Distinct roles of ROCK (Rho-kinase) and MLCK in spatial regulation of MLC phosphorylation for assembly of stress fibers and focal adhesions in 3T3 fibroblasts.
ROCK (Rho-kinase), an effector molecule of RhoA, phosphorylates the myosin binding subunit (MBS) of myosin phosphatase and inhibits the phosphatase activity. This inhibition increases phosphorylation of myosin light chain (MLC) of myosin II, which is suggested to induce RhoA-mediated assembly of stress fibers and focal adhesions. ROCK is also known to directly phosphorylate MLC in vitro; however, the physiological significance of this MLC kinase activity is unknown. It is also not clear whether MLC phosphorylation alone is sufficient for the assembly of stress fibers and focal adhesions. We have developed two reagents with opposing effects on myosin phosphatase. One is an antibody against MBS that is able to inhibit myosin phosphatase activity. The other is a truncation mutant of MBS that constitutively activates myosin phosphatase. Through microinjection of these two reagents followed by immunofluorescence with a specific antibody against phosphorylated MLC, we have found that MLC phosphorylation is both necessary and sufficient for the assembly of stress fibers and focal adhesions in 3T3 fibroblasts. The assembly of stress fibers in the center of cells requires ROCK activity in addition to the inhibition of myosin phosphatase, suggesting that ROCK not only inhibits myosin phosphatase but also phosphorylates MLC directly in the center of cells. At the cell periphery, on the other hand, MLCK but not ROCK appears to be the kinase responsible for phosphorylating MLC. These results suggest that ROCK and MLCK play distinct roles in spatial regulation of MLC phosphorylation.
Totsukawa
G
G
Department of Molecular Biology and Biochemistry, Rutgers University, Nelson Lab, Busch Campus, Piscataway, New Jersey 08855, USA.
Yamakita
Y
Y
Yamashiro
S
S
Hartshorne
D J
DJ
Sasaki
Y
Y
Matsumura
F
F
D016475:3T3 Cells; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001665:Binding Sites; D002448:Cell Adhesion / Q000502:physiology*; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D018994:Myosin Light Chains / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D043263:Myosin-Light-Chain Phosphatase; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D000911:Antibodies, Monoclonal; D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D009219:Myosin-Light-Chain Kinase; D010749:Phosphoprotein Phosphatases; D043263:Myosin-Light-Chain Phosphatase; D020742:rhoA GTP-Binding Protein