Department of Chemistry and Biochemistry, University of the Sciences, Philadelphia, PA, 19104, USA.
Eukaryotic Tissue Culture Facility, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.
Chembiochem. 2019 Nov 18;20(22):2850-2861. doi: 10.1002/cbic.201900266. Epub 2019 Oct 15.
The most prevalent BRAF mutation, V600E, occurs frequently in melanoma and other cancers. Although extensive progress has been made toward understanding the biology of RAF kinases, little in vitro characterization of full-length BRAF is available. Herein, we show the successful purification of active, full-length BRAF from mammalian cells for in vitro experiments. Our biochemical characterization of intact BRAF together with molecular dynamics (MD) simulations of the BRAF kinase domain and cell-based assays demonstrate that BRAF has several unique features that contribute to its tumorigenesis. Firstly, steady-state kinetic analyses reveal that purified BRAF is more active than fully activated wild-type BRAF; this is consistent with the notion that elevated signaling output is necessary for transformation. Secondly, BRAF has a higher potential to form oligomers, despite the fact that the V600E substitution confers constitutive kinase activation independent of an intact side-to-side dimer interface. Thirdly, BRAF bypasses inhibitory P-loop phosphorylation to enforce the necessary elevated signaling output for tumorigenesis. Together, these results provide new insight into the biochemical properties of BRAF , complementing the understanding of BRAF regulation under normal and disease conditions.
最常见的 BRAF 突变 V600E 频繁发生于黑色素瘤和其他癌症中。尽管人们在理解 RAF 激酶的生物学方面取得了广泛的进展，但对于全长 BRAF 的体外表征却知之甚少。在此，我们展示了从哺乳动物细胞中成功纯化出具有活性的全长 BRAF，可用于体外实验。我们对完整 BRAF 的生化特性以及 BRAF 激酶结构域的分子动力学 (MD) 模拟和基于细胞的测定表明，BRAF 具有几个独特的特征，有助于其肿瘤发生。首先，稳态动力学分析表明，纯化的 BRAF 比完全激活的野生型 BRAF 更具活性；这与升高的信号输出对于转化是必需的观点一致。其次，尽管 V600E 取代赋予了独立于完整侧向二聚体界面的组成型激酶激活，但 BRAF 具有更高形成寡聚体的潜力。第三，BRAF 绕过抑制性 P 环磷酸化，以强制产生致癌所需的升高的信号输出。总之，这些结果为 BRAF 的生化特性提供了新的见解，补充了在正常和疾病条件下对 BRAF 调节的理解。
