Abstract
The cytoplasmic region of human epidermal growth factor receptor (EGFR) contains an intrinsic tyrosine kinase (697-955) followed by a 231-residue-long COOH-terminal tail (C-tail), which contains multiple tyrosine residues. To examine the role of the EGFR C-tail in signal transducer and activator of transcription (STAT) activation, a series of EGFR C-tail truncations were constructed. Transient transfection of 293 cells with EGFR lacking the C-tail, i.e. Y974DeltaEGFR or Y992DeltaEGFR, led to EGF-independent or constitutive STAT activation, whereas EGF-dependent STAT activation was restored with truncations made COOH-terminal to the next tyrosine residue, i.e. EGFR-Y1045Delta. Transfection with the-truncated form EGFR-Y954Delta resulted in the loss of STAT activation, suggesting that the sequence between Tyr(974) and Tyr(954) is essential for STAT activation. Phosphopeptide competition analysis revealed multiple tyrosine residues within the C-tail that can act as the docking sites for both Stat1 and Stat3. A region that negatively regulated STAT activation was also identified, extending from Tyr(1114) to Glu(1172), consistent with the ability of this region to recruit a suppressor of cytokine signaling factors SOCS1 and SOCS3. When cotransfected with the full-length EGFR, but not Y992DeltaEGFR, SOCS1 or SOCS3 inhibited STAT activation by EGF in 293 cells. This suggests that both SOCS1 and SOCS3 can negatively regulate EGFR activation, presumably by inducing ubiquitination-dependent EGFR degradation upon ligand binding. These findings may therefore offer clues to how the EGF receptor C-tail regulates STAT activity.
Highlights
The cytoplasmic region of human epidermal growth factor receptor (EGFR) contains an intrinsic tyrosine kinase [697–955] followed by a 231-residue-long COOHterminal tail (C-tail), which contains multiple tyrosine residues
Stat3 DNA binding activity was barely detected under the conditions used, both Stat1 and Stat3 tyrosine phosphorylations were detected (Fig. 1, A and B). These results suggest that EGFR expression is crucial for signal transducer and activator of transcription (STAT) but not for MAP kinase ligand-induced activation
The maximal C-tail region that is essential for STAT activation extends from Tyr954 to Arg973, whereas residues Tyr992 to the carboxyl terminus Ala1186 appear to be inessential for STAT activation but critical for Epidermal growth factor (EGF)-mediated regulation of STAT activation
Summary
The cytoplasmic region of human epidermal growth factor receptor (EGFR) contains an intrinsic tyrosine kinase [697–955] followed by a 231-residue-long COOHterminal tail (C-tail), which contains multiple tyrosine residues. When cotransfected with the full-length EGFR, but not Y992⌬EGFR, SOCS1 or SOCS3 inhibited STAT activation by EGF in 293 cells This suggests that both SOCS1 and SOCS3 can negatively regulate EGFR activation, presumably by inducing ubiquitination-dependent EGFR degradation upon ligand binding. An EGFR-associated inhibitory factor was proposed to block EGF-mediated Stat activation [17] For those cytokine receptors lacking intrinsic kinase activity, removal or mutation of the C-tail tyrosine residues does impair the ability phoretic mobility shift assay; GST, glutathione S-transferase; JAK, Janus kinase; SH2, Src homology 2; MAP, mitogen-activated protein; SOCS, suppressor of cytokine signaling; IFN, interferon; pY, phospho tyrosine. Together these data revealed multiple docking sites for both Stat and Stat within the EGFR C-tail as well as a negative regulatory region for STAT activation. The observation that SOCS1 and SOCS3 were found to interact with EGFR C-tail, together with the knowledge that STAT activation is sensitive to EGFR expression levels, suggests that SOCS1 and SOCS3 inhibit STAT activation, presumably because of their induction of ubiquitinationdependent EGFR proteolytic degradation
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