Protein Phosphorylation Laboratory, Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3LY, UK.
Biochem J. 2011 Oct 15;439(2):287-97. doi: 10.1042/BJ20110678.
The protein kinase TOR (target of rapamycin) is a key regulator of cell growth and metabolism with significant clinical relevance. In mammals, TOR signals through two distinct multi-protein complexes, mTORC1 and mTORC2 (mammalian TOR complex 1 and 2 respectively), the subunits of which appear to define the operational pathways. Rapamycin selectively targets mTORC1 function, and the emergence of specific ATP-competitive kinase inhibitors has enabled assessment of dual mTORC1 and mTORC2 blockade. Little is known, however, of the molecular action of mTORC2 components or the relative importance of targeting this pathway. In the present study, we have identified the mTORC2 subunit Sin1 as a direct binding partner of the PKC (protein kinase C) ε kinase domain and map the interaction to the central highly conserved region of Sin1. Exploiting the conformational dependence for PKC phosphorylation, we demonstrate that mTORC2 is essential for acute priming of PKC. Inducible expression of Sin1 mutants, lacking the PKC-interaction domain, displaces endogenous Sin1 from mTORC2 and disrupts PKC phosphorylation. PKB (protein kinase B)/Akt phosphorylation is also suppressed by these Sin1 mutants, but not the mTORC1 substrate p70(S6K) (S6 kinase), providing evidence that Sin1 serves as a selectivity adaptor for the recruitment of mTORC2 targets. This inducible selective mTORC2 intervention is used to demonstrate a key role for mTORC2 in cell proliferation in three-dimensional culture.
蛋白激酶 TOR（雷帕霉素靶蛋白）是细胞生长和代谢的关键调节因子，具有重要的临床意义。在哺乳动物中，TOR 通过两个不同的多蛋白复合物 mTORC1 和 mTORC2（分别为哺乳动物 TOR 复合物 1 和 2）信号传导，其亚基似乎定义了操作途径。雷帕霉素选择性靶向 mTORC1 功能，而特定的 ATP 竞争性激酶抑制剂的出现使得能够评估双重 mTORC1 和 mTORC2 阻断。然而，对于 mTORC2 成分的分子作用或靶向该途径的相对重要性知之甚少。在本研究中，我们已经确定了 mTORC2 亚基 Sin1 是 PKC（蛋白激酶 C）ε激酶结构域的直接结合伴侣，并将该相互作用映射到 Sin1 的中央高度保守区域。利用 PKC 磷酸化的构象依赖性，我们证明 mTORC2 对于 PKC 的急性启动至关重要。缺乏 PKC 相互作用结构域的 Sin1 突变体的可诱导表达从 mTORC2 中置换内源性 Sin1 并破坏 PKC 磷酸化。这些 Sin1 突变体还抑制 PKB（蛋白激酶 B）/Akt 磷酸化，但不抑制 mTORC1 底物 p70(S6K)（S6 激酶），这提供了证据表明 Sin1 作为募集 mTORC2 靶标的选择性衔接子。这种可诱导的选择性 mTORC2 干预用于证明 mTORC2 在三维培养中的细胞增殖中起关键作用。
