Signal Transduction and Cellular Functions of the Small GTPase Rho and its Effectors

确定了小G蛋白酶Rho的效应子，并确定了从Rho到肌动蛋白细胞骨架重组的信号传导途径。(1)我们发现，在Rho下游被激活的ROCK激酶至少通过三种途径来调节肌动蛋白细胞骨架，一种是磷酸化和肌球蛋白磷酸酶的失活以增加肌球蛋白为基础的收缩力，第二种是磷酸化和LIM-连接酶的活化导致cofilin的肌动蛋白解聚活性的失活，第三种是I型NaH交换剂的磷酸化。我们进一步表明，这些Rho-ROCK通路与高血压的诱发以及肿瘤细胞的侵袭和转移有关。(2)我们确定p140 mDia作为一种新的Rho效应蛋白。该蛋白通过其FH 1区域结合profiin，并通过其FH 2区域调节微管的排列，从而整合肌动蛋白细胞骨架和微管的组织。我们发现，这种行动的mDia是必不可少的Rho诱导诱导和应力纤维的排列。(3)我们鉴定了Citron激酶，并表明它在胞质分裂中作为Rho效应子起作用。我们还建立了一种GTP-Rho的pull-down检测方法，证明ECT 2是一种在胞质分裂中起作用的Rho交换蛋白。(4)我们确定ropporin作为rhophilin的结合蛋白，并表明这两种蛋白质一起定位于精子尾部。我们还发现，ropporin产生同源二聚体，并与另一种精子蛋白AKAP-110结合。

Effectors for the small GTPase Rho were determined, and signal tranuction pathways from Rho to the reorganization of the actin cytoskeleton were identified. (1) We identified that ROCK-kinase, that is activated downstream of Rho, moblizes at least three pathways to regulate the actin cytoskeleton, one being phsporylaiton and inacitvation of myosin phosphatas to increase the myosin-based contractility, the second phosphorylaiton and acivaton of LIM-linase leading to inactivation of actin depolymerizing activity of cofilin, and the third phosphorylation of type I NaH exchanger. We further showed that these Rho-ROCK pathways are impliated in elicitation of hypertension and invasion and metastasis of tumor cells. (2) We identified p140mDia as a novel Rho effector protein. This protein binds profiin through its FH1 region and regulate alignment of microtubules through its FH2 region, and thereby integrate the organization of the actin cytoskeleton and microtubules. We showed that this action of mDia is essential in Rho-induced inducion and alignment of stress fibers. (3) We identified Citron-kinase, and showed that it works as a Rho effector in cytokinesis. We heve also developed a pull-down assay for GTP-Rho, and showed that ECT2 is a Rho exchanger working in cytokinesis. (4) We identified ropporin as a binding protein of rhophilin, and showed that the two proteins together localize in the sperm tail. We also showed that ropporin makes a homo dimer and binds to another sperm protein, AKAP-110.