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.
It is clear from several studies that myosin phosphatase (MP) can be inhibited via a pathway that involves RhoA. However, the mechanism of inhibition is not established. These studies were carried out to test the hypothesis that Rho-kinase (Rho-associated kinase) via phosphorylation of the myosin phosphatase target subunit 1 (MYPT1) inhibited MP activity and to identify relevant sites of phosphorylation. Phosphorylation by Rho-kinase inhibited MP activity and this reflected a decrease in V max . Activity of MP with different substrates also was inhibited by phosphorylation.
We examined the role of regulatory myosin light chain (MLC) phosphorylation of myosin II in cell migration of fibroblasts. Myosin light chain kinase (MLCK) inhibition blocked MLC phosphorylation at the cell periphery, but not in the center. MLCK-inhibited cells did not assemble zyxin-containing adhesions at the periphery, but maintained focal adhesions in the center. They generated membrane protrusions all around the cell, turned more frequently, and migrated less effectively. In contrast, Rho-associated kinase (ROCK) inhibition blocked MLC phosphorylation in the center, but not at the periphery. ROCK-inhibited cells assembled zyxin-containing adhesions at the periphery, but not focal adhesions in the center. They moved faster and more straight. On the other hand, inhibition of myosin phosphatase increased MLC phosphorylation and blocked peripheral membrane ruffling, as well as turnover of focal adhesions and cell migration. Our results suggest that myosin II activated by MLCK at the cell periphery controls membrane ruffling, and that the spatial regulation of MLC phosphorylation plays critical roles in controlling cell migration of fibroblasts.
Phosphorylation of myosin II plays an important role in many cell functions, including smooth muscle contraction. The level of myosin II phosphorylation is determined by activities of myosin light chain kinase and myosin phosphatase (MP). MP is composed of 3 subunits: a catalytic subunit of type 1 phosphatase, PPlc; a targeting subunit, termed myosin phosphatase target subunit, MYPT; and a smaller subunit, M20, of unknown function. Most of the properties of MP are due to MYPT and include binding of PP1c and substrate. Other interactions are discussed. A recent discovery is the existence of an MYPT family and members include, MYPT1, MYPT2, MBS85, MYPT3 and TIMAP. Characteristics of each are outlined. An important discovery was that the activity of MP could be regulated and both activation and inhibition were reported. Activation occurs in response to elevated cyclic nucleotide levels and various mechanisms are presented. Inhibition of MP is a major component of Ca2+-sensitization in smooth muscle and various molecular mechanisms are discussed. Two mechanisms are cited frequently: (1) Phosphorylation of an inhibitory site on MYPT1, Thr696 (human isoform) and resulting inhibition of PP1c activity. Several kinases can phosphorylate Thr696, including Rho-kinase that serves an important role in smooth muscle function; and (2) Inhibition of MP by the protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17). Examples where these mechanisms are implicated in smooth muscle function are presented. The critical role of RhoA/Rho-kinase signaling in various systems is discussed, in particular those vascular smooth muscle disorders involving hypercontractility.
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