Daub M, Jöckel J, Quack T, Weber C K, Schmitz F, Rapp U R, Wittinghofer A, Block C
Abteilung Strukturelle Biologie, Max-Planck-Institut für Molekulare Physiologie, Dortmund, Germany.
Mol Cell Biol. 1998 Nov;18(11):6698-710. doi: 10.1128/MCB.18.11.6698.
Activation of c-Raf-1 (referred to as Raf) by Ras is a pivotal step in mitogenic signaling. Raf activation is initiated by binding of Ras to the regulatory N terminus of Raf. While Ras binding to residues 51 to 131 is well understood, the role of the RafC1 cysteine-rich domain comprising residues 139 to 184 has remained elusive. To resolve the function of the RafC1 domain, we have performed an exhaustive surface scanning mutagenesis. In our study, we defined a high-resolution map of multiple distinct functional epitopes within RafC1 that are required for both negative control of the kinase and the positive function of the protein. Activating mutations in three different epitopes enhanced Ras-dependent Raf activation, while only some of these mutations markedly increased Raf basal activity. One contiguous inhibitory epitope consisting of S177, T182, and M183 clearly contributed to Ras-Raf binding energy and represents the putative Ras binding site of the RafC1 domain. The effects of all RafC1 mutations on Ras binding and Raf activation were independent of Ras lipid modification. The inhibitory mutation L160A is localized to a position analogous to the phorbol ester binding site in the protein kinase C C1 domain, suggesting a function in cofactor binding. Complete inhibition of Ras-dependent Raf activation was achieved by combining mutations K144A and L160A, which clearly demonstrates an absolute requirement for correct RafC1 function in Ras-dependent Raf activation.
Ras介导的c-Raf-1（简称为Raf）激活是有丝分裂信号传导中的关键步骤。Raf的激活始于Ras与Raf调节性N端的结合。虽然Ras与第51至131位残基的结合已得到充分了解，但包含第139至184位残基的RafC1富含半胱氨酸结构域的作用仍不清楚。为了解析RafC1结构域的功能，我们进行了全面的表面扫描诱变。在我们的研究中，我们定义了RafC1内多个不同功能表位的高分辨率图谱，这些表位对于激酶的负调控和该蛋白的正功能都是必需的。三个不同表位中的激活突变增强了Ras依赖的Raf激活，而这些突变中只有一些显著增加了Raf的基础活性。一个由S177、T182和M183组成的连续抑制性表位明显有助于Ras-Raf结合能，代表了RafC1结构域的假定Ras结合位点。所有RafC1突变对Ras结合和Raf激活的影响均与Ras的脂质修饰无关。抑制性突变L160A定位于与蛋白激酶C C1结构域中佛波酯结合位点类似的位置，提示其在辅因子结合中的功能。通过组合K144A和L160A突变实现了对Ras依赖的Raf激活的完全抑制，这清楚地表明在Ras依赖的Raf激活中对正确的RafC1功能有绝对需求。
