rho p21 in Neural Development and Plasticity

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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