Phosphoprotein inhibitor CPI-17 specificity depends on allosteric regulation of protein phosphatase-1 by regulatory subunits.
Inhibition of myosin phosphatase is critical for agonist-induced contractility of vascular smooth muscle. The protein CPI-17 is a phosphorylation-dependent inhibitor of myosin phosphatase and, in response to agonists, Thr-38 is phosphorylated by protein kinase C, producing a >1,000-fold increase in inhibitory potency. Here, we addressed how CPI-17 could selectively inhibit myosin phosphatase among other protein phosphatase-1 (PP1) holoenzymes. PP1 in cell lysates was separated by sequential affinity chromatography into at least two fractions, one bound specifically to thiophospho-CPI-17, and another bound specifically to inhibitor-2. The MYPT1 regulatory subunit of myosin phosphatase was concentrated only in the fraction bound to thiophospho-CPI-17. This binding was eliminated by addition of excess microcystin-LR to the lysate, showing that binding at the active site of PP1 is required. Phospho-CPI-17 failed to inhibit glycogen-bound PP1 from skeletal muscle, composed primarily of PP1 with the striated muscle glycogen-targeting subunit (G(M)) regulatory subunit. Phospho-CPI-17 was dephosphorylated during assay of glycogen-bound PP1, not MYPT1-associated PP1, even though these two holoenzymes have the same PP1 catalytic subunit. Phosphorylation of CPI-17 in rabbit arteries was enhanced by calyculin A but not okadaic acid or fostriecin, consistent with PP1-mediated dephosphorylation. We propose that CPI-17 binds at the PP1 active site where it is dephosphorylated, but association of MYPT1 with PP1C allosterically retards this hydrolysis, resulting in formation of a complex of MYPT1.PP1C.P-CPI-17, leading to an increase in smooth muscle contraction.
Eto
Masumi
M
Center for Cell Signaling, University of Virginia School of Medicine, Charlottesville, VA 22908, USA. me2h@virginia.edu
Kitazawa
Toshio
T
Brautigan
David L
DL
D000494:Allosteric Regulation; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D004791:Enzyme Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D005263:Femoral Artery / Q000378:metabolism; D020035:Holoenzymes / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D041681:NIH 3T3 Cells; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding; D054645:Protein Phosphatase 1; D021122:Protein Subunits; D011506:Proteins / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000097847:Protein Phosphatase Inhibitory Proteins
D004791:Enzyme Inhibitors; D020035:Holoenzymes; D047908:Intracellular Signaling Peptides and Proteins; D009124:Muscle Proteins; D043263:Myosin-Light-Chain Phosphatase; D010749:Phosphoprotein Phosphatases; D010750:Phosphoproteins; D054645:Protein Phosphatase 1; D021122:Protein Subunits; D011506:Proteins; D011993:Recombinant Fusion Proteins; C491521:CPI-17 protein, Oryctolagus cuniculus; C107507:PPP1R14A protein, human; C491520:Ppp1r14a protein, mouse; C024833:protein phosphatase inhibitor-2; D000097847:Protein Phosphatase Inhibitory Proteins