title: Interactions of the subunits of smooth muscle myosin phosphatase.
abstract: Myosin phosphatase from smooth muscle consists of a catalytic subunit (PP1c) and two non-catalytic subunits, M130 and M20. Interactions among PP1c, M20, and various mutants of M130 were investigated. Using the yeast two-hybrid system, PP1c was shown to bind to the NH2-terminal sequence of M130, 1-511. Other interactions were detected, i.e. PP1c to PP1c, M20 to the COOH-terminal fragment of M130, and dimerization of the COOH-terminal fragment of M130. Mutants of M130 were constructed to localize the PP1c and light chain binding regions. Results from the two-hybrid system indicated two binding sites for PP1c on M130: one site in the NH2-terminal 38 residues and a weaker site(s) in the ankyrin repeats region. Inhibition of PP1c activity with phosphorylase a by the M130 mutants also was consistent with the assignment of these two sites. Overlay assays showed binding of phosphorylated light chain to the ankyrin repeats, probably in the COOH-terminal repeats. Activation of PP1c with phosphorylated light chain required binding sites for PP1c and substrate, plus an additional sequence COOH-terminal to the ankyrin repeats. Thus, activation of phosphatase and binding of PP1c and substrate are properties of the NH2-terminal one-third of M130.
authors.affiliation: Muscle Biology Group, University of Arizona, Tucson, Arizona 85721, USA.
authors.forename: K
authors.initials: K
authors.lastname: Hirano
authors.forename: B C
authors.initials: BC
authors.lastname: Phan
authors.forename: D J
authors.initials: DJ
authors.lastname: Hartshorne
mesh_terms: D004789:Enzyme Activation; D004926:Escherichia coli; D017930:Genes, Reporter; D009130:Muscle, Smooth / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D043263:Myosin-Light-Chain Phosphatase; D010749:Phosphoprotein Phosphatases / Q000737:chemistry*; D011487:Protein Conformation; D054645:Protein Phosphatase 1; D012441:Saccharomyces cerevisiae; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D043263:Myosin-Light-Chain Phosphatase; D001616:beta-Galactosidase