Grb2 的 SUMO 化通过增加其与 Sos1 的结合来增强 ERK 活性。,Molecular Cancer

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Grb2 的 SUMO 化通过增加其与 Sos1 的结合来增强 ERK 活性。
Molecular Cancer ( IF 27.7 ) Pub Date : 2014-04-30 , DOI: 10.1186/1476-4598-13-95
Yingying Qu , Qin Chen , Xueping Lai , Changhong Zhu , Cheng Chen , Xian Zhao , Rong Deng , Ming Xu , Haihua Yuan , Yanli Wang , Jianxiu Yu ₁ , Jian Huang

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背景 Grb2（生长因子受体结合蛋白 2）是维持 ERK 活性的关键衔接蛋白，通过将 Sos1（Son of Sevenless 同源物 1）或其他蛋白连接到激活的 RTK，例如 EGFR。目前，除了磷酸化之外，关于 Grb2 的翻译后修饰 (PTM) 的知识很少。由于新出现的证据强调了 SUMOylation（小泛素相关修饰符），一种可逆的 PTM，在调节蛋白质功能方面的重要性，我们想知道 Grb2 是否可以被 SUMOylated 从而影响其功能，尤其是参与 Ras/MEK/ERK 通路的功能。方法使用 Ni2+-NTA 亲和力下拉和体外基于大肠杆菌的 SUMOylation 测定法，通过体内 SUMOylation 测定法分析 Grb2 的 SUMOylation。为了测试 ERK 活性和细胞转化，小鼠成纤维细胞系NIH/3T3和小鼠结肠癌细胞系CMT-93用于Grb2敲低、异位再表达、细胞转化和迁移等实验。免疫沉淀 (IP) 用于寻找与 SUMO 修饰的 Grb2 相互作用的蛋白质。在小鼠中进行异种移植肿瘤模型以验证 Grb2 SUMOylation 在体内调节肿瘤发生。结果 Grb2 可以在赖氨酸 56 (K56) 处被 SUMO1 SUMO 化，其位于 N 端 SH3 结构域和 SH2 结构域之间的接头区域。Grb2 的组合式降低了 ERK 活性并抑制了体外和体内的细胞运动和肿瘤发生，这些都可以通过野生型 Grb2 的稳定异位重新表达而不是突变体 Grb2K56R 来挽救。此外，K56 处的 Grb2 SUMOylation 增加了 Grb2-Sos1 复合物的形成，这依次导致 Ras/MEK/MAPK 通路的激活。结论我们的结果提供证据表明 Grb2 在体内 SUMO 化并且这种修饰通过增加 Grb2-Sos1 复合物的形成来增强 ERK 活性，并因此可能促进细胞运动、转化和肿瘤发生。

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SUMOylation of Grb2 enhances the ERK activity by increasing its binding with Sos1.

BACKGROUND Grb2 (Growth factor receptor-bound protein 2) is a key adaptor protein in maintaining the ERK activity via linking Sos1 (Son of sevenless homolog 1) or other proteins to activated RTKs, such as EGFR. Currently, little knowledge is available concerning the post-translational modification (PTM) of Grb2 except for its phosphorylation. Since emerging evidences have highlighted the importance of SUMOylation (Small ubiquitin-related modifier), a reversible PTM, in modulating protein functions, we wondered if Grb2 could be SUMOylated and thereby influences its functions especially involved in the Ras/MEK/ERK pathway. METHODS SUMOylation of Grb2 was analyzed with the in vivo SUMOylation assay using the Ni2+-NTA affinity pulldown and the in vitro E.coli-based SUMOylation assay. To test the ERK activity and cell transformation, the murine fibroblast cell line NIH/3T3 and the murine colon cancer cell line CMT-93 were used for the experiments including Grb2 knockdown, ectopic re-expression, cell transformation and migration. Immunoprecipitation (IP) was employed for seeking proteins that interact with SUMO modified Grb2. Xenograft tumor model in mice was conducted to verify that Grb2 SUMOylation regulated tumorigenesis in vivo. RESULTS Grb2 can be SUMOylated by SUMO1 at lysine 56 (K56), which is located in the linker region between the N-terminal SH3 domain and the SH2 domain. Knockdown of Grb2 reduced the ERK activity and suppressed cell motility and tumorigenesis in vitro and in vivo, which were all rescued by stable ectopic re-expression of wild-type Grb2 but not the mutant Grb2K56R. Furthermore, Grb2 SUMOylation at K56 increased the formation of Grb2-Sos1 complex, which sequentially leads to the activation of Ras/MEK/MAPK pathway. CONCLUSIONS Our results provide evidences that Grb2 is SUMOylated in vivo and this modification enhances ERK activities via increasing the formation of Grb2-Sos1 complex, and may consequently promote cell motility, transformation and tumorigenesis.

更新日期：2014-04-29

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