Takaya Akiyuki, Ohba Yusuke, Kurokawa Kazuo, Matsuda Michiyuki
Department of Tumor Virology, Research Institute for Microbial Diseases, Osaka University, Yamadaoka, Suita-shi, Osaka 565-0871, Japan.
Mol Biol Cell. 2004 Jun;15(6):2549-57. doi: 10.1091/mbc.e03-11-0857. Epub 2004 Mar 19.
RalA, a member of the Ras-family GTPases, regulates various cellular functions such as filopodia formation, endocytosis, and exocytosis. On epidermal growth factor (EGF) stimulation, activated Ras recruits guanine nucleotide exchange factors (GEFs) for RalA, followed by RalA activation. By using fluorescence resonance energy transfer-based probes for RalA activity, we found that the EGF-induced RalA activation in Cos7 cells was restricted at the EGF-induced nascent lamellipodia, whereas under a similar condition both Ras activation and Ras-dependent translocation of Ral GEFs occurred more diffusely at the plasma membrane. This EGF-induced RalA activation was not observed when lamellipodial protrusion was suppressed by a dominant negative mutant of Rac1, a GTPase-activating protein for Cdc42, inhibitors of phosphatidylinositol 3-kinase, or inhibitors of actin polymerization. On the other hand, EGF-induced lamellipodial protrusion was inhibited by microinjection of the RalA-binding domains of RalBP1 and Sec5. Furthermore, we found that RalA activity was high at the lamellipodia of migrating Madin-Darby canine kidney cells and that the migration of Madin-Darby canine kidney cells was perturbed by the microinjection of RalBP1-RalA-binding domain. Thus, RalA activation is required for the induction of lamellipodia, and conversely, lamellipodial protrusion seems to be required for the RalA activation, suggesting the presence of a positive feedback loop between RalA activation and lamellipodial protrusion. Our observation also demonstrates that the spatial regulation of RalA is conducted by a mechanism distinct from the temporal regulation conducted by Ras-dependent plasma membrane recruitment of Ral guanine nucleotide exchange factors.
RalA是Ras家族GTP酶的成员之一，可调节多种细胞功能，如丝状伪足形成、胞吞作用和胞吐作用。在表皮生长因子（EGF）刺激下，活化的Ras募集RalA的鸟嘌呤核苷酸交换因子（GEF），随后RalA被激活。通过使用基于荧光共振能量转移的RalA活性探针，我们发现Cos7细胞中EGF诱导的RalA激活局限于EGF诱导的新生片状伪足，而在类似条件下，Ras激活和Ral GEFs的Ras依赖性转位在质膜上的发生更为分散。当丝状伪足突出被Rac1（一种Cdc42的GTP酶激活蛋白）的显性负性突变体、磷脂酰肌醇3激酶抑制剂或肌动蛋白聚合抑制剂抑制时，未观察到这种EGF诱导的RalA激活。另一方面，显微注射RalBP1和Sec5的RalA结合结构域可抑制EGF诱导的片状伪足突出。此外，我们发现迁移的Madin-Darby犬肾细胞的片状伪足处RalA活性较高，并且显微注射RalBP1-RalA结合结构域会干扰Madin-Darby犬肾细胞的迁移。因此，RalA激活是诱导片状伪足所必需的，相反，片状伪足突出似乎也是RalA激活所必需的，这表明RalA激活与片状伪足突出之间存在正反馈回路。我们的观察还表明，RalA的空间调节是通过一种不同于由Ras依赖性质膜募集Ral鸟嘌呤核苷酸交换因子进行的时间调节的机制来进行的。
