Weng Z, Thomas S M, Rickles R J, Taylor J A, Brauer A W, Seidel-Dugan C, Michael W M, Dreyfuss G, Brugge J S
ARIAD Pharmaceuticals, Cambridge, Massachusetts 02139.
Mol Cell Biol. 1994 Jul;14(7):4509-21. doi: 10.1128/mcb.14.7.4509-4521.1994.
Src homology 3 (SH3) domains mediate protein-protein interactions necessary for the coupling of cellular proteins involved in intracellular signal transduction. We previously established solution-binding conditions that allow affinity isolation of Src SH3-binding proteins from cellular extracts (Z. Weng, J. A. Taylor, C. E. Turner, J. S. Brugge, and C. Seidel-Dugan, J. Biol. Chem. 268:14956-14963, 1993). In this report, we identified three of these proteins: Shc, a signaling protein that couples membrane tyrosine kinases with Ras; p62, a protein which can bind to p21rasGAP; and heterogeneous nuclear ribonucleoprotein K, a pre-mRNA-binding protein. All of these proteins contain proline-rich peptide motifs that could serve as SH3 domain ligands, and the binding of these proteins to the Src SH3 domain was inhibited with a proline-rich Src SH3 peptide ligand. These three proteins, as well as most of the other Src SH3 ligands, also bound to the SH3 domains of the closely related protein tyrosine kinases Fyn and Lyn. However, Src- and Lyn-specific SH3-binding proteins were also detected, suggesting subtle differences in the binding specificity of the SH3 domains from these related proteins. Several Src SH3-binding proteins were phosphorylated in Src-transformed cells. The phosphorylation of these proteins was not detected in cells transformed by a mutant variant of Src lacking the SH3 domain, while there was little change in tyrosine phosphorylation of other Src-induced phosphoproteins. In addition, the coprecipitation of v-Src with two tyrosyl-phosphorylated proteins with M(r)s of 62,000 and 130,000 was inhibited by incubation with a Src SH3 peptide ligand, suggesting that the binding of these substrate proteins is dependent on interactions with the SH3 domain. These results strongly suggest a role for the Src SH3 domain in the recruitment of substrates to this protein tyrosine kinase, either through direct interaction with the SH3 domain or indirectly through interactions with proteins that bind to the SH3 domain.
Src同源结构域3（SH3）介导细胞内信号转导相关细胞蛋白偶联所必需的蛋白质-蛋白质相互作用。我们之前建立了溶液结合条件，可从细胞提取物中亲和分离出与Src SH3结合的蛋白质（翁Z、泰勒JA、特纳CE、布鲁格JS和塞德尔-杜根C，《生物化学杂志》268:14956 - 14963，1993）。在本报告中，我们鉴定出了其中三种蛋白质：Shc，一种将膜酪氨酸激酶与Ras偶联的信号蛋白；p62，一种可与p21rasGAP结合的蛋白质；以及不均一核核糖核蛋白K，一种前体mRNA结合蛋白。所有这些蛋白质都含有富含脯氨酸的肽基序，可作为SH3结构域配体，并且这些蛋白质与Src SH3结构域的结合被富含脯氨酸的Src SH3肽配体所抑制。这三种蛋白质以及大多数其他Src SH3配体，也与密切相关的蛋白酪氨酸激酶Fyn和Lyn的SH3结构域结合。然而，也检测到了Src和Lyn特异性的SH3结合蛋白，表明这些相关蛋白质的SH3结构域在结合特异性上存在细微差异。几种Src SH3结合蛋白在Src转化的细胞中被磷酸化。在由缺乏SH3结构域的Src突变体转化的细胞中未检测到这些蛋白质的磷酸化，而其他Src诱导的磷蛋白的酪氨酸磷酸化几乎没有变化。此外，与Src SH3肽配体孵育可抑制v-Src与两种分子量分别为62,000和130,000的酪氨酸磷酸化蛋白的共沉淀，表明这些底物蛋白的结合依赖于与SH3结构域的相互作用。这些结果强烈表明，Src SH3结构域在将底物招募到这种蛋白酪氨酸激酶中发挥作用，要么通过与SH3结构域的直接相互作用，要么通过与结合到SH3结构域的蛋白质的间接相互作用。
