BRAF是细胞生长所必需的MAPK信号通路的关键成员,扩散,和差异化。BRAF的失调或突变通常是各种类型癌症的根本原因。RAS,一种作用于BRAF上游的小GTP酶蛋白,已被确定为所有癌症的三分之一的驱动因素。当BRAF通过RAS结合域(RBD)和膜募集与RAS相互作用时,BRAF经历了从非活性的构象变化,将单体自抑制为活性二聚体,随后磷酸化MEK以传播信号。BRAF结构域参与调控机制的特定功能,例如,通过半胱氨酸丰富结构域(CRD)和BRAF的激酶结构域(KD)之间的相互作用来维持自抑制构象。尽管BRAF在细胞信号传导中起着核心作用,其激活步骤的确切顺序和大小尚未得到实验证实。我们采用了下拉测定,开放表面等离子体共振(OpenSPR),和氢-氘交换质谱(HDX-MS)研究调节区在BRAF激活和自抑制中的作用。我们的结果表明,BRAF特异性区(BSR)和CRD在调节BRAF的活性中起着至关重要的作用。此外,我们定量了BRAFN末端结构域与KD之间的自抑制结合亲和力,并揭示了BRAF调控结构域的个体作用.此外,我们量化了RAS结合时BRAF的N端结构域和KD之间的自抑制的缓解,提供RAS结合启动RAF激活的直接证据。此外,我们的研究结果提供了证据,即BSR以RAS同工型特异性方式负调节BRAF激活,并突出了在开发针对RAF-RAS相互作用的抑制剂时考虑特定同工型对的重要性.我们的发现还表明,致癌BRAF-KDD594G突变体对调节域的亲和力较低,暗示致病性BRAF通过降低自身抑制的倾向起作用。总的来说,我们的研究为BRAF激酶的激活机制提供了有价值的见解,并可能有助于指导癌症治疗新治疗策略的开发.
BRAF is a key member in the MAPK signaling pathway essential for cell growth, proliferation, and differentiation. Dysregulation or mutation of BRAF is often the underlying cause of various types of cancer. RAS, a small GTPase protein that acts upstream of BRAF, has been identified as a driver of up to one-third of all cancers. When BRAF interacts with RAS via the RAS binding domain (RBD) and membrane recruitment, BRAF undergoes a conformational change from an inactive, autoinhibited monomer to an active dimer and subsequently phosphorylates MEK to propagate the signal. Despite the central role of BRAF in cellular signaling, the exact order and magnitude of its activation steps has yet to be confirmed experimentally. By studying the inter- and intramolecular interactions of BRAF, we unveil the domain-specific and isoform-specific details of BRAF regulation. We employed pulldown assays, open surface plasmon resonance (OpenSPR), and hydrogen-deuterium exchange mass spectrometry (HDX-MS) to investigate the roles of the regulatory regions in BRAF activation and
autoinhibition. Our results demonstrate that the BRAF specific region (BSR) and cysteine rich domain (CRD) play a crucial role in regulating the activity of BRAF. Moreover, we quantified the autoinhibitory binding affinities between the N-terminal domains and the kinase domain (KD) of BRAF and revealed the individual roles of the BRAF regulatory domains. Additionally, our findings provide evidence that the BSR negatively regulates BRAF activation in a RAS isoform-specific manner. Our findings also indicate that oncogenic BRAF-KDD594G mutant has a lower affinity for the regulatory domains, implicating that pathogenic BRAF acts through decreased propensity for
autoinhibition. Collectively, our study provides valuable insights into the activation mechanism of BRAF kinase and may help to guide the development of new therapeutic strategies for cancer treatment.