Trebino Tarah, Markusic Borna, Nan Haihan, Banerjee Shrhea, Wang Zhihong
Rowan University, 201 Mullica Hill Rd, Glassboro, NJ 08028, USA.
Max Planck Institute of Biophysics, Max-von-Laue Straße 3, 60438 Frankfurt am Main, Germany.
bioRxiv. 2023 Sep 27:2023.04.24.538112. doi: 10.1101/2023.04.24.538112.
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-KD 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.
BRAF是丝裂原活化蛋白激酶（MAPK）信号通路中的关键成员，对细胞生长、增殖和分化至关重要。BRAF的失调或突变通常是各类癌症的根本原因。RAS是一种在BRAF上游起作用的小GTPase蛋白，已被确定为高达三分之一的所有癌症的驱动因素。当BRAF通过RAS结合域（RBD）与RAS相互作用并进行膜募集时，BRAF会经历从无活性的自抑制单体到活性二聚体的构象变化，随后磷酸化MEK以传播信号。尽管BRAF在细胞信号传导中起着核心作用，但其激活步骤的确切顺序和程度尚未通过实验得到证实。通过研究BRAF的分子间和分子内相互作用，我们揭示了BRAF调控的结构域特异性和异构体特异性细节。我们采用下拉实验、开放表面等离子体共振（OpenSPR）和氢-氘交换质谱（HDX-MS）来研究调控区域在BRAF激活和自抑制中的作用。我们的结果表明，BRAF特异性区域（BSR）和富含半胱氨酸结构域（CRD）在调节BRAF活性方面起着关键作用。此外，我们量化了BRAF的N端结构域与激酶结构域（KD）之间的自抑制结合亲和力，并揭示了BRAF调控结构域的各自作用。此外，我们的研究结果证明，BSR以RAS异构体特异性方式负向调节BRAF激活。我们的研究结果还表明，致癌性BRAF-KD突变体对调控结构域的亲和力较低，这意味着致病性BRAF通过降低自抑制倾向发挥作用。总体而言，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策略的开发。
