Myosin light chain phosphatase (PP1M) is composed of three subunits, i.e., M20, MBS, and a catalytic subunit. Whereas MBS is assigned as a myosin binding subunit, the function of M20 is unknown. In the present study, we found that M20 binds to microtubules. The binding activity was revealed by cosedimentation of M20 with microtubules and binding of tubulin to M20 affinity resin. Green fluorescent protein (GFP)-tagged M20 (M20-GFP) was expressed in chicken primary smooth muscle cells and COS-7 cells and was used as a probe for studying the association between M20 and microtubules in living cells. M20-GFP was localized on filamentous structures in both cell types. Colocalization analysis revealed that M20-GFP colocalized with tubulin. Treatment with nocodazole, but not cytochalasin B, abolished the filamentous structure of M20-GFP. These results indicate that M20-GFP associates with microtubules in cells. Microinjection of rhodamine-tubulin into the M20-expressing cells revealed that incorporation of rhodamine-tubulin into microtubules was significantly facilitated by microtubule-associated M20. Consistent with this result, M20 enhanced the rate of tubulin polymerization in vitro and produced elongated microtubules. These results suggest that M20 has a microtubule binding activity and plays a role in regulating microtubule dynamics.

Agonist-induced translocation of RhoA and the spatio-temporal change in myosin regulatory light chain (MLC20) phosphorylation in smooth muscle was clarified at the single cell level. We expressed green fluorescent protein-tagged RhoA in the differentiated tracheal smooth muscle cells and visualized the translocation of RhoA in a living cell with three-dimensional digital imaging analysis. The stimulation of the cells by carbachol initiated the translocation of green fluorescent protein-tagged wild type RhoA to the plasma membrane within a minute. The change in MLC20 phosphorylation level after carbachol stimulation was monitored by using phospho-Ser-19-specific antibody recognizing the phosphorylated MLC20 in single cells. Cells expressing the dominant negative form (T19N) of RhoA significantly suppressed sustained MLC20 phosphorylation during the prolonged phase (>300 s), whereas the maximum phosphorylation level (reached at 10 s after stimulation) of these cells was not significantly different from the control cells. The kinetics of RhoA translocation was consistent with that of sustained myosin phosphorylation, suggesting the involvement of a RhoA pathway. Carbachol stimulation increased myosin phosphorylation within a minute both at the cortical and the central region. On the other hand, during prolonged phase, myosin phosphorylation was sustained at the cortical region of the cells but not at the central fibers. A myosin light chain kinase-specific inhibitor, ML-9, diminished myosin phosphorylation at the central region of the cells after the stimulation but not at the cortical area. On the other hand, Y-27632, a Rho kinase-specific inhibitor, diminished myosin phosphorylation at the cortical region but not the central region. The results clearly show that the myosin light chain kinase pathway and the Rho pathway distinctly change myosin phosphorylation in smooth muscle cells in both a temporal and spatial manner.