Regions of the 110-kDa regulatory subunit M110 required for regulation of myosin-light-chain-phosphatase activity in smooth muscle.
To characterize the in situ interactions between the subunits (regulatory 110 kDa, M110; 21-kDa, M21 and catalytic, 37-kDa, PP1c) of smooth muscle myosin phosphatase (SMPP-1M), we determined, in Triton-X-100-permeabilized rabbit portal vein contracted with microcystin-LR, the ability of the following fragments of M110 to regulate relaxation induced by exogenous PP1c: (a) M110 purified from pig bladder; (b) the 72.5-kDa N-terminal fragment expressed from rat kidney cDNA [glutathione-S-transferase-M110-(11-612)-peptide]; (c) a 58-kDa fragment, the N-terminal degradation product of M110 (M58); (d) two fragments expressed from rat aorta cDNA [M110-(1-309)-peptide and M110-(39-309)-peptide]; a synthetic fragment of M110 [M110-(1-38)-peptide]. The M110/M21 complex accelerated approximately 1.6-fold the rate of dephosphorylation of the myosin P-light chain and also relaxation induced by PP1c. The glutathione-S-transferase-M110-(11-612)-peptide and the M58 fragments, as well as the M110-(1-309)-peptide and, at higher concentration, M110-(1-38)-peptide, had similar effects that did not require the M21 subunit. Arachidonic acid, known to dissociate PP1c from the native holoenzyme and inhibit SMPP-1M activity, inhibited the regulatory action of the M110/M21 complex on PP1c activity and, to a lesser extent that of the glutathione-S-transferase-M110-(11-612)-peptide, but not that of the M58 fragment or of the shorter peptides. We conclude that, consistent with in vitro studies [8], the N-terminal sequence (1-309) of the M110 subunit is also sufficient to enhance the activity of PP1c for myosin in muscle. However, its C-terminal half (downstream from the M58 fragment) is required for inhibition by arachidonic acid. In contrast to the effect of the M110 subunit and its fragments, a peptide, corresponding to part of the PP1c-binding site of the regulatory glycogen-binding subunit from skeletal muscle GM [GM-(63-93)-peptide], specifically slowed the relaxation, induced by flash photolysis of diazo-2, of Triton X-100-permeabilized femoral artery strips, and inhibited the holoenzyme-induced relaxation in the portal vein, suggesting that the GM subunit can compete with the regulatory effect of M110 on PP1c in smooth muscle.
Gailly
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Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Charlottesville 22908, USA.
Wu
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Haystead
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Somlyo
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Cohen
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Cohen
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Somlyo
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D000818:Animals; D016718:Arachidonic Acid / Q000494:pharmacology; D005263:Femoral Artery; D005982:Glutathione Transferase; D006706:Homeostasis; D066298:In Vitro Techniques; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D046911:Macromolecular Substances; D009126:Muscle Relaxation / Q000187:drug effects; D009130:Muscle, Smooth / Q000201:enzymology*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology* / Q000502:physiology; D043263:Myosin-Light-Chain Phosphatase; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D011169:Portal Vein / Q000502:physiology; D054645:Protein Phosphatase 1; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013552:Swine; D001743:Urinary Bladder / Q000201:enzymology
D046911:Macromolecular Substances; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D016718:Arachidonic Acid; D005982:Glutathione Transferase; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D043263:Myosin-Light-Chain Phosphatase