Novel functions and regulatory mechanisms of tumor suppressor Mig-6

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Mig-6 acts as an inhibitor of EGF signaling via binding with the EGF receptor (EGFR). Downregulated expression of the Mig-6 gene is observed in breast carcinomas, in which it correlates with reduced overall survival. Mig-6-deficient mice show hyperactivation of endogenous EGFR and develop spontaneous tumors in various organs. Therefore, Mig-6 is an important tumor suppressor. However, its post-translational modifications and regulatory mechanisms have not been elucidated. Here, we investigated the phosphorylation of human Mig-6 and found that Chk1 phosphorylated Mig-6 in vivo as well as in vitro. Moreover, EGF stimulation promoted phosphorylation of Mig-6 without DNA damage and the phosphorylation was inhibited by depletion of Chk1. EGF also increased Ser280-phosphorylated Chk1, a cytoplasmic-tethering form, via PI3K pathway. Mass spectrometric analyses suggested that Ser 251 of Mig-6 was a major phosphorylation site by Chk1 in vitro and in vivo. Substitution of Ser 251 to alanine increased inhibitory activity of Mig-6 against EGFR activation. Moreover, EGF-dependent activation of EGFR and cell growth were inhibited by Chk1-depletion, and were rescued by co-depletion of Mig-6. Our results suggest that Chk1 phosphorylates Mig-6 on Ser 251, resulting in the inhibition of Mig-6, and that Chk1 acts a positive regulator of EGF signaling. This is a novel function of Chk1.