Differential Association and Localization of Myosin Phosphatase Subunits During Agonist-Induced Signal Transduction in Smooth Muscle

Abstract

Abstract-It has been known for some time that agonist-induced contractions of vascular smooth muscle are often associated with a sensitization of the contractile apparatus to intracellular Ca 2ϩ . One mechanism that has been suggested to explain Ca 2ϩ sensitization is inhibition of myosin phosphatase activity. In the present study, we tested the hypothesis that differential localization of the phosphatase might be associated with its inhibition. Quantitative confocal microscopy of freshly dissociated, fully contractile smooth muscle cells was used in parallel with measurements of myosin light chain and myosin phosphatase phosphorylation. The results indicate that, in the smooth muscle cells, the catalytic and targeting subunits of the phosphatase are dissociated from each other in an agonist-specific manner and that the dissociation is accompanied by a slower rate of myosin phosphorylation. Targeting of myosin phosphatase to the cell membrane precedes the dissociation of subunits and is associated with phosphorylation of the targeting subunit at a Rho-associated kinase (ROK) phosphorylation site. The phosphorylation and membrane translocation of the targeting subunit are inhibited by a ROK inhibitor. This dissociation of subunits may provide a mechanism for the decreased phosphatase activity of phosphorylated myosin phosphatase. (Circ Res. 2002;90:546-553.)

Myosin phosphatase target subunit: Many roles in cell function

“…The translocation to the membrane was accompanied by phosphorylation of MYPT1 at T697 and was prevented by inhibition of ROK. The dissociation of MP would reduce phosphatase activity toward P-myosin and this was consistent with a sustained contractile response to PGF2α, compared to a more phasic response with phenylephrine [23]. This was the first report that documented the possible role of MYPT1 phosphorylation in translocation.…”

Transduction of the N-Terminal Fragments of MYPT1 Enhances Myofilament Ca 2+ Sensitivity in an Intact Coronary Artery

“…A dissociation of PP1c from the holoenzyme was reported to play an important role in inhibiting the myosin phosphatase activity. 35,36 However, the augmenting effect of TAT-MYPT1 fragments was demonstrated to be reversible and reproducible (Figure 1e). It is thus unlikely that exogenous MYPT1 fragments caused substantial dissociation of PP1c from the holoenzyme and thereby inhibited the myosin phosphatase activity.…”

TheCaenorhabditis elegansnonmuscle myosin genesnmy-1andnmy-2function as redundant components of thelet-502/Rho-binding kinase andmel-11/myosin phosphatase pathway during embryonic morphogenesis

“…4). Similar to MYPT during smooth muscle contraction (Shin et al, 2002), MEL-11 moves from the cytoplasm to the membrane, away from the contractile apparatus, when contraction begins (Figs 5 and 6). After elongation is complete, low levels of cytoplasmic MEL-11 reappear.…”

“…6E-H). The vertebrate MEL-11 homolog, MYPT, moves to the plasma membrane after phosphorylation by ROK, where it remains sequestered from actin/myosin to allow contraction (Shin et al, 2002). To determine if MEL-11 was similarly influenced by let-502 activity, we examined MEL-11 localization in let-502 mutants.…”