Forlani Greta, Baldassa Simona, Lavagni Paola, Sturani Emmapaola, Zippel Renata
Department of Biomolecular Sciences and Biotechnology, University of Milan, Italy.
FEBS J. 2006 May;273(10):2127-38. doi: 10.1111/j.1742-4658.2006.05226.x.
RasGRF is a family of guanine nucleotide exchange factors with dual specificity for both Ras and Rac GTPases. In this study, using mouse brain extracts, we show that both RasGRF1 and RasGRF2 interact with microtubules in an in vitro microtubule assembly system and this binding is very tight. To characterize this association, recombinant purified proteins containing different regions of RasGRF1 were tested for their ability to bind microtubules preassembled from pure tubulin. Only the DHPH2 tandem directly associates with microtubules, whereas the isolated DH or PH2 domains do not, indicating that the entire DHPH2 region is required for this association. The interaction occurs with high affinity (Kd approximately = 2 microM) and with a stoichiometry, at saturating conditions, of one DHPH2 molecule for two tubulin dimers. Competition experiments support the hypothesis that the DHPH2 module is largely responsible for RasGRF1-microtubule interaction. In vivo colocalization of RasGRF1 and microtubules was also observed by fluorescence confocal microscopy in nonneuronal cells after stimulation with an oxidative stress agent and in highly differentiated neuron-like cells. Identification of microtubules as new binding partners of RasGRF1 may help to elucidate the signaling network in which RasGRF1 is involved.
RasGRF是一类对Ras和Rac GTP酶具有双重特异性的鸟嘌呤核苷酸交换因子家族。在本研究中，我们使用小鼠脑提取物表明，在体外微管组装系统中，RasGRF1和RasGRF2均与微管相互作用，且这种结合非常紧密。为了表征这种关联，我们测试了含有RasGRF1不同区域的重组纯化蛋白与由纯微管蛋白预组装的微管结合的能力。只有DHPH2串联直接与微管结合，而分离的DH或PH2结构域则不结合，这表明整个DHPH2区域是这种关联所必需的。这种相互作用以高亲和力（Kd约 = 2 microM）发生，在饱和条件下，一个DHPH2分子与两个微管蛋白二聚体的化学计量比为1:2。竞争实验支持了DHPH2模块在很大程度上负责RasGRF1-微管相互作用的假设。在用氧化应激剂刺激后，通过荧光共聚焦显微镜在非神经元细胞以及高度分化的神经元样细胞中也观察到了RasGRF1与微管在体内的共定位。将微管鉴定为RasGRF1的新结合伙伴可能有助于阐明RasGRF1所涉及的信号网络。
