Biscardi J S, Maa M C, Tice D A, Cox M E, Leu T H, Parsons S J
Department of Microbiology and Cancer Center, Box 441, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA.
J Biol Chem. 1999 Mar 19;274(12):8335-43. doi: 10.1074/jbc.274.12.8335.
Accumulating evidence indicates that interactions between the epidermal growth factor receptor (EGFR) and the nonreceptor tyrosine kinase c-Src may contribute to an aggressive phenotype in multiple human tumors. Previous work from our laboratory demonstrated that murine fibroblasts which overexpress both these tyrosine kinases display synergistic increases in DNA synthesis, soft agar growth, and tumor formation in nude mice, and increased phosphorylation of the receptor substrates Shc and phospholipase gamma as compared with single overexpressors. These parameters correlated with the ability of c-Src and EGFR to form an EGF-dependent heterocomplex in vivo. Here we provide evidence that association between c-Src and EGFR can occur directly, as shown by receptor overlay experiments, and that it results in the appearance of two novel tyrosine phosphorylations on the receptor that are seen both in vitro and in vivo following EGF stimulation. Edman degradation analyses and co-migration of synthetic peptides with EGFR-derived tryptic phosphopeptides identify these sites as Tyr845 and Tyr1101. Tyr1101 lies within the carboxyl-terminal region of the EGFR among sites of receptor autophosphorylation, while Tyr845 resides in the catalytic domain, in a position analogous to Tyr416 of c-Src. Phosphorylation of Tyr416 and homologous residues in other tyrosine kinase receptors has been shown to be required for or to increase catalytic activity, suggesting that c-Src can influence EGFR activity by mediating phosphorylation of Tyr845. Indeed, EGF-induced phosphorylation of Tyr845 was increased in MDA468 human breast cancer cells engineered to overexpress c-Src as compared with parental MDA 468 cells. Furthermore, transient expression of a Y845F variant EGFR in murine fibroblasts resulted in an ablation of EGF-induced DNA synthesis to nonstimulated levels. Together, these data support the hypothesis that c-Src-mediated phosphorylation of EGFR Tyr845 is involved in regulation of receptor function, as well as in tumor progression.
越来越多的证据表明，表皮生长因子受体（EGFR）与非受体酪氨酸激酶c-Src之间的相互作用可能在多种人类肿瘤中导致侵袭性表型。我们实验室之前的研究表明，同时过表达这两种酪氨酸激酶的小鼠成纤维细胞在DNA合成、软琼脂生长以及裸鼠肿瘤形成方面表现出协同增加，并且与单一过表达细胞相比，受体底物Shc和磷脂酶γ的磷酸化增加。这些参数与c-Src和EGFR在体内形成依赖于表皮生长因子（EGF）的异源复合物的能力相关。在此我们提供证据表明，c-Src与EGFR之间的结合可以直接发生，如受体覆盖实验所示，并且这会导致受体上出现两种新的酪氨酸磷酸化，在EGF刺激后的体外和体内均可观察到。埃德曼降解分析以及合成肽与EGFR衍生的胰蛋白酶磷酸肽的共迁移确定这些位点为Tyr845和Tyr1101。Tyr1101位于EGFR的羧基末端区域内的受体自身磷酸化位点之中，而Tyr845位于催化结构域中，处于与c-Src的Tyr416类似的位置。已表明其他酪氨酸激酶受体中Tyr416及其同源残基的磷酸化对于催化活性是必需的或可增加催化活性，这表明c-Src可以通过介导Tyr845的磷酸化来影响EGFR活性。事实上，与亲本MDA 468细胞相比，在经过基因工程改造以过表达c-Src的MDA468人乳腺癌细胞中，EGF诱导的Tyr845磷酸化增加。此外，在小鼠成纤维细胞中瞬时表达Y845F变体EGFR导致EGF诱导的DNA合成减少至未刺激水平。总之，这些数据支持这样的假说，即c-Src介导的EGFR Tyr845磷酸化参与受体功能的调节以及肿瘤进展。
