An In Vitro BRAF Activation Assay Elucidates Molecular Mechanisms Driving the Disassembly of the BRAF Autoinhibited State

The RAF kinases (ARAF, BRAF and CRAF) are essential components of the RAS-ERK signaling pathway, which controls vital cellular processes and is frequently dysregulated in human disease. Notably, mutations that alter BRAF function are prominent drivers of human cancer and certain RASopathy disorders, making BRAF an important target for therapeutic intervention. Despite extensive research, several aspects of BRAF regulation remain unclear. In this study, we developed an in vitro BRAF activation assay using purified autoinhibited BRAF:14-3-32:MEK complexes. Our results show that fully processed, active-state KRAS alone can promote dimer-dependent BRAF activation. Moreover, we found that phosphatidylserine (PS)-containing liposomes synergized with KRAS to promote BRAF activation, achieving activity levels comparable to those observed with BRAF proteins that constitutively dimerize. In contrast, the SMP phosphatase complex had only a minimal effect on BRAF catalytic activity in this system but mediated the dephosphorylation of the negative regulatory pS365 14-3-3 binding site in a manner that was accelerated by the presence of KRAS alone or KRAS and 30% PS liposomes. Finally, we show that inhibitors blocking the BRAF RBD:KRAS interaction were able to suppress the in vitro activation of BRAF, underscoring the critical role of RAS binding in initiating the disassembly of the BRAF autoinhibited state. Thus, this assay provides valuable insights into the steps required for BRAF activation and can serve as an effective screening tool for identifying compounds that may inhibit this process and have therapeutic potential.

The files in this data set include images of immunoblots and quantification of the BRAF kinase assays.

RAF激酶（RAF kinases，包含ARAF、BRAF与CRAF）是RAS-ERK信号通路的核心组成元件，该通路调控诸多关键细胞生命进程，且在人类疾病中常发生失调。值得注意的是，可改变BRAF功能的突变是人类癌症及部分RASopathy综合征的重要驱动因素，这使得BRAF成为治疗干预的关键靶点。尽管已有大量研究积累，但BRAF调控的诸多环节仍未完全明确。本研究利用纯化的自抑制状态BRAF:14-3-3₂:MEK复合物，构建了体外BRAF激活检测体系。实验结果显示，仅完全活化的活性状态KRAS即可介导依赖二聚化的BRAF激活。此外，我们发现携带磷脂酰丝氨酸（PS）的脂质体可与KRAS协同促进BRAF激活，其激活效果可与组成型二聚化BRAF蛋白所达到的活性水平相当。与之相反，SMP磷酸酶复合物在该体系中对BRAF的催化活性仅具有极微弱的影响，但可介导负调控位点pS365所在的14-3-3结合位点的去磷酸化过程；且仅KRAS存在，或KRAS与30% PS脂质体共同存在时，该去磷酸化过程会被显著加速。最后，我们证实，可阻断BRAF的RBD与KRAS相互作用的抑制剂能够抑制BRAF的体外激活，这进一步明确了RAS结合在启动BRAF自抑制状态解离过程中的关键作用。综上，本检测体系可为BRAF激活所需的关键步骤提供宝贵的机制见解，同时可作为筛选可抑制该过程并具有治疗潜力的化合物的有效工具。

本数据集包含免疫印迹图像及BRAF激酶实验的定量分析结果。