Department of Molecular Medicine and Surgery, Karolinska Institutet, SE-17177 Stockholm, Sweden.
J Biol Chem. 2011 Jan 7;286(1):480-90. doi: 10.1074/jbc.M110.173526. Epub 2010 Oct 28.
The c-KIT receptor tyrosine kinase mediates the cellular response to stem cell factor (SCF). Whereas c-KIT activity is important for the proliferation of hematopoietic cells, melanocytes and germ cells, uncontrolled c-KIT activity contributes to the growth of diverse human tumors. Suppressor of cytokine signaling 6 (SOCS6) is a member of the SOCS family of E3 ubiquitin ligases that can interact with c-KIT and suppress c-KIT-dependent pathways. Here, we analyzed the molecular mechanisms that determine SOCS6 substrate recognition. Our results show that the SH2 domain of SOCS6 is essential for its interaction with c-KIT pY568. The 1.45-Å crystal structure of SOCS6 SH2 domain bound to the c-KIT substrate peptide (c-KIT residues 564-574) revealed a highly complementary and specific interface giving rise to a high affinity interaction (K(d) = 0.3 μm). Interestingly, the SH2 binding pocket extends to substrate residue position pY+6 and envelopes the c-KIT phosphopeptide with a large BG loop insertion that contributes significantly to substrate interaction. We demonstrate that SOCS6 has ubiquitin ligase activity toward c-KIT and regulates c-KIT protein turnover in cells. Our data support a role of SOCS6 as a feedback inhibitor of SCF-dependent signaling and provides molecular data to account for target specificity within the SOCS family of ubiquitin ligases.
c-KIT 受体酪氨酸激酶介导细胞对干细胞因子 (SCF) 的反应。虽然 c-KIT 活性对于造血细胞、黑素细胞和生殖细胞的增殖很重要，但不受控制的 c-KIT 活性会促进多种人类肿瘤的生长。细胞因子信号转导抑制因子 6（SOCS6）是 SOCS 家族 E3 泛素连接酶的成员，可与 c-KIT 相互作用并抑制 c-KIT 依赖性途径。在这里，我们分析了决定 SOCS6 底物识别的分子机制。我们的结果表明，SOCS6 的 SH2 结构域对于其与 c-KIT pY568 的相互作用是必不可少的。SOCS6 SH2 结构域与 c-KIT 底物肽（c-KIT 残基 564-574）结合的 1.45-Å 晶体结构显示出高度互补和特异性的界面，导致高亲和力相互作用（K(d) = 0.3 μm）。有趣的是，SH2 结合口袋延伸到底物残基位置 pY+6，并包围 c-KIT 磷酸肽，具有较大的 BG 环插入，对底物相互作用有重要贡献。我们证明 SOCS6 对 c-KIT 具有泛素连接酶活性，并调节细胞中的 c-KIT 蛋白周转。我们的数据支持 SOCS6 作为 SCF 依赖性信号的反馈抑制剂的作用，并提供了分子数据来解释 SOCS 家族泛素连接酶中的靶标特异性。
