Dissociation of LPA-induced cytoskeletal contraction from stress fiber formation by differential localization of RhoA.

Addition of lysophosphatidic acid (LPA) to serum-deprived N1E-115 neuronal cells results in rapid f-actin assembly accompanied by neurite retraction and rounding of the cell body due to contraction of the cortical actin cytoskeleton. LPA action is mimicked by activated RhoA, while it is blocked by dominant-negative RhoA (N19RhoA) and the Rho-inactivating C3 toxin. Using immunofluorescence analysis and high speed centrifugation we show that activated RhoA is localized to the plasma membrane. Wild-type RhoA and N19RhoA, however, are mainly cytosolic. We find that LPA-induced shape changes are preceded by translocation of RhoA from the cytosol to the cell periphery. LPA also stimulates translocation of inactive N19RhoA in the absence of ensuing shape changes. When membrane localization of RhoA is prevented by lovastatin, an inhibitor of protein isoprenylation, or by CAAX motif mutation, cytoskeletal contraction is blocked. However, the assembly of f-actin into stress fibers is not affected under these conditions. The effects of both LPA and activated RhoA are blocked by tyrosine kinase inhibitors (herbimycin, genistein, tyrphostin), but not by dominant-negative Src. We conclude that: (1) LPA-induced cytoskeletal contraction, but not stress fiber formation, requires translocation of RhoA from the cytosol to the plasma membrane; (2) translocation of RhoA occurs independently of its activation; and (3), a non-Src tyrosine kinase is involved in RhoA-stimulated contractility.