ERM PROTEIN-BASED MOLECULAR MECHANISM UNDERLYING THE REGULATION OF CELLULAR MORPHOGENESIS AND PROLIFERATION

细胞质中的休眠ERM （ezrin/radixin/moesin）蛋白以rho依赖的方式被伴随的苏氨酸磷酸化激活，并被招募到质膜上，在那里它们作为肌动蛋白丝/质膜交联剂形成微绒毛。Rho相关激酶是Rho的直接效应，在体外化学计量苏氨酸磷酸化ERM蛋白，使ERM蛋白作为交联剂保持活性状态。PI4P5K， Rho的另一个直接效应，产生磷脂酰肌醇4,5-二磷酸（PIP_2），在体外激活ERM蛋白。然后，我们研究了体内rho依赖性ERM蛋白的激活是否由rho相关激酶和/或PI4P5K介导。对血清饥饿的NIH3T3细胞使用苏氨酸磷酸化ERM蛋白（CPERMs）特异性mAb进行免疫荧光显微镜观察。转染组成活性RhoA (V14RhoA)，而不转染Rac1或Cdc42，可以增加cperm的水平，cperm只集中在微绒毛中。这种v14rhoa诱导的ERM蛋白的激活不能通过转染组成活性的rho相关激酶来模拟，也不能被Y-27632（一种特异性抑制剂）或包括rho相关激酶在内的ROCK激酶抑制。相反，PI4P5K的过表达，而不是其激酶死亡突变体，像V14RhoA一样激活了ERM蛋白。这些PI4P5K诱导的表型不受Y-27632或显性阴性RhoA （N19RhoA）的影响。这些发现表明，PI4P5K而不是ROCK激酶参与体内ERM蛋白的rho依赖性激活。我们推测pi4p5k介导的PIP2的产生主要激活ERM蛋白，并且ERM蛋白的活性状态是通过尚未确定的激酶对其苏氨酸磷酸化来维持的。这个激活过程会导致微绒毛的形成。

Dormant ERM (ezrin/radixin/moesin) proteins in the cytoplasm are activated with concomitant threonine-phosphorylation in a Rho-dependent manner, and recruited to the plasma membranes, where they function as actin filament/plasma membrane cross-linkers to form microvilli. Rho-associated kinase, a direct effector of Rho, stoichiometrically threonine-phosphorylates ERM proteins in vitro, which keeps ERM proteins at the active state as cross-linkers. PI4P5K, another direct effector of Rho, produces phosphatidylinositol 4,5-bisphosphate (PIP_2), which activates ERM proteins in vitro. We then have examined whether the Rho-dependent activation of ERM proteins is mediated by Rho-associated kinases and/or PI4P5K in vivo. Immunofluorescence microscopy of serum-starved NIH3T3 cells with a mAb specific for threonine-phosphorylated ERM proteins (CPERMs) was performed. Transfection of a constitutively active RhoA (V14RhoA), but not Rac1 or Cdc42, increased the level of CPERMs, which were exclusively concentrated in microvilli. This V14RhoA-induced activation of ERM proteins was not mimicked by transfection of constitutively active Rho-associated kinase, and was not suppressed by Y-27632, a specific inhibitor or ROCK kinases including Rho-associated kinase. In contrast, overexpression of PI4P5K, but not its kinase-dead mutant, activated ERM proteins as V14RhoA did. These PI4P5K-induced phenotypes were not affected by Y-27632 or dominant-negative RhoA (N19RhoA) These findings indicate that PI4P5K but not ROCK kinases are involved in the Rho-dependent activation of ERM proteins in vivo. We speculate that PI4P5K-mediated production of PIP2 primarily activates ERM proteins, and that the active state of ERM proteins is maintained through their threonine-phosphorylation by not-yet-determined kinases. This activation process would result in the microvilli formation.