RHOA-BINDING KINASE-ALPHA TRANSLOCATION IS FACILITATED BY THE COLLAPSE OF THE VIMENTIN INTERMEDIATE FILAMENT NETWORK

The regulation of morphological changes in eukaryotic cells is a compl ex process involving major components of the cytoskeleton including ac tin microfilaments, microtubules, and intermediate filaments (IFs), Th e putative effector of RhoA, RhoA-binding kinase alpha (ROK alpha), is a serine/threonine kinase that has been implicated in the reorganizat ion of actin filaments and in myosin contractility, Here, we show that ROK alpha also directly affects the structural integrity of IFs. Over expression of active ROK alpha, like that of RhoA, caused the collapse of filamentous vimentin, a type III IF. A RhoA-binding-deficient, kin ase-inactive ROK alpha inhibited the collapse of vimentin Ifs induced by RhoA in HeLa cells. In vitro, ROK alpha bound and phosphorylated vi mentin at its head-rod domain, thereby inhibiting the assembly of vime ntin, ROK alpha colocalized predominantly with the filamentous vimenti n network, which remained intact in serum-starved cells. Treatment of cells with vinblastine, a microtubule-disrupting agent, also resulted in filamentous vimentin collapse and concomitant ROK alpha translocati on to the cell periphery. ROK alpha translocation did not occur when t he vimentin network remained intact in vinblastine-treated cells at 4 degrees C or in the presence of the dominant-negative RhoAN19 mutant, Transient translocation of ROK alpha was also observed in cells subjec ted to heat shock, which caused the disassembly of the vimentin networ k. Thus, the translocation of ROK alpha to the cell periphery upon ove rexpression of RhoAV14 or growth factor treatment is associated with d isassembly of vimentin IFs. These results indicate that Rho effecters known to act on microfilaments may be involved in regulating the assem bly of IFs. Vimentin when phosphorylated also exhibits reduced affinit y for the inactive ROK alpha. The translocation of ROK alpha from Ifs to the cell periphery upon action by activated RhoA and ROK alpha sugg ests that ROK alpha may initiate its own cascade of activation.