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

我们最近发现的小GTbromM-Ras诱导PC 12细胞的神经元分化。我们研究了负责分化的信号转导途径。M-Ras参与cAMP诱导的分化。M-Ras被cAMP激活的cAMP-GEF激活，进而诱导转录因子CREB的激活。因此，这种分化途径是：cAMP → cAMP-GEF → M-Ras →？→ CREB。该途径与Ras和Papl参与的那些途径不同。由于CREB对长时记忆至关重要，因此M-Ras可能参与包括长时记忆在内的高级脑功能的调节。为了通过产生M-Ras基因敲除小鼠来评估这种可能性，我们构建了M-Ras基因靶向载体。利用酵母双杂交系统鉴定了M-Ras的靶蛋白。这些包括Nore 1和Rgl，它们被认为是Ras的靶蛋白。我们正在研究这些蛋白质是否参与Ras的靶蛋白。我们正在研究这些蛋白质是否参与CREB诱导的神经元分化。RhoD诱导的应力纤维和局灶性粘连的解体和抑制细胞迁移在培养的细胞。它也干扰胞质分裂，但不与核分裂，导致在培养细胞和爪蟾胚胎多核。这些现象是由RhoD向RhoA的反式异构化引起的。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通过螯合RhoA的靶蛋白来拮抗RhoA。我们进一步克隆了Tc 10和新的Rho家族蛋白RhoT，它们都在肌肉组织中高度表达并引起微刺形成。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.