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 neur onal cells results in rapid f-actin assembly accompanied by neurite re traction and rounding of the cell body due to contraction of the corti cal actin cytoskeleton, LPA action is mimicked by activated RhoA, whil e it is blocked by dominant-negative RhoA (N19RhoA) and the Rho-inacti vating C3 toxin, Using immunofluorescence analysis and high speed cent rifugation we show that activated RhoA is localized to the plasma memb rane, Wildtype RhoA and N19RhoA, however, are mainly cytosolic, We fin d that LPA-induced shape changes are preceded by translocation of RhoA from the cytosol to the cell periphery, LPA also stimulates transloca tion of inactive N19RhoA in the absence of ensuing shape changes, When membrane localization of RhoA is prevented by lovastatin, an inhibito r of protein isoprenylation, or by CAAX motif mutation, cytoskeletal c ontraction is blocked, However, the assembly of f-actin into stress fi bers is not affected under these conditions, The effects of both LPA a nd activated RhoA are blocked by tyrosine kinase inhibitors (herbimyci n, genistein, tyrphostin), but not by dominant-negative Src, We conclu de that: (1) LPA-induced cytoskeletal contraction, but not stress fibe r formation, requires translocation of RhoA from the cytosol to the pl asma membrane; (2) translocation of RhoA occurs independently of its a ctivation; and (3), a non-Src tyrosine kinase is involved in RhoA-stim ulated contractility.