Involvement of pp60c-src with two major signaling pathways in human breast cancer.

The phosphotyrosine residues of receptor tyrosine kinases serve as unique binding sites for proteins involved in intracellular signaling, which contain SRC homology 2 (SH2) domains. Since overexpression or activation of the pp60c-src kinase has been reported in a number of human tumors, including primary human breast carcinomas, we examined the interactions of the SH2 and SH3 domains of human SRC with target proteins in human carcinoma cell lines. Glutathione S-transferase fusion proteins containing either the SH2, SH3, or the entire SH3/SH2 region of human SRC were used to affinity purify tyrosine-phosphorylated proteins from human breast carcinoma cell lines. We show here that in human breast carcinoma cell lines, the SRC SH2 domain binds to activated epidermal growth factor receptor (EGFR) and p185HER2/neu. SRC SH2 binding to EGFR was also observed in a nontumorigenic cell line after hormone stimulation. Endogenous pp60c-src was found to tightly associate with tyrosine-phosphorylated EGFR. Association of the SRC SH2 with the EGFR was blocked by tyrosyl phosphopeptides containing the sequences surrounding tyrosine-530, the regulatory site in the SRC C terminus, or sequences surrounding the major sites of autophosphorylation in the EGFR. These results raise the possibility that association of pp60c-src with these receptor tyrosine kinases is an integral part of the signaling events mediated by these receptors and may contribute to malignant transformation. IMAGES:

受体酪氨酸激酶的磷酸酪氨酸残基，是参与细胞内信号转导的蛋白质的特异性结合位点，此类蛋白质携带有Src同源2（SH2）结构域。鉴于已有研究证实，pp60c-src激酶的过表达与激活可在多种人类肿瘤中被检测到，其中包括原发性人类乳腺癌，本研究针对人Src的SH2与SH3结构域在人类癌细胞系中与其靶蛋白的相互作用展开了分析。我们采用携带有完整人Src SH2结构域、SH3结构域，或是完整SH3/SH2区域的谷胱甘肽S-转移酶融合蛋白，从人乳腺癌细胞系中亲和纯化酪氨酸磷酸化蛋白。本研究结果显示，在人乳腺癌细胞系中，Src的SH2结构域可结合激活型表皮生长因子受体（EGFR）与p185HER2/neu。经激素刺激后的非致瘤性细胞系中，同样可观测到Src SH2结构域与EGFR的结合。研究发现，内源性pp60c-src可与酪氨酸磷酸化的EGFR形成紧密结合复合物。Src SH2结构域与EGFR的结合可被两类酪氨酸磷酸肽竞争性阻断：一类包含Src C端调控位点酪氨酸530周边的氨基酸序列，另一类包含EGFR主要自磷酸化位点周边的氨基酸序列。上述研究结果提示，pp60c-src与这类受体酪氨酸激酶的结合，是上述受体介导的信号转导过程的核心组成部分，或可推动恶性转化的发生。IMAGES: