rho p21 in Neural Development and Plasticity

rho p21 在神经发育和可塑性中的作用

• 批准号: 07044248
• 负责人: KAKIZUKA Akira
• 金额: $ 7.04万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for international Scientific Research
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07044248/
• 关键词: rho p21; p160ROCK; focal adhesion; intergrin; p140mDia; actin polymerization; Neural plasticity; cell adhesion; rho p21; ボツリヌスC_3酵素; PKN; P160^<ROCK>; 細胞形態変化; SH_3

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

我们利用配体覆盖印迹和酵母双杂交系统分离了新的Rho效应分子p160 ROCK、ROCK-II、Rhotekin和p140 mDia。我们现在正在表征这些效应分子的功能。p160 ROCK诱导应力纤维和粘着斑的形成，在转染实验中观察到。构建了p160 ROCK显性失活突变体，并与显性活性突变体RhoA Val 14-RhoA共转染HeLa细胞，该突变体抑制了Val 14-RhoA表型，即Rho诱导的应力纤维和粘着斑的形成。这些结果表明，p160 ROCK在Rho下游起作用，介导应力纤维和局灶性粘连的形成。我们还利用酵母双杂交系统分离了Rhotekin。Rhotekin的Rho结合结构域显示出与PKN和Rhophilin中的Rho结合结构域的同源性，定义了Rho结合的新的共有序列。Rhotekin与Rho结合，Rho GT3活性降低 ...更多信息 这种束缚。最近，我们又利用酵母双杂交系统分离到了p140 mDia。p140 mDia在其N-末端具有Rho结合结构域，在蛋白质的中间具有聚脯氨酸区域。p140 mDia通过其多聚脯氨酸区域与profilin（调节肌动蛋白聚合）结合。在免疫荧光分析中，p140 mDia定位于胞质分裂过程中的分裂沟和胞膜附近。我们的Rho效应分子的cDNA已被发送给世界上许多研究人员，并进行了几次合作。例如，维滕贝格博士的小组（马克斯普朗克研究所）正在使用X射线衍射确定我们的Rho效应器的3D结构。西蒙兹博士（ONYX制药公司）用显微注射等细胞学技术帮助我们，沃里博士（拉霍亚癌症基金会）正在通过整合蛋白分析细胞粘附，这是一种涉及p160 ROCK的功能。我们还与穆伦纳博士合作研究神经突收缩对细胞外刺激的反应。少

项目成果

Tim Reid, T.et al.: "Rhotekin, a new putative target for Rho bearing homology to a serine-threonine kinase, PKN,and rhophilin in the Rho-binding domain" Journal of Biological Chemistry. 271. 13556-1356- (1996)

Tim Reid, T.et al.：“Rhotekin，Rho 的新推定靶标，与 Rho 结合域中的丝氨酸-苏氨酸激酶、PKN 和 rhophilin 同源”《生物化学杂志》。

Watanabe,N.et al.: "p140mDia,a mammalian homologue of Drosophila diaphanous,is a target protein for Rho small GTPase and is a ligand for profilin" EMBO J.(in press)

Watanabe,N.et al.：“p140mDia，果蝇透明的哺乳动物同源物，是 Rho 小 GTP 酶的靶蛋白，并且是 profilin 的配体” EMBO J.（正在印刷中）

Narumiya,S.: "The small GTPase Rho:Cellular Functions and Signal Trunsduction" J.Biochemistry, 14 (1996)

Narumiya,S.：“小 GTP 酶 Rho：细胞功能和信号截断”J.Biochemistry，14 (1996)

Reid,T.et al.: "Rhotekin,a new putative target for Rho bearing homology to a serine-threonine kinase,PKN,and rhophilin in the Rho-binding domain" Journal of Biological Chemistry. 271. 13556-13560 (1996)

Reid,T.等人：“Rhotekin，Rho 的新推定靶标，与 Rho 结合域中的丝氨酸-苏氨酸激酶、PKN 和 rhophilin 具有同源性”《生物化学杂志》。

Fujisawa,K et al.: "Identification of the Rho-binding Domain of p160ROCK,a Rho associated coiled-coil Containing Protein Kinase" Journal of Biological Chemistry. 271. 23022-23028 (1996)

Fujisawa,K 等人：“p160ROCK 的 Rho 结合域的鉴定，一种含有蛋白激酶的 Rho 相关卷曲螺旋”《生物化学杂志》。