Distinct Binding Preferences Between Ras and Raf Family Members and the Impact on Oncogenic Ras Signaling by Terrell et al

The Ras GTPases are frequently mutated in human cancer and although the Raf kinases are essential effectors of Ras signaling, the tumorigenic properties of specific Ras-Raf complexes are not well characterized. This study examines the ability of individual Ras and Raf proteins to interact in live cells using bioluminescence resonance energy transfer (BRET) technology. The BRET system allows quantitative measurements to be obtained under conditions that preserve crucial features of Ras and Raf regulation, including lipid processing, intracellular trafficking, membrane microdomain targeting, and protein phosphorylation. Strikingly, our findings demonstrate pronounced binding preferences between the Ras and Raf family members. We find that while C-Raf and K-Ras can bind with high affinity to all Ras or Raf family members respectively, H-Ras displays preferential binding to C-Raf whereas B-Raf exhibits a striking selectivity for K-Ras. The B-Raf selectivity is mediated by the acidic, N-terminal segment of B-Raf and requires the K-Ras polybasic region for high-affinity binding. This study also finds that C-Raf is required for mutant H-Ras-driven signaling and that events inducing stable B-Raf/C-Raf dimer formation, such as Raf inhibitor treatment or certain B-Raf mutations, allow mutant H-Ras to engage B-Raf with increased affinity to promote ERK activation and tumorigenesis.Ras GTP酶（Ras GTPases）在人类癌症中高频突变，尽管Raf激酶是Ras信号通路的关键效应分子，但特定Ras-Raf复合物的致瘤特性尚未得到充分阐释。本研究借助生物发光共振能量转移（bioluminescence resonance energy transfer, BRET）技术，探究了活细胞内单个Ras与Raf蛋白的相互作用能力。BRET系统可在保留Ras与Raf关键调控特征的条件下实现定量检测，这些特征包括脂质修饰、胞内转运、膜微区靶向以及蛋白质磷酸化。值得注意的是，本研究结果揭示了Ras与Raf家族成员间显著的结合偏好性：C-Raf与K-Ras分别可与所有Raf或Ras家族成员以高亲和力结合，但H-Ras倾向于优先结合C-Raf，而B-Raf则对K-Ras表现出极强的选择性。B-Raf的这种选择性由其N端酸性区段介导，且需要K-Ras的多碱基区域以实现高亲和力结合。本研究同时发现，C-Raf是突变型H-Ras驱动的信号通路所必需的；而诱导B-Raf/C-Raf形成稳定二聚体的事件（如Raf抑制剂处理或特定B-Raf突变），可使突变型H-Ras以更高亲和力结合B-Raf，进而促进ERK激活与肿瘤发生。