Weng Z, Taylor J A, Turner C E, Brugge J S, Seidel-Dugan C
ARIAD Pharmaceuticals, Cambridge, Massachusetts 02139.
J Biol Chem. 1993 Jul 15;268(20):14956-63.
The Src homology 3 (SH3) domain, located in the amino-terminal, noncatalytic half of pp60src, is highly conserved among members of the Src family of tyrosine kinases. SH3 domains have also been identified in a variety of proteins otherwise unrelated to protein-tyrosine kinases. The presence of SH3 domains in proteins with diverse functions suggests this domain may be important for directing protein-protein interactions necessary for protein function or cellular localization. To explore possible interactions between the SH3 domain and cellular proteins, we have established conditions for the isolation of proteins that bind in solution to the Src SH3 domain. A 67-amino acid fragment of c-Src containing either the entire glutathione S-transferase-SH3 domain (GST-SH3) or the SH3 domain from the neuronal form of c-Src (GST-SH3+) was expressed as a glutathione S-transferase fusion protein. The GST fusion proteins were incubated with lysates from [35S]methionine-labeled Balb/c 3T3 cells or v-Src-transformed Balb/c 3T3 cells. We found that GST-SH3, but not wild-type GST, specifically interacted with multiple cellular proteins, whereas GST-SH3+ only weakly associated with a small subset of these proteins. The majority of the SH3-binding proteins were found in particulate and detergent-insoluble cell fractions. Anti-phosphotyrosine immunoblots of the SH3-binding proteins revealed that several of the SH3-binding proteins are phosphorylated on tyrosine in v-Src-transformed cells. In addition, a number of the SH3-binding proteins were phosphorylated on serine and/or threonine in in vitro kinase assays, suggesting that one or more of the SH3-binding proteins has kinase activity. We identified paxillin, a vinculin-binding protein, as one of the Src SH3-binding proteins. This finding strongly supports the hypothesis that SH3 domains may be involved in subcellular localization of proteins to cytoskeleton and/or cellular membranes.
位于pp60src氨基末端非催化区的Src同源3（SH3）结构域在酪氨酸激酶Src家族成员中高度保守。在与蛋白酪氨酸激酶无关的多种蛋白质中也发现了SH3结构域。具有不同功能的蛋白质中存在SH3结构域，这表明该结构域可能对于指导蛋白质功能或细胞定位所需的蛋白质-蛋白质相互作用很重要。为了探索SH3结构域与细胞蛋白之间可能的相互作用，我们建立了在溶液中分离与Src SH3结构域结合的蛋白质的条件。包含整个谷胱甘肽S-转移酶-SH3结构域（GST-SH3）或来自神经元形式c-Src的SH3结构域（GST-SH3 +）的c-Src的67个氨基酸片段被表达为谷胱甘肽S-转移酶融合蛋白。将GST融合蛋白与来自[35S]甲硫氨酸标记的Balb / c 3T3细胞或v-Src转化的Balb / c 3T3细胞的裂解物一起孵育。我们发现GST-SH3而非野生型GST与多种细胞蛋白特异性相互作用，而GST-SH3 +仅与这些蛋白的一小部分弱相关。大多数SH3结合蛋白存在于颗粒状和去污剂不溶性细胞组分中。对SH3结合蛋白的抗磷酸酪氨酸免疫印迹显示，在v-Src转化的细胞中，几种SH3结合蛋白在酪氨酸上被磷酸化。此外，在体外激酶测定中，许多SH3结合蛋白在丝氨酸和/或苏氨酸上被磷酸化，这表明一种或多种SH3结合蛋白具有激酶活性。我们鉴定出桩蛋白（一种纽蛋白结合蛋白）是Src SH3结合蛋白之一。这一发现有力地支持了以下假设：SH3结构域可能参与蛋白质向细胞骨架和/或细胞膜的亚细胞定位。
