Tian X, Feig L A
Department of Biochemistry, Tufts University School of Medicine, Boston, MA 02111, USA.
J Biol Chem. 2001 Dec 14;276(50):47248-56. doi: 10.1074/jbc.M107407200. Epub 2001 Sep 17.
Sos and Ras-GRF are two families of guanine nucleotide exchange factors that activate Ras proteins in cells. Sos proteins are ubiquitously expressed and are activated in response to cell-surface tyrosine kinase stimulation. In contrast, Ras-GRF proteins are expressed primarily in central nervous system neurons and are activated by calcium/calmodulin binding and by phosphorylation. Although both Sos1 and Ras-GRF1 activate the Ras proteins Ha-Ras, N-Ras, and Ki-Ras, only Ras-GRF1 also activates the functionally distinct R-Ras GTPase. In this study, we determined which amino acid sequences in these exchange factors and their target GTPases are responsible for this signaling specificity difference. Analysis of chimeras and individual amino acid exchanges between Sos1 and Ras-GRF1 revealed that the critical amino acids reside within an 11-amino acid segment of their catalytic domains between the second and third structurally conserved regions (amino acids (aa) 828-838 in Sos1 and 1057-1067 in Ras-GRF1) of Ras guanine nucleotide exchange factors. In Sos1, this segment is in helix B, which is known to interact with the switch 2 region of Ha-Ras. Interestingly, a similar analysis of Ha-Ras and R-Ras chimeras did not identify the switch 2 region of Ha-Ras as encoding specificity. Instead, we found a more distal protein segment, helix 3 (aa 91-103 in Ha-Ras and 117-129 in R-Ras), which interacts instead primarily with helix K (aa 1002-1016) of Sos1. These findings suggest that specificity derives from the fact that R-Ras-specific amino acids in the region analogous to Ha-Ras helix 3 prevent a functional interaction with Sos1 indirectly, possibly by preventing an appropriate association of its switch 2 region with helix B of Sos1. Although previous studies have shown that helix B of Sos1 and helix 3 of Ha-Ras are involved in promoting nucleotide exchange on Ras proteins, this study highlights the importance of these regions in establishing signaling specificity.
Sos和Ras-GRF是两类鸟嘌呤核苷酸交换因子家族，它们可在细胞中激活Ras蛋白。Sos蛋白广泛表达，并在细胞表面酪氨酸激酶刺激下被激活。相比之下，Ras-GRF蛋白主要在中枢神经系统神经元中表达，并通过钙/钙调蛋白结合和磷酸化而被激活。虽然Sos1和Ras-GRF1都能激活Ras蛋白Ha-Ras、N-Ras和Ki-Ras，但只有Ras-GRF1还能激活功能上不同的R-Ras GTP酶。在本研究中，我们确定了这些交换因子及其靶标GTP酶中的哪些氨基酸序列导致了这种信号特异性差异。对Sos1和Ras-GRF1之间的嵌合体和单个氨基酸交换的分析表明，关键氨基酸位于Ras鸟嘌呤核苷酸交换因子的第二和第三结构保守区域之间（Sos1中的第828 - 838位氨基酸和Ras-GRF1中的第1057 - 1067位氨基酸）的催化结构域内的一个11个氨基酸的片段中。在Sos1中，该片段位于螺旋B中，已知其与Ha-Ras的开关2区域相互作用。有趣的是，对Ha-Ras和R-Ras嵌合体的类似分析并未确定Ha-Ras的开关2区域编码特异性。相反，我们发现了一个更远端的蛋白片段，螺旋3（Ha-Ras中的第91 - 103位氨基酸和R-Ras中的第117 - 129位氨基酸），它主要与Sos1的螺旋K（第1002 - 1016位氨基酸）相互作用。这些发现表明，特异性源于这样一个事实，即类似于Ha-Ras螺旋3区域中的R-Ras特异性氨基酸可能通过阻止其开关2区域与Sos1的螺旋B进行适当结合，从而间接阻止与Sos1的功能相互作用。虽然先前的研究表明Sos1的螺旋B和Ha-Ras的螺旋3参与促进Ras蛋白上的核苷酸交换，但本研究强调了这些区域在建立信号特异性中的重要性。
