title: Differential localization of myosin and myosin phosphatase subunits in smooth muscle cells and migrating fibroblasts.
abstract: Myosin II light chains (MLC20) are phosphorylated by a Ca2+/calmodulin-activated kinase and dephosphorylated by a phosphatase that has been purified as a trimer containing the delta isoform of type 1 catalytic subunit (PP1C delta), a myosin-binding 130-kDa subunit (M130) and a 20-kDa subunit. The distribution of M130 and PP1C as well as myosin II was examined in smooth muscle cells and fibroblasts by immunofluorescence microscopy and immunoblotting after differential extraction. Myosin and M130 colocalized with actin stress fibers in permeabilized cells. However, in nonpermeabilized cells the staining for myosin and M130 was different, with myosin mostly at the periphery of the cell and the M130 appearing diffusely throughout the cytoplasm. Accordingly, most M130 was recovered in a soluble fraction during permeabilization of cells, but the conditions used affected the solubility of both M130 and myosin. The PP1C alpha isoform colocalized with M130 and also was in the nucleus, whereas the PP1C delta isoform was localized prominently in the nucleus and in focal adhesions. In migrating cells, M130 concentrated in the tailing edge and was depleted from the leading half of the cell, where double staining showed myosin II was present. Because the tailing edge of migrating cells is known to contain phosphorylated myosin, inhibition of myosin LC20 phosphatase, probably by phosphorylation of the M130 subunit, may be required for cell migration.
authors.affiliation: Center for Cell Signaling, University of Virginia Health Sciences Center, Charlottesville 22908, USA.
authors.forename: K
authors.initials: K
authors.lastname: Murata
authors.forename: K
authors.initials: K
authors.lastname: Hirano
authors.forename: E
authors.initials: E
authors.lastname: Villa-Moruzzi
authors.forename: D J
authors.initials: DJ
authors.lastname: Hartshorne
authors.forename: D L
authors.initials: DL
authors.lastname: Brautigan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001671:Biochemistry / Q000379:methods; D015153:Blotting, Western; D002465:Cell Movement / Q000502:physiology; D002642:Chick Embryo; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D007527:Isoenzymes; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D018994:Myosin Light Chains / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase; D009218:Myosins / Q000737:chemistry / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D054645:Protein Phosphatase 1; D011506:Proteins / Q000276:immunology; D011817:Rabbits; D051381:Rats; D013347:Subcellular Fractions
chemical_list: D007527:Isoenzymes; D018994:Myosin Light Chains; D011506:Proteins; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D043263:Myosin-Light-Chain Phosphatase; D009218:Myosins