Rac-induced increase of phosphorylation of myosin regulatory light chain in HeLa cells.
The pathways by which activation of the small GTP-binding protein Rac causes cytoskeletal changes are not fully understood but are likely to involve both assembly of new actin filaments and reorganization of actin filaments driven by the actin-dependent ATPase activity of myosin II. Here we show that expression of active RacQ61 in growing HeLa cells, in addition to inducing ruffling, substantially enhances the level of phosphorylation of serine-19 of the myosin II regulatory light chain (MLC), which would increase actomyosin II ATPase and motor activities. Phosphorylated myosin was localized to RacQ61-induced ruffles and stress fibers. RacQ61-induced phosphorylation of MLC was reduced by a maximum of about 38% by an inhibitor (Tat-PAK) of p21-activated kinase (PAK), about 35% by an inhibitor (Y-27632) of Rho kinase, 51% by Tat-PAK plus Y-27632, and 10% by an inhibitor (ML7) of myosin light chain kinase. Staurosporine, a non-specific inhibitor of serine/threonine kinases, reduced RacQ61-induced phosphorylation of MLC by about 58%, at the maximum concentration that did not kill cells. Since Rac activates PAK and PAK can phosphorylate MLC, these data strongly suggest that PAK is responsible for a significant fraction of RacQ61-induced MLC phosphorylation. To our knowledge, this is the first evidence that active Rac causes phosphorylation of MLC in cells, thus implicating activation of the ATPase activity of actomyosin II as one of the ways by which Rac may induce cytoskeletal changes.
Brzeska
Hanna
H
Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892, USA.
Szczepanowska
Joanna
J
Matsumura
Fumio
F
Korn
Edward D
ED
D008841:Actin Cytoskeleton / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D048430:Cell Shape / Q000502:physiology; D022081:Cell Surface Extensions / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains / Q000378:metabolism*; D024462:Myosin Type II / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D022502:Stress Fibers / Q000378:metabolism; D015854:Up-Regulation / Q000502:physiology*; D054462:p21-Activated Kinases; D020744:rac GTP-Binding Proteins / Q000378:metabolism*; D054460:rho-Associated Kinases
D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains; D012694:Serine; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D054460:rho-Associated Kinases; D009219:Myosin-Light-Chain Kinase; D000251:Adenosine Triphosphatases; D024462:Myosin Type II; D020744:rac GTP-Binding Proteins