Overexpression of both cellular Src (c-Src) and the epidermal growth factor receptor (EGFR) occurs in many of the same human tumors, suggesting that they may functionally interact and contribute to the progression of cancer. Indeed, in murine fibroblasts, overexpression of c-Src has been shown to potentiate the mitogenic and tumorigenic capacity of the overexpressed EGFR. Potentiation correlated with the ability of c-Src to physically associate with the activated EGFR and the appearance of two unique in vivo phosphorylations on the receptor (Tyr-845 and Tyr-1101). Using stable cell lines of C3H10T 1 ⁄2 murine fibroblasts that contain kinase-deficient (K؊) c-Src and overexpressed wildtype EGFR, we show that the kinase activity of c-Src is required for both the biological synergy with the receptor and the phosphorylations on the receptor, but not for the association of c-Src with the receptor. In transient transfection assays, not only epidermal growth factor but also serum-and lysophosphatidic acid-induced DNA synthesis was ablated in a dominant-negative fashion by a Y845F mutant of the EGFR, indicating that c-Src-induced phosphorylation of Y845 is critical for the mitogenic response to both the EGFR and a G protein-coupled receptor (lysophosphatidic acid receptor). Unexpectedly, the Y845F mutant EGFR was found to retain its full kinase activity and its ability to activate the adapter protein SHC and extracellular signal-regulated kinase ERK2 in response to EGF, demonstrating that the mitogenic pathway involving phosphorylation of Y845 is independent of ERK2-activation. The application of these findings to the development of novel therapeutics for human cancers that overexpress c-Src and EGFR is discussed.Considerable evidence has accumulated in recent years to suggest that cellular Src (c-Src) and members of the epidermal growth factor (EGF) receptor (EGFR) family are critical elements in the etiology of multiple human cancers. Both kinases are found overexpressed in many of the same types of tumors, including glioblastomas and carcinomas of the colon, breast, and lung (1-4), raising the question of whether they functionally interact to promote the growth of these malignancies. In breast cancer, overexpression of EGFR family members is estimated to occur in 60% or more of the cases (5), and overexpression of the family member HER2͞NEU, has been associated with a poor prognosis for the disease (6). Recent reports have also described overexpression of c-Src in a significant majority of patients with breast cancer, a frequency that approaches 100% (1). Studies to assess the oncogenic potential of each kinase have shown that the EGFR is tumorigenic when overexpressed in cultured fibroblasts and activated by ligand (7,8), but overexpression of c-Src alone is insufficient for malignant transformation (9, 10).A possible role for c-Src in tumorigenesis was revealed when it was demonstrated in C3H10T 1 ⁄2 murine fibroblasts that co-overexpression of c-Src and the EGFR resulted in a synergistic increase in EGF-ind...
Both the non-receptor tyrosine kinase, c-Src, and members of the epidermal growth factor (EGF) receptor family are overexpressed in high percentages of human breast cancers. Because these molecules are plasma membrane-associated and involved in mitogenesis, it has been speculated that they function in concert with one another to promote breast cancer development and progression. Evidence to date supports a model wherein c-Src potentiates the survival, proliferation and tumorigenesis of EGF receptor family members, in part by associating with them. Phosphorylation of the EGF receptor by c-SRC is also critical for mitogenic signaling initiated by the EGF receptor itself, as well as by several G-protein coupled receptors (GPCRs), a cytokine receptor, and the estrogen receptor. Thus, c-Src appears to have pleiotropic effects on cancer cells by modulating the action of multiple growth-promoting receptors.
Overexpression of the epidermal growth factor receptor (EGFR) and its association with the tyrosine kinase, c-Src, is correlated with increased cellular proliferation and tumorigenesis. Previous studies have shown that EGFR and c-Src co-overexpression and association leads to the c-Src-mediated phosphorylation of tyrosine 845 of the EGFR and that mutation of Tyr(845) ablates epidermal growth factor (EGF)-induced DNA synthesis. Here, we investigate the contribution of the signal transducers and activators of transcription (STAT5b) in the signaling pathways regulated by EGFR and c-Src overexpression in human breast tumor cell lines as well as in a mouse fibroblast model (C3H10T1/2). We demonstrate that 1) activation of STAT5b by EGF requires overexpression of the EGFR, 2) co-overexpression of c-Src alone does not result in EGF-induced activation of STAT5b but enhances that seen in EGFR-overexpressing cells, and 3) EGF-induced tyrosine phosphorylation of STAT5b requires Tyr(845) of the EGFR. Furthermore, the stable overexpression of a kinase-defective c-Src in the context of EGFR overexpression results in a decrease in the tyrosine phosphorylation of STAT5b in response to EGF and a more dramatic decrease in EGF-induced transcriptional activation of STAT5b, suggesting an integral role for c-Src in the physiological actions of STAT5b. Using a dominant negative STAT5b, we provide evidence that one such physiological action is to mediate EGF-induced DNA-synthesis. Finally, the use of site-specific tyrosine mutants demonstrates that EGF-induced phosphorylation of STAT5b involves not only tyrosine 699 of STAT5b, which is required for its transcriptional activation, but also three previously identified tyrosines in the C terminus of STAT5b (Tyr(725)/Tyr(740)/Tyr(743)).
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