Phosphorylation of Y845 on the Epidermal Growth Factor Receptor Mediates Binding to the Mitochondrial Protein Cytochrome c Oxidase Subunit II

Abstract

When co-overexpressed, the epidermal growth factor receptor (EGFR) and c-Src cooperate to cause synergistic increases in EGF-induced DNA synthesis, soft agar colony growth, and tumor formation in nude mice. This synergy is dependent upon c-Src-mediated phosphorylation of a unique tyrosine on the EGFR, namely, tyrosine 845 (Y845). Phenylalanine substitution of Y845 (Y845F) was found to inhibit EGF-induced DNA synthesis without affecting the catalytic activity of the receptor or its ability to phosphorylate Shc or activate mitogen-activated protein kinase. These results suggest that synergism may occur through alternate signaling pathways mediated by phosphorylated Y845 (pY845). One such pathway involves the transcription factor Stat5b. Here we describe another pathway that involves cytochrome c oxidase subunit II (CoxII). CoxII was identified as a specific binding partner of a pY845-containing peptide in a phage display screen. EGFdependent binding of CoxII to the wild type but not to the mutant Y845F-EGFR was confirmed by coimmunoprecipitation experiments. This association also required the kinase activity of c-Src. Confocal microscopy, as well as biochemical fractionation, indicated that the EGFR translocates to the mitochondria after EGF stimulation, where it colocalizes with CoxII. Such translocation required the catalytic activity of the receptor but not phosphorylation of Y845. However, ectopic expression of the Y845F-EGFR prevented the EGF from protecting MDA-MB-231 breast cancer cells from adriamycin-induced apoptosis, whereas two mutants of Stat5b, a dominant-interfering mutant (DNstat5b) and a tyrosine mutation at 699 (Y699F-Stat5b) did not. Taken together, these data suggest that, through the ability of EGFR to translocate to the mitochondria, the binding of proteins such as CoxII to pY845 on the EGFR may positively regulate survival pathways that contribute to oncogenesis.The epidermal growth factor receptor (EGFR) is a critical regulator of many normal cellular processes, including cell growth, differentiation, survival, and migration. The EGFR also is implicated in pathological processes of cellular transformation and oncogenesis due to its overexpression in many types of cancers (including 20 to 30% of all breast cancers) (2) and its ability to induce morphological transformation of cultured cells and tumor formation in nude mice (11, 37). Many different signaling pathways have been discovered to mediate the effects of EGF, the most notable being Ras-mitogen-activated protein kinase (MAPK) and the phosphatidylinositol 3-kinase pathways (reviewed in reference 9). In these pathways EGF binds to the EGFR to induce dimerization, catalytic activation, and autophosphorylation of tyrosines in the C-terminal tail of the EGFR. These phosphorylated tyrosines provide docking sites for adapter proteins such as Grb2, Shc, and Gab2 to link the receptor to the Ras-MAPK, as well as phosphatidylinositol 3-kinase pathways. Interestingly, activation of both pathways has been implicated in both normal and pathological...

Neurotensin stimulates mitogenesis of prostate cancer cells through a novel c-Src/Stat5b pathway

“…As inhibition of DNA synthesis was not completely abrogated by mutant Y699F or DN Stat5b, other effectors downstream of Tyr 845 may be involved in the signaling pathway. One possibility is the CoxII, which we have previously demonstrated to bind to EGFR Tyr 845 in breast cancer cells using a phage display screen (Boerner et al, 2004).…”

“…EGFR Tyr 845 is activated by NT in a c-Src-dependent manner In breast cancer cells, Tyr 845 of the EGFR has been identified as a c-Src-dependent phosphorylation site that is required for cell proliferation by multiple agonists Boerner et al, 2004Boerner et al, , 2005. We therefore tested whether this phosphorylation site was involved in transactivation of the EGFR by NT in prostate cancer cells.…”

“…C-terminally truncated forms of Stat5b have been shown to inhibit transcriptional activation by WT Stat5b (Moriggl et al, 1996). Construction of a mutant EGFR harboring a phenylalanine substitution for Tyr 845 (Y845F) has been described previously (Boerner et al, 2004). Appropriate vector DNA for Stat5b (pCMV) or Y845F (pcDNA) experiments were used as negative controls.…”

“…As a result of these and other studies, phospho-Tyr 845 is emerging as a candidate, novel target of the EGFR, and efforts are underway to develop small molecule inhibitors of phospho-Tyr 845 , which will disrupt its interaction with its downstream effectors, for example, signal transducer and activator of transcription 5b (Stat5b) (Kloth et al, 2003) and cytochrome c oxidase subunit II (CoxII) (Boerner et al, 2004). Importantly, roles for neither Tyr 845 nor Stat5b/CoxII in NE-related prostate cancer progression have been reported.…”

Journey to the Center of the Mitochondria Guided by the Tail Anchor of Protein Tyrosine Phosphatase 1B

“…PTP1B's potential presence at the mitochondria could be important for regulation of the mitochondrial phosphotyrosine proteome 34 , with important targets including several enzymes in the electron transport chain 35 , Src family kinases that localize to the mitochondria 33,[36][37][38][39][40] , or other well-established substrates of PTP1B that also have been detected at the mitochondria like the EGF receptors ErbB1 41,42 and ErbB2 43 and the tyrosine phosphatase SHP2 33,40,44 . PTP1B's mitochondrial localization, though, should be confirmed first (in particular, for more general cell lines), before further speculating on how it may reach this organelle or how its interaction there with putative as well as known substrates might affect basic mitochondrial functions.…”