Phosphoprotein inhibitor CPI-17 specificity depends on allosteric regulation of protein phosphatase-1 by regulatory subunits
AUTOR(ES)
Eto, Masumi
FONTE
National Academy of Sciences
RESUMO
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 (GM) 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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=428442Documentos Relacionados
- Differential Regulation of Glycogenolysis by Mutant Protein Phosphatase-1 Glycogen-targeting Subunits*
- Nitric oxide-induced biphasic mechanism of vascular relaxation via dephosphorylation of CPI-17 and MYPT1
- Protein phosphatase 1 regulation by inhibitors and targeting subunits
- Carboxyl methylation regulates phosphoprotein phosphatase 2A by controlling the association of regulatory B subunits
- Binding Specificity of Protein Phosphatase 2A Core Enzyme for Regulatory B Subunits and T Antigens