The RAS–RAF pathway is one of the most commonly dysregulated in human cancers^1,2,3. Despite decades of study, understanding of the molecular mechanisms underlying dimerization and activation⁴ of the kinase RAF remains limited. Recent structures of inactive RAF monomer⁵ and active RAF dimer^5,6,7,8 bound to 14-3-3^9,10 have revealed the mechanisms by which 14-3-3 stabilizes both RAF conformations via specific phosphoserine residues. Prior to RAF dimerization, the protein phosphatase 1 catalytic subunit (PP1C) must dephosphorylate the N-terminal phosphoserine (NTpS) of RAF¹¹ to relieve inhibition by 14-3-3, although PP1C in isolation lacks intrinsic substrate selectivity. SHOC2 is as an essential scaffolding protein that engages both PP1C and RAS to dephosphorylate RAF NTpS^11,12,13, but the structure of SHOC2 and the architecture of the presumptive SHOC2–PP1C–RAS complex remain unknown. Here we present a cryo-electron microscopy structure of the SHOC2–PP1C–MRAS complex to an overall resolution of 3 Å, revealing a tripartite molecular architecture in which a crescent-shaped SHOC2 acts as a cradle and brings together PP1C and MRAS. Our work demonstrates the GTP dependence of multiple RAS isoforms for complex formation, delineates the RAS-isoform preference for complex assembly, and uncovers how the SHOC2 scaffold and RAS collectively drive specificity of PP1C for RAF NTpS. Our data indicate that disease-relevant mutations affect complex assembly, reveal the simultaneous requirement of two RAS molecules for RAF activation, and establish rational avenues for discovery of new classes of inhibitors to target this pathway.

RAS–RAF 通路是人类癌症中最常见的失调通路之一^1,2,3 。尽管经过数十年的研究，对激酶 RAF 二聚化和激活⁴的分子机制的了解仍然有限。与 14-3-3 ^9,10结合的非活性 RAF 单体⁵和活性 RAF 二聚体^5,6,7,8的最新结构揭示了 14-3-3 通过特定磷酸丝氨酸残基稳定两种 RAF 构象的机制。在 RAF 二聚化之前，蛋白磷酸酶 1 催化亚基 (PP1C) 必须使 RAF ¹¹的 N 末端磷酸丝氨酸 (NTpS) 去磷酸化，以减轻 14-3-3 的抑制，尽管分离的 PP1C 缺乏内在的底物选择性。 SHOC2 是一种重要的支架蛋白，可结合 PP1C 和 RAS 使 RAF NTpS ^11,12,13去磷酸化，但 SHOC2 的结构和假定的 SHOC2-PP1C-RAS 复合物的结构仍然未知。在这里，我们展示了 SHOC2-PP1C-MRAS 复合物的冷冻电子显微镜结构，整体分辨率为 3 Å，揭示了三重分子结构，其中新月形的 SHOC2 充当摇篮，将 PP1C 和 MRAS 结合在一起。我们的工作证明了多种 RAS 同工型对复合物形成的 GTP 依赖性，描述了 RAS 同工型对复杂组装的偏好，并揭示了 SHOC2 支架和 RAS 如何共同驱动 PP1C 对 RAF NTpS 的特异性。我们的数据表明，与疾病相关的突变会影响复杂的组装，揭示了 RAF 激活同时需要两个 RAS 分子，并为发现针对该途径的新型抑制剂建立了合理的途径。