Unique N-region determines low basal activity and limited inducibility of A-RAF kinase - The role of N-region in the evolutionary divergence of RAF kinase function in vertebrates
Unique N-region determines low basal activity and limited inducibility of A-RAF kinase - The role of N-region in the evolutionary divergence of RAF kinase function in vertebrates
复制标题
DOI：
10.1074/jbc.m702429200
复制
发表时间：
2007-09-07
影响因子：
4.8
通讯作者：
Rapp, Ulf R.
中科院分区：
文献类型：
--
作者：
Baljuls, Angela;Mueller, Thomas;Rapp, Ulf R.
在哺乳动物中，丝氨酸/苏氨酸激酶的RAF家族由三个成员A-、B-和C-RAF组成。RAF同种型的一个突出特征是基础和诱导型激酶活性的差异。为了阐明这些差异的性质，我们研究了N区（负电荷调节区）内非保守残基的作用。相对于A-RAF中高度保守的丝氨酸299和C-RAF中的丝氨酸338的位置-3和+1的非保守氨基酸迄今为止尚未被认为是调节残基。在这里，我们证明了这些残基在RAF激活过程中的重要作用。A-RAF中的酪氨酸296被精氨酸取代导致组成型活性激酶。相反，甘氨酸300被丝氨酸取代（模拟B-和C-RAF）以抑制方式起作用。与这些数据一致，在C-RAF（S339 G突变体）的类似位置引入甘氨酸导致组成型活性C-RAF激酶。基于B- RAF催化结构域的三维结构，并使用A-和C-RAF的N-区的序列，我们通过分子模拟寻找这两个部分之间的假定接触点。C-RAF的N-区残基丝氨酸339和催化结构域的精氨酸398之间的紧密相互作用被鉴定并提出抑制RAF蛋白的激酶活性，因为这种相互作用的消除有助于RAF活化。此外，A-RAF中的酪氨酸296有利于N区区段的空间取向，这使得能够与催化结构域更紧密地接触，而C-RAF中该位置处的谷氨酰胺残基消除了这种相互作用。考虑到这一观察结果，我们认为，酪氨酸296，这是唯一的A-RAF，是一个主要的决定因素，低激活效力的RAF亚型。
In mammals the RAF family of serine/threonine kinases consists of three members, A-, B-, and C-RAF. A prominent feature of RAF isoforms regards differences in basal and inducible kinase activities. To elucidate the nature of these differences, we studied the role of the nonconserved residues within the N-region (Negative-charge regulatory region). The nonconserved amino acids in positions -3 and +1 relative to the highly conserved serine 299 in A- RAF and serine 338 in C-RAF have so far not been considered as regulatory residues. Here we demonstrate the essential role of these residues in the RAF activation process. Substitution of tyrosine 296 in A- RAF to arginine led to a constitutively active kinase. In contrast, substitution of glycine 300 by serine ( mimicking B- and C-RAF) acts in an inhibitory manner. Consistent with these data, the introduction of glycine in the analogous position of C-RAF (S339G mutant) led to a constitutively active C-RAF kinase. Based on the three-dimensional structure of the catalytic domain of B- RAF and using the sequences of the N-regions of A- and C-RAF, we searched by molecular modeling for the putative contact points between these two moieties. A tight interaction between the N-region residue serine 339 of C-RAF and arginine 398 of the catalytic domain was identified and proposed to inhibit the kinase activity of RAF proteins, because abrogation of this interaction contributes to RAF activation. Furthermore, tyrosine 296 in A- RAF favors a spatial orientation of the N-region segment, which enables a tighter contact to the catalytic domain, whereas a glutamine residue at this position in C-RAF abrogates this interaction. Considering this observation, we suggest that tyrosine 296, which is unique for A- RAF, is a major determinant of the low activating potency of this RAF isoform.