rho p21 in Neural Development and Plasticity

我们利用配体覆盖印迹和酵母双杂交系统分离出了新的Rho效应分子p160ROCK、ROCK-II、Rhotekin和p140mDia。我们现在正在描述这些效应分子的功能。转染实验中观察到p160ROCK诱导应力纤维的形成和局灶粘连。构建p160ROCK显性阴性突变体，与显性活性突变体RhoA Val14-RhoA共转染HeLa细胞。p160ROCK的显性阴性突变体抑制了Val14-RhoA表型，即Rho诱导的应力纤维和局灶粘连的形成。这些结果表明p160ROCK作用于Rho下游，介导应力纤维的形成和局灶粘连。我们还分离了红景天与酵母双杂交体系。Rhotekin的Rho结合域与PKN和Rhophilin的Rho结合域具有同源性，为Rho结合定义了一个新的共识序列。Rhotekin与Rho结合，Rho GTPase活性降低。最近，我们还分离了酵母双杂交系统的p140mDia。p140mDia在其n端有一个rho结合结构域，在蛋白质的中间有一个聚脯氨酸区域。p140mDia通过其聚脯氨酸区与profilin（它调节肌动蛋白聚合）结合。在免疫荧光分析中，p140mDia定位于靠近膜的周围，以及细胞分裂时的卵裂沟。我们参加了两次国际会议，展示了这些成果，交流了新的信息。我们的Rho效应分子cDNA已发送给世界上许多研究人员，并进行了多次合作。例如，wittenhofer博士的团队（Max-Planck研究所）正在使用x射线衍射确定我们的Rho效应的3d结构。symonds博士（ONYX制药公司）用显微注射等细胞学技术帮助我们，vuori博士（La Jolla癌症基金会）正在通过intergrin分析细胞粘附性，这是一种涉及p160ROCK的功能。我们还与moolenaar博士合作研究神经突收缩对额外细胞刺激的反应。少

We have isolated new Rho effector molecules, p160ROCK,ROCK-II,Rhotekin and p140mDia using ligand overlay blotting and yeast two hybrid system. We are now characterizing functions of these effector molecules.p160ROCK induces formation of stress fibers and focal adhesions, as observed in transfection experiments. A dominant negative mutant of p160ROCK was constructed, and co-transfected in HeLa cells together with Val14-RhoA,a dominant active mutant RhoA.This dominant negative mutant of p160ROCK suppressed the Val14-RhoA phenotype, namely the Rho induced stress fibers and focal adhesion formation. These results demonstrate that p160ROCK works downstream of Rho, mediating the formation of stress fibers and focal adhesions. We have also isolated Rhotekin with yeast two hybrid system. The Rho binding domain of Rhotekin shows homology to the Rho binding domains in PKN and Rhophilin, defining a new consensus sequence for Rho binding. Rhotekin binds to Rho and Rho GTPase activity is decreased … More by this binding. Recently, we also isolated p140mDia with yeast two hybrid system. p140mDia has a Rho-binding domain in its N-terminus and polyproline regions in the middle of the protein. p140mDia binds to profilin (which regulates actin polymerization)through its poly-proline regions. In immunofluorescence analysis, p140mDia was localized at the periphery near the membrane, and at the cleavage furrow during cytokinesis.We participated in two international meeting, presenting these results and exchanging new informations. Our cDNA of Rho effector molecules have been sent to numerous researchers in the world and several collaborations have been undertaken. For example Dr.Wittenghofer's group (Max-Planck institute) is determining the 3D-structure of our Rho effectors using X-ray diffraction. Dr.Symonds (ONYX pharmaceutics) helped us with cytological techniques such as microinjection, and Dr.Vuori (La Jolla Cancer Foundation) is analyzing cell adhesion through intergrin, a function that involves p160ROCK.We are also collaborating with Dr.Moolenaar on neurite retraction in response to extra cellular stimulation. Less