INTERACTION OF SMOOTH-MUSCLE MYOSIN PHOSPHATASE WITH PHOSPHOLIPIDS

The 130 kDa myosin-binding subunit (MBS) of smooth muscle myosin phosp hatase was detected in cytoskeletal, cytosolic, and membrane fractions of T24 cells. Also, MBS was distributed between cytoplasm and plasmal emma in mitotic REF52 cells. These observations prompted this study of the interaction(s) of phospholipids with myosin phosphatase. Using a sedimentation assay, gizzard myosin phosphatase bound to vesicles of a cidic phospholipids, i.e. phosphatidylserine (PS), phosphatidylinosito l, and phosphatidic acid (PA). Neutral phospholipids did not bind. Bin ding of PS to myosin phosphatase also was demonstrated by electrophore sis under nondenaturing conditions. Preferential binding of PA, compar ed to that of the other acidic phospholipids, was indicated. Interacti on of acidic phospholipids with myosin phosphatase inhibited phosphata se activity toward phosphorylated myosin. The extent of PS binding wit h myosin phosphatase decreased on increasing ionic strength and Mg2+ c oncentration. MBS (M130/M133) and M20 were phosphorylated by protein k inase A to 3 and 1 mol of P/(mol of subunit), respectively. Phosphoryl ation of the holoenzyme decreased phospholipid binding with recovery o f phosphatase activity. Using limited proteolysis of the holoenzyme an d various mutants, it was shown that phospholipid binding was associat ed with the C-terminal part of MBS, Ser 667-Ile 1004, and M20. The pho sphorylation site involved in regulation of phospholipid binding is wi thin the C-terminal MBS sequence. These results suggest that myosin ph osphatase may interact with membranes and that phosphorylation by prot ein kinase A could modify this interaction. This mechanism could be im portant in localization of myosin phosphatase and in targeting substra tes at different loci.