G protein‐mediated Ca2+ sensitization of airway smooth muscle contraction was investigated with respect to the relative importance of Rho‐associated coiled coil forming protein kinase (ROCK) and protein kinase C (PKC). We examined the effects of Y‐27632, a ROCK inhibitor, and GF 109203X, a PKC inhibitor, on guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS)‐induced contraction in α‐toxin‐ or β‐escin‐permeabilized rabbit trachea. Although pre‐treatment with Y‐27632 dose‐dependently inhibited GTPγS (10 μM)‐induced Ca2+ sensitization of α‐toxin‐permeabilized trachea, a Y‐27632‐insensitive component (approximately 16% of the maximum contraction) was retained during the early phase of the GTPγS response in the presence of Y‐27632 (100 μM). GF 109203X (5 μM) abolished 1 μM 4β‐phorbol 12, 13‐dibutyrate (PDBu)‐induced, but only partially inhibited the GTPγS‐induced Ca2+ sensitization. A combination of Y‐27632 (100 μM) and GF 109203X (5 μM) totally abolished the GTPγS response. GTPγS caused only a small contraction in the absence of Ca2+. Wortmannin (30 μM), a myosin light chain kinase (MLCK) inhibitor, completely inhibited Ca2+‐induced contraction. ATP‐triggered contraction of the strip which had been treated with calyculin A (1 μM), a phosphatase inhibitor, in rigor solutions was markedly slowed by worthmannin (30 μM), but not by Y‐27632 (100 μM), in the presence of GTPγS and Ca2+. GTPγS, but not PDBu, contracted the β‐escin‐permeabilized trachea in the absence of Ca2+, but the presence of Ca2+‐independent MLCK. We conclude that ROCK plays a primary role in G‐protein‐mediated Ca2+ sensitization, which requires MLCK activity, with minor contribution of PKC to the early phase of contraction, and PDBu utilizes conventional PKC(s) in airway smooth muscle. British Journal of Pharmacology (1999) 128, 925–933; doi:

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In addition to its kinase activity, myosin light chain kinase has an actin-binding activity, which results in bundling of actin filaments [Hayakawa et al., Biochem. Biophys. Res. Commun. 199, 786-791, 1994]. There are two actin-binding sites on the kinase: calcium- and calmodulin-sensitive and insensitive sites [Ye et al., J. Biol. Chem. 272, 32182-32189, 1997]. The calcium/calmodulin-sensitive, actin-binding site is located at Asp2-Pro41 and the insensitive site is at Ser138-Met213. The cyanogen bromide fragment, consisting of Asp2-Met213, is furnished with both sites and is the actin-binding core of myosin light chain kinase. Cross-linking between the two sites assembles actin filaments into bundles. Breaking of actin-binding at the calcium/calmodulin-sensitive site by calcium/calmodulin disassembles the bundles.

No abstract

Myosin light chain kinase (MLCK) phosphorylates the light chain of smooth muscle myosin enabling its interaction with actin. This interaction initiates smooth muscle contraction. MLCK has another role that is not attributable to its phosphorylating activity, i.e., it inhibits the ATP-dependent movement of actin filaments on a glass surface coated with phosphorylated myosin. To analyze the inhibitory effect of MLCK, the catalytic domain of MLCK was obtained with or without the regulatory sequence adjacent to the C-terminal of the domain, and the inhibitory effect of the domain was examined by the movement of actin filaments. All the domains work so as to inhibit actin filament movement whether or not the regulatory sequence is included. When the domain includes the regulatory sequence, calmodulin in the presence of calcium abolishes the inhibition. Since the phosphorylation reaction is not involved in regulating the movement by MLCK, and a catalytic fragment that shows no kinase activity also inhibits movement, the kinase activity is not related to inhibition. Higher concentrations of MLCK inhibit the binding of actin filaments to myosin-coated surfaces as well as their movement. We discuss the dual roles of the domain, the phosphorylation of myosin that allows myosin to cross-bridge with actin and a novel function that breaks cross-bridging.