底物（Cas），而是c-cbl原癌基因的蛋白产物。T细胞活化后，Cas未发生酪氨酸磷酸化，而Cbl则高度磷酸化。活化T细胞裂解物中的Crk免疫沉淀物含有酪氨酸磷酸化的Cbl。这种结合是由Crk的SH2结构域介导的，体外实验中，Cbl与编码Crk-SH2结构域的谷胱甘肽S-转移酶（GST）表达构建体的融合蛋白产物之间的相互作用证明了这一点。此外，磷酸肽结合研究表明，GST-Crk SH2结构域与对应于Cbl第770 - 781位氨基酸的酪氨酸磷酸化肽具有高亲和力结合。Cbl是一种蛋白酪氨酸激酶（PTK）底物，在包括TCR在内的众多细胞表面受体结合后会发生磷酸化。线虫秀丽隐杆线虫的遗传学研究数据表明，一种类似Cbl的分子Sli-1是Let-23信号通路的负调节因子。由于来自Let-23通路的信号会影响Let-60（秀丽隐杆线虫中的Ras同源物）通路的活化状态，Crk与Cbl之间的活化依赖性结合可能代表了另一种导致Ras相关通路的TCR产生的信号。
