Activation of ERM proteins in vivo by Rho involves phosphatidylinositol 4-phosphate 5-kinase and not ROCK kinases

When activated, ERM (ezrin, radixin, moesin) proteins are recruited to the plasma membrane, with concomitant carboxy-terminal threonine phosphorylation, where they crosslink actin filaments to the plasma membrane to form microvilli (reviewed in [1-5]). Here, we report that, when NIH3T3 or HeLa cells were transfected with a constitutively active mutant of the small GTPase RhoA (V14RhoA), microvilli were induced and the level of carboxy-terminal threonine-phosphorylated ERM proteins (CPERM) [6,7] increased similar to 30-fold. This increase was not observed following transfection of constitutively active forms of two other Rho family GTPases, Rad and Cdc42, or of a direct effector of Rho, Rho-kinase (also known as ROK alpha or ROCK-II) [8-10]. The V14RhoA-induced phosphorylation of ERM proteins was not suppressed by Y-27832, a specific inhibitor of ROCK kinases including Rho-kinase [11], Overexpression of another direct effector of Rho, phosphatidylinositol 4-phosphate 5-kinase (PI4P5K) type I alpha [12-14], but not a kinase-inactive mutant [15], increased similar to sixfold the level of CPERM, and induced microvilli, Together with the previous finding that the PI4P5K product phosphatidylinositol 4,5-bisphosphate (PIP2) activates ERM proteins in vitro [16], our data suggest that PIP2, and not ROCK kinases, is involved in the RhoA-dependent activation of ERM proteins in vivo. The active state of ERM proteins is maintained through threonine phosphorylation by as yet undetermined kinases, leading to microvillus formation. (C) 1999 Elsevier Science Ltd. All rights reserved.