REQUIREMENT FOR RHO-MEDIATED MYOSIN LIGHT-CHAIN PHOSPHORYLATION IN THROMBIN-STIMULATED CELL ROUNDING AND ITS DISSOCIATION FROM MITOGENESIS

Thrombin treatment causes a dose-dependent rounding of 1321N1 astrocyt oma cells, This cytoskeletal response is rapid, peaking 2 h after thro mbin stimulation, and reverses by 50% after 24 h, The thrombin recepto r peptide SFLLRNP also induces cell rounding, whereas other G; protein -linked receptor agonists such as carbachol, lysophosphatidic acid, or bradykinin fail to do so. Results of studies using pharmacological in hibitors do not support a requirement for phosphatidylinositol 3-kinas e, mitogen-activated protein kinase, or Ca2+ mobilization in this resp onse. Inhibition of protein kinase C or tyrosine kinase produces minim al blockade, Pertussis toxin treatment is also without effect. However , thrombin-induced rounding is fully blocked by the C3 toxin from Clos tridium botulinum, which specifically ADP-ribosylates and inactivates the small G protein Rho, Thrombin also leads to a rapid, 2.4-fold incr ease in P-32 incorporation into myosin Light chain while carbachol doe s not, Myosin phosphorylation, like cell rounding is inhibited by inac tivation of Rho with C3 exoenzyme, suggesting that myosin phosphorylat ion is necessary for this cytoskeletal response. This is supported by the observation that thrombin-induced rounding is also blocked by the myosin light chain kinase inhibitor KT5926. However, treatment with KT 5926 fails to inhibit mitogenesis, Thus, cell rounding is not prerequi site to thrombin-induced DNA synthesis, We conclude that stimulation o f the heterotrimeric G protein-coupled thrombin receptor in 1321N1 cel ls activates Rho-dependent pathways for both DNA synthesis and cell ro unding, the cytoskeletal response being mediated in part through incre ases in myosin phosphorylation.