No abstract

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.

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To characterize the in situ interactions between the subunits (regulatory 110 kDa, MI,,,; 21-kDa, M,, and catalytic, 37-kDa, PPl,.) of smooth muscle myosin phosphatase (SMPP-I M), we determined, in Triton-X-l 00-permeabilized rabbit portal vein contracted with microcystin-LR, the ability of the following fragments of M I , , , to regulate relaxation induced by exogenous PPI,: (a) M I , , , purified from pig bladder; (b) the 72.5-kDa N-terminal fragment expressed from rat kidney cDNA [glutathione-S-transferase-MI(1 1-61 2)-peptide]; (c) a 58-kDa fragment, the N-terminal degradation product of M,,,, (M5J; (d) two fragments expressed from rat aorta cDNA [M, ,,,-(l - of the M I ,,, subunit is also sufficient to enhance the activity of PPI, for myosin in muscle. However, its C-terminal half (downstream from the M,, fragment) is required for inhibition by arachidonic acid. In contrast to the effect of the MI,,, subunit and its fragments, a peptide, corresponding to part of the PP1,-binding site of the regulatory glycogen-binding subunit from skeletal muscle G , [G,,-(63 -93)-peptide], specifically slowed the relaxation, induced by tlash 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 G, subunit can compete with the regulatory effect of M , , , , on PPI, in smooth muscle.Keywords: protein phosphatase; smooth muscle myosin phosphatase; smooth muscle; calcium sensitization; arachidonic acid.Contraction of smooth muscle is activated by phosphorylation and inactivated by dephosphorylation of the 20-kDa regulatory light chain of myosin (MLC,,,). Dephosphorylation of MLC,,, is regulated by a guanine-nucleotide-binding-proteincoupled mechanism and reflects the activity of smooth muscle myosin phosphatase (SMPP-IM), a heterotrimcr of a 110-kDa regulatory subunit (MI ,,, MLC?,, and also binds to myosin [3-51, 'targeting' the holoenzyme SMPP-IM to its substrate. Similarly, in skeletal muscle, PPI,. can he 'targeted' by the G , regulatory subunit to glycogen 16, 71. To understand the mechanisms underlying the regulation of these processes, Johnson et al. 181, identified, in v i m , the sites on G , and MI,,, that bind to PPI, and showed that the binding of these subunits to PPI,. is mutually exclusive. The purpose of our study was to determine whether the mechanisms deduced from experiments conducted with isolated proteins in solution also operate in a muscle cell environment of filamentous myosin and other proteins involved in contraction. We show here that the N-terminal 309-amino-acid sequence of M I ,,, and, at higher concentration, the M I ,,,-(I -38)-peptide can increase, in permeabilized fibers, the activity of PP1, towards MLC,,, to a level indistinguishable from that of the holoenzyme. We also show that a peptide corresponding to the PP1,-binding site of G,, that can displace MI,,, from PP1,. in vitro [S], inhibits, in permeabilized muscle, the acceleration of PP1 ,.-induced relaxation of smooth muscle by M...

This review summarizes the role of MgADP in force maintenance by dephosphorylated cross-bridges in smooth muscle and a potential physiological role for telokin. In tonic, compared with phasic, smooth muscles the affinity of cross-bridges in approximately 5 times higher for MgADP and the apparent second-order rate constant for MgATP is approximately 3 times lower. This gives rise to a large population of dephosphorylated cross-bridges in tonic smooth muscle. Such cross-bridges are thought to be major determinants of the different relaxation kinetics of the two types of smooth muscle and contribute to force maintenance at low levels of MLC20 phosphorylation, termed 'catch-like state' (Somlyo & Somlyo 1967) or 'latch' (Dillon et al. 1981). The molecular basis of the different affinities for MgADP and MgATP between tonic and phasic smooth muscle myosin was explored by exchange of essential myosin light chain (LC17) isoforms. In phasic bladder smooth muscle the exchange of LC17b for LC17a caused a significant decrease in the unloaded shortening velocity of non-phosphorylated, slowly cycling cross-bridges, suggesting that the LC17 isoforms contribute to the nucleotide affinity of latch bridges. The role of telokin in Ca(2+)-desensitization in phasic smooth muscle is reviewed. Telokin, the independently expressed C-terminus of myosin light chain kinase, is extensively phosphorylated during forskolin- and 8-br-cGMP-induced relaxation in situ. Telokin accelerated dephosphorylation of the regulatory myosin light chain and relaxed rabbit ileum smooth muscle. The results suggest that telokin contributes to cAMP and/or cGMP kinase-mediated Ca(2+)-desensitization of phasic smooth muscles.