Regulation of ROCK serine/threonine kinases by Rnd proteins

In mammals, cells need to move during development, wound healing and in response to infection. Cell movement is driven by the cytoskeleton, principally by filaments made of actin molecules. Part of cell movement involves contractile forces, which are generated by myosin filaments moving along actin filaments. Similar interactions between myosin and actin filaments generate muscle contraction. The ability of myosin filaments to move along actin filaments is increased by a protein called ROCK-I. Several human diseases could be treated by chemicals that inhibit ROCK-I, including heart disease, Alzheimers' disease and some cancers. We have identified a protein in cells, called RhoE, that binds to and inhibits ROCK-I, and thus reduces the interaction of myosin filaments with actin filaments. We want to understand more about how RhoE inhibits ROCK-I, and predict that this will help in the design of new chemicals to inhibit ROCK-I.

ROCK-I and ROCK-II are serine/threonine kinases that phosphorylate a number of proteins involved in regulating the actin cytoskeleton and actomyosin-based contractility. They thereby contribute to muscle contraction, cell migration, phagocytosis, and cell-cell junction integrity. RhoE is an atypical Rho family GTP-binding protein that binds to and inhibits ROCK-I but not ROCK-II. ROCK-I in turn phosphorylates RhoE and thereby induces its translocation from intracellular membrane to the cytosol and increases its stability. We propose to investigate the molecular basis for RhoE inhibition of ROCK-I, by mapping the regions of RhoE required for ROCK-I inhibition. In addition, we will determine where RhoE interacts with and inhibits ROCK-I in a variety of mammalian cell types, and how this interaction is regulated by extracellular stimuli. We will also investigate how phosphorylation alters RhoE localization and leads to increased stability, and whether RhoE interaction with other proteins affects its ability to inhibit ROCK-I. Furthermore, we will investigate whether there are other post-translational modifications on RhoE apart from phosphorylation, and if so how they affect its function. Finally, we will compare the responses of mammalian cells to RhoE with those to its closest relatives, Rnd1 and Rnd2, which do not bind to or inhibit ROCK-I/II.