Department of Chemistry, Texas A&M University, College Station, TX 77843.
Proc Natl Acad Sci U S A. 2024 Aug 20;121(34):e2405986121. doi: 10.1073/pnas.2405986121. Epub 2024 Aug 15.
RAS GTPases associate with the biological membrane where they function as molecular switches to regulate cell growth. Recent studies indicate that RAS proteins oligomerize on membranes, and disrupting these assemblies represents an alternative therapeutic strategy. However, conflicting reports on RAS assemblies, ranging in size from dimers to nanoclusters, have brought to the fore key questions regarding the stoichiometry and parameters that influence oligomerization. Here, we probe three isoforms of RAS [Kirsten Rat Sarcoma viral oncogene (KRAS), Harvey Rat Sarcoma viral oncogene (HRAS), and Neuroblastoma oncogene (NRAS)] directly from membranes using mass spectrometry. We show that KRAS on membranes in the inactive state (GDP-bound) is monomeric but forms dimers in the active state (GTP-bound). We demonstrate that the small molecule BI2852 can induce dimerization of KRAS, whereas the binding of effector proteins disrupts dimerization. We also show that RAS dimerization is dependent on lipid composition and reveal that oligomerization of NRAS is regulated by palmitoylation. By monitoring the intrinsic GTPase activity of RAS, we capture the emergence of a dimer containing either mixed nucleotides or GDP on membranes. We find that the interaction of RAS with the catalytic domain of Son of Sevenless (SOS) is influenced by membrane composition. We also capture the activation and monomer to dimer conversion of KRAS by SOS. These results not only reveal the stoichiometry of RAS assemblies on membranes but also uncover the impact of critical factors on oligomerization, encompassing regulation by nucleotides, lipids, and palmitoylation.
RAS GTPases 与生物膜结合，在那里它们作为分子开关调节细胞生长。最近的研究表明，RAS 蛋白在膜上寡聚化，破坏这些组装体代表了一种替代的治疗策略。然而，关于 RAS 组装体的大小不一，从二聚体到纳米团簇的矛盾报告，提出了关于影响寡聚化的化学计量和参数的关键问题。在这里，我们使用质谱法直接从膜上探测三种 RAS 同工型（Kirsten Rat Sarcoma 病毒癌基因（KRAS）、Harvey Rat Sarcoma 病毒癌基因（HRAS）和神经母细胞瘤癌基因（NRAS））。我们表明，处于非活性状态（GDP 结合）的膜上 KRAS 是单体，但在活性状态（GTP 结合）下形成二聚体。我们证明，小分子 BI2852 可以诱导 KRAS 二聚化，而效应蛋白的结合会破坏二聚化。我们还表明，RAS 二聚化依赖于脂质组成，并揭示了 NRAS 的寡聚化受棕榈酰化调节。通过监测 RAS 的固有 GTPase 活性，我们在膜上捕获了含有混合核苷酸或 GDP 的二聚体。我们发现，RAS 与 Son of Sevenless（SOS）的催化结构域的相互作用受膜组成的影响。我们还捕获了 SOS 对 KRAS 的激活和单体到二聚体的转化。这些结果不仅揭示了 RAS 组装体在膜上的化学计量，还揭示了关键因素对寡聚化的影响，包括核苷酸、脂质和棕榈酰化的调节。
