Phosphorylation of IRS4 by CK1γ2 promotes its degradation by CHIP through the ubiquitin/lysosome pathway.
Phosphorylation of IRS4 by CK1γ2 promotes its degradation by CHIP through the ubiquitin/lysosome pathway.
复制标题
CK1gamma2 对 IRS4 的磷酸化促进其通过泛素/溶酶体途径被 CHIP 降解。
作者：
Li X;Zhong L;Wang Z;Chen H;Liao D;Zhang R;Zhang H;Kang T
IRS4是胰岛素受体底物蛋白家族的一员，即使在缺乏胰岛素或生长因子的情况下也能诱导组成性PI3K/AKT超活化和细胞增殖，并促进肿瘤发生，但其调控仅在转录水平上进行了探索。研究方法：使用Scansite预测可能调节IRS4功能的潜在蛋白激酶，并使用质谱法鉴定IRS4的E3连接酶。通过免疫沉淀法进行蛋白质相互作用，并通过放线菌酮处理测量蛋白质稳定性。体外激酶试验检测酪蛋白激酶1 γ 2（CK1 γ 2）对IRS 4的磷酸化作用。分别采用集落形成实验和荷瘤小鼠体内实验评价肿瘤细胞生长情况。采用免疫组织化学方法检测骨肉瘤组织中IRS 4和CK1 γ 2蛋白表达，并进行χ 2检验。双尾Student t检验或Mann-Whitney U检验用于比较亚组之间的差异。结果如下：在体内外，CK 1 γ 2可使IRS4的Ser859位磷酸化，从而促进Hsc70相互作用蛋白（CHIP）的羧基端通过泛素/溶酶体途径对IRS4进行多聚泛素化和降解。使用骨肉瘤细胞系，IRS4的CK1 γ 2异位非磷酸化突变体触发更高水平的p-Akt，并在体外和裸鼠中显示更快的细胞增殖和肿瘤生长。此外，在骨肉瘤细胞系和组织样品中观察到CK1 γ 2和IRS 4之间的蛋白水平呈负相关。结论：IRS 4作为CHIP的新底物，在翻译后水平受到CK 1 γ 2的负调节，特异性CK 1 γ 2激动剂可能是治疗骨肉瘤患者的潜在有效策略。
IRS4, a member of the insulin receptor substrate protein family, can induce constitutive PI3K/AKT hyperactivation and cell proliferation even in the absence of insulin or growth factors and promote tumorigenesis, but its regulation has only been explored at the transcriptional level. Methods: Scansite was used to predict the potential protein kinases that may regulate the functions of IRS4, and mass spectrometry was used to identify the E3 ligase for IRS4. The protein interaction was carried out by immunoprecipitation, and protein stability was measured by cycloheximide treatment. In vitro kinase assay was used to determine the phosphorylation of IRS4 by casein kinase 1γ2 (CK1γ2). Colony formation assay and xenograft-bearing mice were employed to assess the cancer cell growth in vitro and in vivo, respectively. Immunohistochemistry was performed to examine protein levels of both IRS4 and CK1γ2 in osteosarcoma specimens and their relationship was evaluated by χ2 test. Two-tailed Student's t-test or the Mann-Whitney U test were used to compare the differences between subgroups. Results: IRS4 was phosphorylated at Ser859 by CK1γ2 in vitro and in vivo, which promoted the polyubiquitination and degradation of IRS4 through the ubiquitin/lysosome pathway by the carboxyl terminus of Hsc70-interacting protein(CHIP). Using osteosarcoma cell lines, the ectopic nonphosphorylated mutant of IRS4 by CK1γ2 triggered higher level of p-Akt and displayed faster cell proliferation and cancer growth in vitro and in nude mice. In addition, a negative correlation in protein levels between CK1γ2 and IRS4 was observed in osteosarcoma cell lines and tissue samples. Conclusions: IRS4, as a new substrate of CHIP, is negatively regulated by CK1γ2 at the posttranslational level, and specific CK1γ2 agonists may be a potentially effective strategy for treating patients with osteosarcoma.