INTERACTIONS OF THE SUBUNITS OF SMOOTH-MUSCLE MYOSIN PHOSPHATASE

Myosin phosphatase from smooth muscle consists of a catalytic subunit (PP1c) and two non-catalytic subunits, M130 and M20, Interactions amon g PP1c, M20, and various mutants of M130 were investigated. Using the yeast two hybrid system, PP1c was shown to bind to the NH2-terminal se quence of M130, 1-511. Other interactions were detected, i.e. PP1c to PP1c, M20 to the COOH-terminal fragment of M130, and dimerization of t he COOH-terminal fragment of M130. Mutants of M130 were constructed to localize the PP1c and light chain binding regions, Results from the t wo-hybrid system indicated two binding sites for PP1c on M130: one sit e in the NH2-terminal 38 residues and a weaker site(s) in the ankyrin repeats region, Inhibition of PP1c activity with phosphorylase a by th e M130 mutants also was consistent with the assignment of these two si tes. Overlay assays showed binding of phosphorylated light chain to th e ankyrin repeats, probably in the COOH-terminal repeats. Activation o f PP1c with phosphorylated light chain required binding sites for PP1c and substrate, plus an additional sequence COOH-terminal to the ankyr in repeats. Thus, activation of phosphatase and binding of PP1c and su bstrate are properties of the NH2-terminal one-third of M130.