Ravichandran K S, Lorenz U, Shoelson S E, Burakoff S J
Division of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115.
Mol Cell Biol. 1995 Feb;15(2):593-600. doi: 10.1128/MCB.15.2.593.
The adapter protein Shc has been implicated in Ras signaling via many receptors, including the T-cell antigen receptor (TCR), B-cell antigen receptor, interleukin-2 receptor, interleukin-3 receptor, erythropoietin receptor, and insulin receptor. Moreover, transformation via polyomavirus middle T antigen is dependent on its interaction with Shc and Shc tyrosine phosphorylation. One of the mechanisms of TCR-mediated, tyrosine kinase-dependent Ras activation involves the simultaneous interaction of phosphorylated Shc with the TCR zeta chain and with a second adapter protein, Grb2. Grb2, in turn, interacts with the Ras guanine nucleotide exchange factor mSOS, thereby leading to Ras activation. Although it has been reported that in fibroblasts Grb2 and mSOS constitutively associate with each other and that growth factor stimulation does not alter the levels of Grb2:mSOS association, we show here that TCR stimulation leads to a significant increase in the levels of Grb2 associated with mSOS. This enhanced Grb2:mSOS association, which occurs through an SH3-proline-rich sequence interaction, is regulated through the SH2 domain of Grb2. The following observations support a role for Shc in regulating the Grb2:mSOS association: (i) a phosphopeptide corresponding to the sequence surrounding Tyr-317 of Shc, which displaces Shc from Grb2, abolished the enhanced association between Grb2 and mSOS; and (ii) addition of phosphorylated Shc to unactivated T cell lysates was sufficient to enhance the interaction of Grb2 with mSOS. Furthermore, using fusion proteins encoding different domains of Shc, we show that the collagen homology domain of Shc (which includes the Tyr-317 site) can mediate this effect. Thus, the Shc-mediated regulation of Grb2:mSOS association may provide a means for controlling the extent of Ras activation following receptor stimulation.
衔接蛋白Shc已被证明可通过多种受体参与Ras信号传导，这些受体包括T细胞抗原受体（TCR）、B细胞抗原受体、白细胞介素-2受体、白细胞介素-3受体、促红细胞生成素受体和胰岛素受体。此外，多瘤病毒中T抗原介导的转化依赖于其与Shc的相互作用以及Shc的酪氨酸磷酸化。TCR介导的、酪氨酸激酶依赖性Ras激活的机制之一涉及磷酸化的Shc与TCR ζ链以及与另一种衔接蛋白Grb2的同时相互作用。反过来，Grb2与Ras鸟嘌呤核苷酸交换因子mSOS相互作用，从而导致Ras激活。尽管有报道称在成纤维细胞中Grb2和mSOS彼此组成性结合，并且生长因子刺激不会改变Grb2:mSOS结合水平，但我们在此表明TCR刺激会导致与mSOS结合的Grb2水平显著增加。这种通过富含SH3脯氨酸序列相互作用发生的增强的Grb2:mSOS结合是通过Grb2的SH2结构域调节的。以下观察结果支持Shc在调节Grb2:mSOS结合中的作用：（i）对应于Shc的Tyr-317周围序列的磷酸肽，它将Shc从Grb2上置换下来，消除了Grb2与mSOS之间增强的结合；（ii）将磷酸化的Shc添加到未激活的T细胞裂解物中足以增强Grb2与mSOS的相互作用。此外，使用编码Shc不同结构域的融合蛋白，我们表明Shc的胶原同源结构域（包括Tyr-317位点）可以介导这种效应。因此，Shc介导的Grb2:mSOS结合调节可能为控制受体刺激后Ras激活的程度提供一种手段。
