Novel functions and regulatory mechanisms of tumor suppressor Mig-6

米格-6通过与EGF受体（EGFR）结合，作为EGF信号传导的抑制剂。在乳腺癌中观察到Mig-6基因表达下调，这与总生存率降低有关。mig -6缺陷小鼠表现出内源性EGFR的过度激活，并在各器官发生自发性肿瘤。因此，米格-6是一种重要的肿瘤抑制因子。然而，其翻译后修饰和调控机制尚未阐明。在这里，我们研究了人类米格-6的磷酸化，发现Chk1在体内和体外都磷酸化了米格-6。此外，EGF刺激促进了Mig-6的磷酸化而没有DNA损伤，磷酸化被Chk1的缺失所抑制。EGF还通过PI3K途径增加了ser280磷酸化的Chk1，这是一种细胞质拴系形式。质谱分析表明，Mig-6的Ser 251是Chk1在体内和体外磷酸化的主要位点。Ser 251取代丙氨酸增加了Mig-6对EGFR激活的抑制活性。此外，egf依赖性的EGFR激活和细胞生长受到chk1缺失的抑制，并通过Mig-6的共同缺失得到恢复。我们的研究结果表明，Chk1磷酸化了Ser 251上的米格-6，导致米格-6的抑制，并且Chk1是EGF信号传导的积极调节因子。这是Chk1的一个新函数。

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.