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Interaction of smooth muscle myosin phosphatase with phospholipids
1First Department of Internal Medicine, Mie University School of Medicine, Tsu, Japan. m-ito@clin.medic.mie-u.ac.jp
Abstract:
The 130 kDa myosin-binding subunit (MBS) of smooth muscle myosin phosphatase was detected in cytoskeletal, cytosolic, and membrane fractions of T24 cells. Also, MBS was distributed between cytoplasm and plasmalemma 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 acidic phospholipids, i.e. phosphatidylserine (PS), phosphatidylinositol, and phosphatidic acid (PA). Neutral phospholipids did not bind. Binding of PS to myosin phosphatase also was demonstrated by electrophoresis under nondenaturing conditions. Preferential binding of PA, compared to that of the other acidic phospholipids, was indicated. Interaction of acidic phospholipids with myosin phosphatase inhibited phosphatase activity toward phosphorylated myosin. The extent of PS binding with myosin phosphatase decreased on increasing ionic strength and Mg2+ concentration. MBS (M130/M133) and M20 were phosphorylated by protein kinase A to 3 and 1 mol of P/(mol of subunit), respectively. Phosphorylation of the holoenzyme decreased phospholipid binding with recovery of phosphatase activity. Using limited proteolysis of the holoenzyme and various mutants, it was shown that phospholipid binding was associated with the C-terminal part of MBS, Ser 667-Ile 1004, and M20. The phosphorylation site involved in regulation of phospholipid binding is within the C-terminal MBS sequence. These results suggest that myosin phosphatase may interact with membranes and that phosphorylation by protein kinase A could modify this interaction. This mechanism could be important in localization of myosin phosphatase and in targeting substrates at different loci.
Insights
Smooth muscle myosin phosphatase interacts with acidic phospholipids, which inhibits its activity. Phosphorylation by protein kinase A reduces this interaction, potentially regulating enzyme localization.
Area of Science:
- Biochemistry
- Cell Biology
Background:
- The myosin-binding subunit (MBS) of smooth muscle myosin phosphatase is found in various cellular compartments, including membranes.
- This cellular localization suggests potential interactions between myosin phosphatase and membrane components.
Purpose of the Study:
- To investigate the interaction between myosin phosphatase and phospholipids.
- To determine how this interaction affects enzyme activity and regulation.
Main Methods:
- Sedimentation assays and nondenaturing electrophoresis were used to study phospholipid binding.
- Limited proteolysis and analysis of mutants identified regions involved in binding and phosphorylation.
- Protein kinase A was used to phosphorylate specific subunits.
Main Results:
- Myosin phosphatase selectively binds to acidic phospholipids (phosphatidylserine, phosphatidylinositol, phosphatidic acid), inhibiting its activity.
- Binding affinity is influenced by ionic strength and Mg2+ concentration.
- Phosphorylation of MBS and M20 subunits by protein kinase A reduces phospholipid binding and restores phosphatase activity.
- Phospholipid binding is associated with the C-terminal region of MBS and the M20 subunit.
Conclusions:
- Myosin phosphatase interacts with cellular membranes via acidic phospholipids.
- Phosphorylation by protein kinase A modulates this membrane interaction, potentially controlling enzyme localization and substrate targeting.