Abstract
Myosin II association with actin, which triggers contraction, is regulated by orchestrated waves of phosphorylation/dephosphorylation of the myosin regulatory light chain. Blocking myosin regulatory light chain phosphorylation with small molecule inhibitors alters the shape, adhesion, and migration of many types of smooth muscle and cancer cells. Dephosphorylation is mediated by myosin phosphatase (MP), a complex that consists of a catalytic subunit (protein phosphatase 1c, PP1c), a large subunit (myosin phosphatase targeting subunit, MYPT), and a small subunit of unknown function. MYPT functions by targeting PP1c onto its substrate, phosphorylated myosin II. Using RNA interference, we show here that stability of PP1c beta and MYPT1 is interdependent; knocking down one of the subunits decreases the expression level of the other. Associated changes in cell shape also occur, characterized by flattening and spreading accompanied by increased cortical actin, and cell numbers decrease secondary to apoptosis. Of the three highly conserved isoforms of PP1c, we show that MYPT1 binding is restricted to PP1c beta, and, using chimeric analysis and site-directed mutations, that the central region of PP1c beta confers the isoform-specific binding. This finding was unexpected because the MP crystal structure has been solved and was reported to identify the variable, C-terminal domain of PP1c beta as being the region key for isoform-specific interaction with MYPT1. These findings suggest a potential screening strategy for cardiovascular and cancer therapeutic agents based on destabilizing MP complex formation and function.
Highlights
Stonybrook.edu. 3 The abbreviations used are: MP, myosin phosphatase; MYPT1, myosin phosphatase targeting subunit 1; PP1, protein phosphatase 1; CHO, Chinese hamster ovary; shRNA, small hairpin RNA; GFP, green fluorescent protein; co-IP, co-immunoprecipitation
Specificity and significance of the interaction were examined in CHO cells for the endogenous proteins using a shRNA approach (Fig. 2). shRNA-mediated knockdown of PP1c  using two different targeting sequences (Fig. 2A, lanes 2 and 3) resulted in decreased expression of MYPT1, implying that MYPT1 stability requires a successful complex formation that cannot be performed by other potential partners extant in the cells
We report the surprising finding that the isoform-specific interaction of PP1c  with MYPT1 is driven by interactions with just a few nonconserved residues in a central region of PP1c  that is otherwise 96% identical to PP1c ␥, rather than by interactions with the nonconserved N and C termini
Summary
Stonybrook.edu. 3 The abbreviations used are: MP, myosin phosphatase; MYPT1, myosin phosphatase targeting subunit 1; PP1, protein phosphatase 1; CHO, Chinese hamster ovary; shRNA, small hairpin RNA; GFP, green fluorescent protein; co-IP, co-immunoprecipitation. Specificity and significance of the interaction were examined in CHO cells for the endogenous proteins using a shRNA approach (Fig. 2).
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