Regulation of cytoskeletons and cell differentiation by novel small GTPases, M-Ras and RhoD

我们最近发现的小分子GTP酶M-RAS能诱导PC12细胞分化。我们研究了导致分化的信号转导途径。M-RAS参与了cAMP诱导的分化。M-RAS由cAMP-gef激活，cAMP激活，进而诱导转录因子CREB的激活。因此，该分化途径为：cAMP-→、cAMP-→、M-RAS、→？→、CREB。这一途径不同于RAS和PAPL参与的途径。由于CREB对长时记忆是必不可少的，M-RAS可能参与了包括长时记忆在内的高级脑功能的调节。为了通过建立M-RAS基因敲除小鼠来评估这种可能性，我们构建了M-RAS基因打靶载体。我们还利用酵母双杂交系统鉴定了M-RAS的靶蛋白。其中包括被认为是RAS靶蛋白的Nore-1和Rg1。我们正在研究这些蛋白是否参与RAS的靶蛋白。我们正在研究这些蛋白是否参与了CREB诱导的神经元分化。RhoD诱导培养细胞中应激纤维的解体和灶性粘连，并抑制细胞迁移。它还干扰胞质分裂，但不干扰核分裂，导致培养细胞和非洲爪哇胚胎出现多核。这些现象是由于Rhod对RhoA的拮抗作用引起的。我们通过双杂交系统鉴定了Rhod结合蛋白，其中一些是RhoA的靶蛋白。结果表明，Rhod通过截留RhoA的靶蛋白来拮抗RhoA。我们进一步克隆了Tc10和新的Rho家族蛋白RhoT，这两个蛋白都在肌肉组织中高效表达，并引起微刺的形成。我们正在研究它们在肌肉细胞分化中的作用。

The small GTPase M-Ras, which we have identified recently, induces neuronal differentiation in PC12 cells. We examined signal transduction pathways responsible for the differentiation. M-Ras is involved in the cAMP-induced differentiation. M-Ras was activated by cAMP-GEF, which is activated by cAMP, and then induced the activation of the transcription factor CREB. Consequently, this differentiation pathway is : cAMP → cAMP-GEF → M-Ras → ? → CREB. This pathway is distinct from those in which Ras and Papl are involved. Since CREB is essential for the long-term memory, M-Ras might participate in the regulation of higher-order brain functions including the long-term memory. To assess this possibility by generating M-Ras gene knockout mice, we constructed M-Ras gene targeting vector. We also identified target proteins for M-Ras by yeast two-hybrid system. These include Nore 1 and Rgl, which are regarded as target proteins for Ras. We are investigating whether these proteins participate in the target proteins for Ras. We are investigating whether these proteins participate in the CREB-induced neuronal differentiation.RhoD induced disassembly of the stress fibers and focal adhesions and suppressed cell migration in cultured cells. It also interfered with cytokinesis but not with nuclear division, resulting in multinucleation in cultured cells and Xenopus embryos. These phenomena were brought about by antagonization of RhoD to RhoA. We identified by two-hybrid system RhoD-binding proteins, some of which were target proteins for RhoA. The results suggest that RhoD antagonizes RhoA by sequestering the target proteins for RhoA. We further cloned Tc10 and the novel Rho family protein, RhoT, both of which were highly expressed in muscle tissues and caused microspike formation. We are examining their roles in muscle cell differentiation.