Src Phosphorylation of the Epidermal Growth Factor Receptor at Novel Sites Mediates Receptor Interaction with Src and P85α

Abstract

Following ligand binding, the epidermal growth factor receptor (EGF-R) autophosphorylates itself on tyrosine residues located in its carboxyl terminus; in vitro, three sites are highly phosphorylated, while two other sites are phosphorylated to lesser extents. In the presence of the Src protein-tyrosine kinase, in vitro phosphorylation of the minor autophosphorylation sites was increased, and four additional residues were phosphorylated. Following EGF stimulation, two (Tyr-891 and Tyr-920) were found to be phosphorylated in a colorectal cell line (DLD-1) and in a breast tumor cell line (MCF7). The remaining in vitro sites were not found to be highly phosphorylated in vivo. The sequences surrounding Tyr-891 and Tyr-920 match the reported consensus binding sequences for the SH2 domains of Src and the regulatory domain of phosphatidylinositol 3-kinase (p85 alpha), respectively. In vitro, both of these proteins were found to bind to Src-phosphorylated EGF-R with approximately 100-fold greater affinity than to autophosphorylated EGF-R, demonstrating that Src creates new sites for SH2 binding. Furthermore, Csk-inactivated Src was activated by interaction with Src-phosphorylated EGF-R but not by autophosphorylated EGF-R. Upon EGF treatment of MCF7 or three colorectal carcinoma cell lines (WiDr, DLD-1, and LS174T), the EGF-R coimmunoprecipitated with both p85 alpha and Src. Evidence is also presented that suggests that an EGF-R-related protein, ErbB2, may be involved in similar Src-mediated interactions. These data demonstrate that EGF-R is phosphorylated in vivo at non-autophosphorylation sites and that these novel sites can act as docking sites for Src, P85 alpha, and potentially other SH2-containing proteins. In addition, the data suggest a tyrosine phosphatase-independent mechanism for the elevation of Src activity in cells exposed to growth factors. Overexpression of Src, EGF-R, and/or ErbB2 in breast and colorectal tumor cells suggests the potential that such interactions may contribute to the transformed phenotype of these carcinomas.

Phosphorylation of the Src substrate Sam68 by Cdc2 during mitosis

“…lamins, microtubule-associated proteins, histone H-1, nucleolin, high-mobility group proteins, caldesmon, vimentin, and Src kinase) (Chackalaparampil and Shalloway, 1988;Shenoy et al, 1989;Morgan et al, 1989;King et al, 1994;Laird et al, 1995;Minshull et al, 1994;Lee et al, 1995;Yamakita et al, 1994;O'Connell et al, 1994;Peter et al, 1991;Chou et al, 1990;Peter et al, 1990;Meijer et al, 1991;Yamashiro et al, 1990;Gottesfeld et al, 1994). During mitosis Src, which is activated following its phosphorylation by Cdc2 and dephosphorylation at Tyr 527, phosphorylates its only known mitotic substrate Sam68 (Chackalaparampil and Shalloway, 1988;Shenoy et al, 1989Shenoy et al, , 1992Morgan et al, 1989;Bagrodia et al, 1991;Stover et al, 1994;Taylor and Shalloway, 1994;Fumagalli et al, 1994). This suggests that Cdc2 might regulate Sam68 indirectly through the activation of Src.…”

“…Cellular Src is activated during mitosis in a two-step process involving the phosphorylation of its N-terminal unique domain by the Cdc2/cyclin B complex and dephosphorylation of its inhibitory C-terminal tyrosine residue 527 (Chackalaparampil and Shalloway, 1988;Shenoy et al, 1989Shenoy et al, , 1992Morgan et al, 1989;Bagrodia et al, 1991;Shenoy et al, 1992;Stover et al, 1994). However there is limited information regarding its speci®c substrates during this period.…”

Inhibition of pp60c-Src reduces Bcl-XL expression and reverses the transformed phenotype of cells overexpressing EGF and HER-2 receptors

“…pp60 c-Src (c-Src) has been shown to associate directly with the platelet derived growth factor receptor (PDGFR) (Broome and Hunter, 1996) and CSF-1 receptor (Courtneidge et al, 1993). Src was also reported to interact with EGFR (Sato et al, 1995;Stover et al, 1995), and to phosphorylate the HER-2/ neu and IGF-1 receptors (Stover et al, 1995;Peterson et al, 1996). In these cases, c-Src phosphorylates unique phosphorylation sites on these receptors that serve as docking sites for downstream signal transducers like p85, the regulatory subunit of PI 3'-kinase and Shc (Stover et al, 1995).…”

“…Src was also reported to interact with EGFR (Sato et al, 1995;Stover et al, 1995), and to phosphorylate the HER-2/ neu and IGF-1 receptors (Stover et al, 1995;Peterson et al, 1996). In these cases, c-Src phosphorylates unique phosphorylation sites on these receptors that serve as docking sites for downstream signal transducers like p85, the regulatory subunit of PI 3'-kinase and Shc (Stover et al, 1995). Thus, activation of Src may bypass the need for activation of growth factor receptors by growth factor.…”

Activation of Src in human breast tumor cell lines: elevated levels of phosphotyrosine phosphatase activity that preferentially recognizes the Src carboxy terminal negative regulatory tyrosine 530

“…Src has also been shown to associate in vitro and in vivo with activated forms of the epidermal growth factor receptor (EGFR) and Neu/HER2 receptor (Luttrell et al, 1994;Maa et al, 1995;Oude Weernink et al, 1994;Stover et al, 1995), both of which have been correlated with poor prognosis in the 20 ± 30% of breast cancer cases in which these receptors are found to be ampli®ed or overexpressed (Berger et al, 1988;Kraus et al, 1993;Slamon et al, 1987Slamon et al, , 1989Varley et al, 1987). Overexpression of the neu proto-oncogene in the mammary glands of transgenic mice has been shown to cause tumors, following a latency period of 200 ± 400 days (Guy et al, 1992).…”