Basis for the isoform-specific interaction of myosin phosphatase subunits protein phosphatase 1c beta and myosin phosphatase targeting subunit 1.
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.
Scotto-Lavino
Elizabeth
E
Department of Pharmacology, Stony Brook University, Stony Brook, New York 11794-5140, USA.
Garcia-Diaz
Miguel
M
Du
Guangwei
G
Frohman
Michael A
MA
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005786:Gene Expression Regulation; D007527:Isoenzymes; D016297:Mutagenesis, Site-Directed; D018994:Myosin Light Chains; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D054645:Protein Phosphatase 1 / Q000235:genetics / Q000378:metabolism*; D055550:Protein Stability; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism
D007527:Isoenzymes; D018994:Myosin Light Chains; D021122:Protein Subunits; D054645:Protein Phosphatase 1; D043263:Myosin-Light-Chain Phosphatase