B- and C-RAF Display Essential Differences in Their Binding to Ras
B- and C-RAF Display Essential Differences in Their Binding to Ras
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B-和 C-RAF 在与 Ras 的结合方面显示出本质差异
DOI：
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发表时间：
2007
影响因子：
4.8
通讯作者：
U. Rapp
中科院分区：
文献类型：
--
作者：
A. Fischer;M. Hekman;J. Kuhlmann;I. Rubio;S. Wiese;U. Rapp
RAF激酶向质膜的募集最初被认为是由Ras蛋白通过与RAF Ras结合域（RBD）的相互作用介导的。报道RAF激酶对特定膜脂具有高亲和力的数据支持了一种新的模型，即Ras-RAF相互作用可能在空间上限制在膜平面上。尽管已经详细研究了Ras与分离的RAF RBD结合的偶联特性，但对于加工后的Ras与功能性和全长RAF激酶的相互作用知之甚少。在这里，我们提出了一个定量分析法酰化和非法酰化的H-Ras在存在和不存在脂质环境下与全长B-和C-RAF的结合特性。尽管分离的RBD片段对法酰化和非法酰化的H-Ras都具有高亲和力，但全长RAF激酶在Ras结合方面显示出根本差异。与需要法酰化H-Ras的C-RAF相反，细胞质B-RAF与法酰化和非法酰化的H-Ras有效结合，并且具有更高的亲和力。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，发现在富含半胱氨酸的结构域存在时，只有法尼基化形式的H-Ras结合具有高结合率。B-RAF的极端N端被证明是促进脂质非依赖性Ras与B-RAF结合的原因，因为该区域的截断导致一种蛋白质改变了其激酶特性，类似于C-RAF。使用PC12和COS7细胞的体内研究支持体外结果。使用标记Ras和RAF的共定位测量记录了B-和C-RAF在与Ras的关联方面的本质差异。综上所述，这些数据表明，与C-RAF相比，B-RAF的激活可能同时发生在质膜和细胞质环境中。
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.
影响因子：
16
作者：
Dougherty, MK;Müller, J;Morrison, DK
通讯作者：
Morrison, DK