Muthuswamy S K, Muller W J
Department of Biology, McMaster University, Hamilton, Ontario, Canada.
Oncogene. 1995 Nov 2;11(9):1801-10.
Overexpression and amplification of the erbB-2 (neu) is thought to play a major role in mammary cancer. Although studies suggest that Neu is directly involved in the genesis of mammary tumors, the molecular mechanism by which Neu induces tumors is not well understood. Recently, we have demonstrated that the activity of c-Src tyrosine kinase is elevated in Neu-induced mammary tumors and this elevated activity correlates with its capacity to physically associate with Neu. To explore whether other members of the c-Src family are activated in these mammary tumors, we measured the in vitro kinase activity of the c-Yes and Fyn kinases in protein extracts derived from mammary tumor tissue and morphological normal adjacent tissue. These analyses revealed that c-Yes kinase activity was elevated in Neu-induced tumors by comparison to the adjacent tissue. By contrast, no significant activation of the Fyn kinase was noted in these tumors. Activation of c-Yes tyrosine kinase correlated with the capacity of c-Yes to associate with Neu in vivo in lysates derived from primary tumor samples. Studies with Rat.2 fibroblasts overexpressing activated Neu revealed that c-Src requires the presence of tyrosine phosphorylated Neu for its ability to interact with Neu in vivo. Moreover, analyses using radiolabeled c-Yes SH2 fusion protein revealed that this interaction is likely occurring in a direct fashion. Although both c-Src and c-Yes kinase associate with Neu in vivo, a tyrosine phosphorylated protein of 89 kd (p89) was found associated with c-Src but not with c-Yes in cell lysates derived from mammary epithelial cells transformed by either Neu or PyV middle T antigen. Furthermore, this tyrosine phosphorylated protein was not detected in c-Src complexes derived from fibroblasts transformed by either Neu or PyV middle T. These observations suggest that p89 associates with c-Src only in mammary epithelial cells and not in fibroblasts.
erbB-2（neu）的过表达和扩增被认为在乳腺癌中起主要作用。尽管研究表明Neu直接参与乳腺肿瘤的发生，但其诱导肿瘤的分子机制尚不清楚。最近，我们已经证明c-Src酪氨酸激酶的活性在Neu诱导的乳腺肿瘤中升高，并且这种升高的活性与其与Neu物理结合的能力相关。为了探究c-Src家族的其他成员在这些乳腺肿瘤中是否被激活，我们测量了从乳腺肿瘤组织和形态学正常的相邻组织中提取的蛋白质提取物中c-Yes和Fyn激酶的体外激酶活性。这些分析表明，与相邻组织相比，c-Yes激酶活性在Neu诱导的肿瘤中升高。相比之下，在这些肿瘤中未观察到Fyn激酶的显著激活。c-Yes酪氨酸激酶的激活与c-Yes在源自原发性肿瘤样本的裂解物中在体内与Neu结合的能力相关。对过表达活化Neu的Rat.2成纤维细胞的研究表明，c-Src在体内与Neu相互作用的能力需要酪氨酸磷酸化的Neu的存在。此外，使用放射性标记的c-Yes SH2融合蛋白的分析表明这种相互作用可能以直接方式发生。尽管c-Src和c-Yes激酶在体内均与Neu结合，但在由Neu或PyV中间T抗原转化的乳腺上皮细胞的细胞裂解物中，发现一种89 kd的酪氨酸磷酸化蛋白（p89）与c-Src结合而不与c-Yes结合。此外，在由Neu或PyV中间T转化的成纤维细胞衍生的c-Src复合物中未检测到这种酪氨酸磷酸化蛋白。这些观察结果表明，p89仅在乳腺上皮细胞中与c-Src结合，而不在成纤维细胞中结合。
