Salcini A E, McGlade J, Pelicci G, Nicoletti I, Pawson T, Pelicci P G
Istituto Clinica Medica I, Policlinico Monteluce, University of Perugia, Italy.
Oncogene. 1994 Oct;9(10):2827-36.
The mammalian SHC gene encodes three overlapping proteins which all contain a carboxy-terminal SH2 domain. Shc proteins are phosphorylated on tyrosine by a variety of receptor and cytoplasmic tyrosine kinases. Phosphorylated Shc proteins form a complex with the SH2-SH3 containing Grb2 protein which is implicated in the regulation of Ras, suggesting that Shc is involved in the intracellular transmission of growth signals from activated tyrosine kinases to Ras. Overexpression of Shc proteins in cultured fibroblasts induces a transformed phenotype. We now report that, in vitro, the high affinity binding of Grb2 to Shc proteins requires phosphorylation of Shc at Tyr317, which lies within the high affinity binding motif for the Grb2 SH2 domain, pYVNV, where Asn at the +2 position is crucial for complex formation. In vivo, Tyr317 is the major, but not the only, site for Shc phosphorylation, and is the sole Shc high affinity binding site for Grb2. Mutant Shc proteins with substitution of the Tyr317 by Phe lose the capacity to be highly phosphorylated on tyrosine upon growth factor receptor activation, to bind Grb2 and to induce neoplastic transformation. In contrast, Shc proteins that have an extensive aminoterminal deletion, but retain the Tyr317 site and the SH2 domain conserve the capacity to be phosphorylated, to bind to Grb2 and to induce cell transformation. These data indicate that the formation of the Shc-Grb2 complex is a crucial event in the transformation induced by overexpression of Shc and support the notion that Shc proteins can deliver activation signals to RAS.
哺乳动物的SHC基因编码三种重叠蛋白，它们都含有一个羧基末端的SH2结构域。Shc蛋白可被多种受体酪氨酸激酶和细胞质酪氨酸激酶磷酸化。磷酸化的Shc蛋白与含有SH2-SH3结构域的Grb2蛋白形成复合物，而Grb2蛋白与Ras的调节有关，这表明Shc参与了从活化的酪氨酸激酶到Ras的生长信号的细胞内传递。在培养的成纤维细胞中过表达Shc蛋白会诱导转化表型。我们现在报告，在体外，Grb2与Shc蛋白的高亲和力结合需要Shc在Tyr317位点磷酸化，该位点位于Grb2 SH2结构域的高亲和力结合基序pYVNV内，其中+2位的Asn对于复合物形成至关重要。在体内，Tyr317是Shc磷酸化的主要位点，但不是唯一位点，并且是Shc与Grb2的唯一高亲和力结合位点。用Phe替代Tyr317的突变型Shc蛋白在生长因子受体激活后失去了在酪氨酸上高度磷酸化、结合Grb2和诱导肿瘤转化的能力。相反，具有广泛氨基末端缺失但保留Tyr317位点和SH2结构域的Shc蛋白保留了磷酸化、结合Grb2和诱导细胞转化的能力。这些数据表明，Shc-Grb2复合物的形成是Shc过表达诱导转化中的关键事件，并支持Shc蛋白可以向RAS传递激活信号的观点。
