Rho-kinase-mediated contraction of isolated stress fibers

It is widely accepted that actin filaments and the conventional double-head ed myosin interact to generate force for many types of nonmuscle cell motil ity, and that this interaction occurs when the myosin regulatory light chai n (MLC) is phosphorylated by MLC kinase (MLCK) together with calmodulin and Ca2+. However, recent studies indicate that Rho-kinase is also involved in regulating the smooth muscle and nonmuscle cell contractility. We have rec ently isolated reactivatable stress fibers from cultured cells and establis hed them as a model system For actomyosin-based contraction in nonmuscle ce lls. Here, using isolated stress fibers, we show that Rho-kinase mediates M LC phosphorylation and their contraction in the absence of Ca2+. More rapid and extensive stress fiber contraction was induced by MLCK than was by Rho -kinase. When the activity of Rho-kinase but not MLCK was inhibited, cells not only lost their stress fibers and focal adhesions but also appeared to lose cytoplasmic tension. Our study suggests that actomyosin-based nonmuscl e contractility is regulated by two kinase systems: the Ca2+-dependent MLCK and the Rho-kinase systems. We propose that Ca2+ is used to generate rapid contraction, whereas Rho-kinase plays a major role in maintaining sustaine d contraction in cells.