Molecular Mechanism of Telokin-mediated Disinhibition of Myosin Light Chain Phosphatase and cAMP/cGMP-induced Relaxation of Gastrointestinal Smooth Muscle

• Published in: The Journal of biological chemistry 2012-06, Vol.287 (25), p.20975-20985
• Main Authors: Khromov, Alexander S., Momotani, Ko, Jin, Li, Artamonov, Mykhaylo V., Shannon, John, Eto, Masumi, Somlyo, Avril V.
• Format: Article
• Language: English
• Subjects: Animals; Cell Signaling; Cyclic AMP (cAMP); Cyclic AMP - genetics; Cyclic AMP - metabolism; Cyclic GMP (cGMP); Cyclic GMP - genetics; Cyclic GMP - metabolism; Cyclic Nucleotides; Gastrointestinal Tract - metabolism; Mice; Mice, Knockout; Muscle Relaxation - physiology; Muscle, Smooth - cytology; Muscle, Smooth - metabolism; Mutation, Missense - physiology; Myosin Light Chains - genetics; Myosin Light Chains - metabolism; Myosin Phosphatase; Myosin-Light-Chain Kinase - genetics; Myosin-Light-Chain Kinase - metabolism; Myosin-Light-Chain Phosphatase; Peptide Fragments - genetics; Peptide Fragments - metabolism; Phosphatase; Phosphorylation - physiology; rho-Associated Kinases - genetics; rho-Associated Kinases - metabolism; Signal Transduction; Smooth Muscle; Telokin
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.341479

Phospho-telokin is a target of elevated cyclic nucleotide concentrations that lead to relaxation of gastrointestinal and some vascular smooth muscles (SM). Here, we demonstrate that in telokin-null SM, both Ca2+-activated contraction and Ca2+ sensitization of force induced by a GST-MYPT1(654–880) fragment inhibiting myosin light chain phosphatase were antagonized by the addition of recombinant S13D telokin, without changing the inhibitory phosphorylation status of endogenous MYPT1 (the regulatory subunit of myosin light chain phosphatase) at Thr-696/Thr-853 or activity of Rho kinase. Cyclic nucleotide-induced relaxation of force in telokin-null ileum muscle was reduced but not correlated with a change in MYPT1 phosphorylation. The 40% inhibited activity of phosphorylated MYPT1 in telokin-null ileum homogenates was restored to nonphosphorylated MYPT1 levels by addition of S13D telokin. Using the GST-MYPT1 fragment as a ligand and SM homogenates from WT and telokin KO mice as a source of endogenous proteins, we found that only in the presence of endogenous telokin, thiophospho-GST-MYPT1 co-precipitated with phospho-20-kDa myosin regulatory light chain 20 and PP1. Surface plasmon resonance studies showed that S13D telokin bound to full-length phospho-MYPT1. Results of a protein ligation assay also supported interaction of endogenous phosphorylated MYPT1 with telokin in SM cells. We conclude that the mechanism of action of phospho-telokin is not through modulation of the MYPT1 phosphorylation status but rather it contributes to cyclic nucleotide-induced relaxation of SM by interacting with and activating the inhibited full-length phospho-MYPT1/PP1 through facilitating its binding to phosphomyosin and thus accelerating 20-kDa myosin regulatory light chain dephosphorylation. Phospho-telokin is a cyclic nucleotide-dependent protein kinase substrate that leads to smooth muscle relaxation. Phospho-telokin activates inhibited phosphorylated myosin phosphatase. Phospho-telokin binds to the phosphorylated myosin phosphatase facilitating its binding to phosphomyosin and myosin dephosphorylation. This mechanism may play a role in cyclic nucleotide-mediated relaxation of telokin expressing smooth muscles in the gut and vasculature.