Mittar Dev, Sehajpal Prabodh K, Lander Harry M
Department of Molecular Biology and Biochemistry, Guru Nanak Dev University, Amritsar 143005, India.
Biochem Biophys Res Commun. 2004 Sep 10;322(1):203-9. doi: 10.1016/j.bbrc.2004.07.107.
Rap1 and Ral, the small GTPases belonging to the Ras superfamily, have recently attracted much attention; Ral because of Ral-specific guanine nucleotide exchange factors which are regulated by direct binding to Ras and Rap1 because of its proposed role as an antagonist of Ras signaling. We have previously demonstrated that nitric oxide (NO) activates Ras and proposed the structural basis of interaction between NO and Ras. In the present study we have shown that NO activates Rap1 and Ral in a time- and concentration-dependent manner. Using activation-specific probes for Rap1 and Ral, it was found that the NO-generating compounds SNP and SNAP could activate both Rap1 and Ral in Jurkat and PC12 cell lines. To investigate the involvement of Ras in NO mediated activation of Rap1 and Ral, we used PC12 cell lines expressing either the Ras mutant C118S (Cys118 mutated to Ser) or N17 (GDP-locked and inactive). We had previously shown that NO fails to activate Ras in these mutant cell lines. However, here it was found that Rap1 and Ral were activated by NO in these cell lines. The evidence presented in this study unambiguously demonstrates the existence of Ras-independent pathways for NO mediated activation of Rap1 and Ral.
Rap1和Ral是属于Ras超家族的小GTP酶，最近备受关注；Ral是因为Ral特异性鸟嘌呤核苷酸交换因子受与Ras的直接结合调控，而Rap1则因其作为Ras信号拮抗剂的假定作用而受到关注。我们之前已证明一氧化氮（NO）可激活Ras，并提出了NO与Ras相互作用的结构基础。在本研究中，我们表明NO以时间和浓度依赖性方式激活Rap1和Ral。使用针对Rap1和Ral的激活特异性探针，发现产生NO的化合物SNP和SNAP可在Jurkat和PC12细胞系中激活Rap1和Ral。为了研究Ras在NO介导的Rap1和Ral激活中的作用，我们使用了表达Ras突变体C118S（Cys118突变为Ser）或N17（GDP锁定且无活性）的PC12细胞系。我们之前已表明NO在这些突变细胞系中无法激活Ras。然而，在此发现NO在这些细胞系中激活了Rap1和Ral。本研究提供的证据明确证明了存在不依赖Ras的途径用于NO介导的Rap1和Ral激活。
