Acceleration of myosin light chain dephosphorylation and relaxation of smooth muscle by telokin. Synergism with cyclic nucleotide-activated kinase.
Incorporation of 32P into telokin, a smooth muscle-specific, 17-18-kDa, acidic (pI 4.2-4.4) protein, was increased by forskolin (20 microM) in intact rabbit ileum smooth muscle (ileum) and by 8-bromo-cyclic GMP (100 microM) in alpha-toxin-permeabilized ileum. Native telokin (5-20 microM), purified from turkey gizzard, and recombinant rabbit telokin, expressed in Escherichia coli and purified to >90% purity, induced dose-dependent relaxation, associated with a significant decrease in regulatory myosin light chain phosphorylation, without affecting the rate of thiophosphorylation of regulatory myosin light chain of ileum permeabilized with 0.1% Triton X-100. Endogenous telokin was lost from ileum during prolonged permeabilization (>20 min) with 0.1% Triton X-100, and the time course of loss was correlated with the loss of 8-bromo-cyclic GMP-induced calcium desensitization. Recombinant and native gizzard telokins were phosphorylated, in vitro, by the catalytic subunit of cAMP-dependent protein kinase, cGMP-dependent protein kinase, and p42/44 mitogen-activated protein kinase; the recombinant protein was also phosphorylated by calmodulin-dependent protein kinase II. Exogenous cGMP-dependent protein kinase (0.5 microM) activated by 8-bromo-cyclic GMP (50 microM) phosphorylated recombinant telokin (10 microM) when added concurrently to ileum depleted of its endogenous telokin, and their relaxant effects were mutually potentiated. Forskolin (20 microM) also increased phosphorylation of telokin in intact ileum. We conclude that telokin induces calcium desensitization in smooth muscle by enhancing myosin light chain phosphatase activity, and cGMP- and/or cAMP-dependent phosphorylation of telokin up-regulates its relaxant effect.
Wu
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Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Charlottesville, Virginia 22906-0011, USA.
Haystead
T A
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Nakamoto
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Somlyo
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Somlyo
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D000818:Animals; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D009124:Muscle Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D009126:Muscle Relaxation; D009130:Muscle, Smooth / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D010446:Peptide Fragments; D010455:Peptides; D010766:Phosphorylation; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000378:metabolism
D009124:Muscle Proteins; D010446:Peptide Fragments; D010455:Peptides; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D005576:Colforsin; C016276:8-bromocyclic GMP; C060640:telokin; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D009219:Myosin-Light-Chain Kinase; D006152:Cyclic GMP