Fischer Andreas, Hekman Mirko, Kuhlmann Jürgen, Rubio Ignacio, Wiese Stefan, Rapp Ulf R
Institut für Medizinische Strahlenkunde und Zellforschung, University of Wuerzburg, 97078 Wuerzburg, Germany.
J Biol Chem. 2007 Sep 7;282(36):26503-16. doi: 10.1074/jbc.M607458200. Epub 2007 Jul 16.
Recruitment of RAF kinases to the plasma membrane was initially proposed to be mediated by Ras proteins via interaction with the RAF Ras binding domain (RBD). Data reporting that RAF kinases possess high affinities for particular membrane lipids support a new model in which Ras-RAF interactions may be spatially restricted to the plane of the membrane. Although the coupling features of Ras binding to the isolated RAF RBD were investigated in great detail, little is known about the interactions of the processed Ras with the functional and full-length RAF kinases. Here we present a quantitative analysis of the binding properties of farnesylated and nonfarnesylated H-Ras to both full-length B- and C-RAF in the presence and absence of lipid environment. Although isolated RBD fragments associate with high affinity to both farnesylated and nonfarnesylated H-Ras, the full-length RAF kinases revealed fundamental differences with respect to Ras binding. In contrast to C-RAF that requires farnesylated H-Ras, cytosolic B-RAF associates effectively and with significantly higher affinity with both farnesylated and nonfarnesylated H-Ras. To investigate the potential farnesyl binding site(s) we prepared several N-terminal fragments of C-RAF and found that in the presence of cysteine-rich domain only the farnesylated form of H-Ras binds with high association rates. The extreme N terminus of B-RAF turned out to be responsible for the facilitation of lipid independent Ras binding to B-RAF, since truncation of this region resulted in a protein that changed its kinase properties and resembles C-RAF. In vivo studies using PC12 and COS7 cells support in vitro results. Co-localization measurements using labeled Ras and RAF documented essential differences between B- and C-RAF with respect to association with Ras. Taken together, these data suggest that the activation of B-RAF, in contrast to C-RAF, may take place both at the plasma membrane and in the cytosolic environment.
RAF激酶向质膜的募集最初被认为是由Ras蛋白通过与RAF Ras结合结构域（RBD）相互作用介导的。有数据表明RAF激酶对特定膜脂具有高亲和力，这支持了一种新模型，即Ras-RAF相互作用可能在膜平面上受到空间限制。尽管对Ras与分离的RAF RBD结合的偶联特征进行了详细研究，但对于加工后的Ras与功能性全长RAF激酶之间的相互作用知之甚少。在此，我们对法尼基化和非法尼基化的H-Ras在有无脂质环境下与全长B-RAF和C-RAF的结合特性进行了定量分析。尽管分离的RBD片段与法尼基化和非法尼基化的H-Ras都具有高亲和力结合，但全长RAF激酶在Ras结合方面显示出根本差异。与需要法尼基化H-Ras的C-RAF不同，胞质B-RAF与法尼基化和非法尼基化的H-Ras都能有效结合且亲和力显著更高。为了研究潜在的法尼基结合位点，我们制备了几个C-RAF的N端片段，发现仅在富含半胱氨酸结构域存在时，法尼基化形式的H-Ras才以高结合率结合。结果表明，B-RAF的极端N端负责促进脂质非依赖性的Ras与B-RAF结合，因为该区域的截断导致一种改变其激酶特性且类似于C-RAF的蛋白质。使用PC12和COS7细胞的体内研究支持了体外结果。使用标记的Ras和RAF进行的共定位测量记录了B-RAF和C-RAF在与Ras结合方面的本质差异。综上所述，这些数据表明，与C-RAF不同，B-RAF的激活可能在质膜和胞质环境中都发生。
