Biochemistry of smooth muscle myosin light chain kinase.

The smooth muscle isoform of myosin light chain kinase (MLCK) is a Ca(2+)-calmodulin-activated kinase that is found in many tissues. It is particularly important for regulating smooth muscle contraction by phosphorylation of myosin. This review summarizes selected aspects of recent biochemical work on MLCK that pertains to its function in smooth muscle. In general, the focus of the review is on new findings, unresolved issues, and areas with the potential for high physiological significance that need further study. The review includes a concise summary of the structure, substrates, and enzyme activity, followed by a discussion of the factors that may limit the effective activity of MLCK in the muscle. The interactions of each of the many domains of MLCK with the proteins of the contractile apparatus, and the multi-domain interactions of MLCK that may control its behaviors in the cell are summarized. Finally, new in vitro approaches to studying the mechanism of phosphorylation of myosin are introduced.

Direct evidence for functional smooth muscle myosin II in the 10S self-inhibited monomeric conformation in airway smooth muscle cells.

The 10S self-inhibited monomeric conformation of myosin II has been characterized extensively in vitro. Based upon its structural and functional characteristics, it has been proposed to be an assembly-competent myosin pool in equilibrium with filaments in cells. It is known that myosin filaments can assemble and disassemble in nonmuscle cells, and in some smooth muscle cells, but whether or not the disassembled pool contains functional 10S myosin has not been determined. Here we address this question using human airway smooth muscle cells (hASMCs). Using two antibodies against different epitopes on smooth muscle myosin II (SMM), two distinct pools of SMM, diffuse, and stress-fiber-associated, were visualized by immunocytochemical staining. The two SMM pools were functional in that they could be interconverted in two ways: (i) by exposure to 10S- versus filament-promoting buffer conditions, and (ii) by exposure to a peptide that shifts the filament-10S equilibrium toward filaments in vitro by a known mechanism that requires the presence of the 10S conformation. The effect of the peptide was not due to a trivial increase in SMM phosphorylation, and its specificity was demonstrated by use of a scrambled peptide, which had no effect. Based upon these data, we conclude that hASMCs contain a significant pool of functional SMM in the 10S conformation that can assemble into filaments upon changing cellular conditions. This study provides unique direct evidence for the presence of a significant pool of functional myosin in the 10S conformation in cells.

Kinetic and motor functions mediated by distinct regions of the regulatory light chain of smooth muscle myosin.

To understand the importance of selected regions of the regulatory light chain (RLC) for phosphorylation-dependent regulation of smooth muscle myosin (SMM), we expressed three heavy meromyosins (HMMs) containing the following RLC mutants; K12E in a critical region of the phosphorylation domain, GTDP(95-98)/AAAA in the central hinge, and R160C a putative binding residue for phosphorylated S19. Single-turnover actin-activated Mg(2+)-ATPase (V(max) and K(ATPase)) and in vitro actin-sliding velocities were examined for both unphosphorylated (up-) and phosphorylated (p-) states. Turnover rates for the up-state (0.007-0.030 s(-1)) and velocities (no motion) for all constructs were not significantly different from the up-wild type (WT) indicating that they were completely turned off. The apparent binding constants for actin in the presence of ATP (K(ATPase)) were too weak to measure as expected for fully regulated constructs. For p-HMM containing GTDP/AAAA, we found that both ATPase and motility were normal. The data suggest that the native sequence in the central hinge between the two lobes of the RLC is not required for turning the HMM off and on both kinetically and mechanically. For p-HMM containing R160C, all parameters were normal, suggesting that R160C is not involved in coordination of the phosphorylated S19. For p-HMM containing K12E, the V(max) was 64% and the actin-sliding velocity was approximately 50% of WT, suggesting that K12 is an important residue for the ability to sense or to promote the conformational changes required for kinetic and mechanical activation.

Characterization of tightly associated smooth muscle myosin-myosin light-chain kinase-calmodulin complexes.

A current popular model to explain phosphorylation of smooth muscle myosin (SMM) by myosin light-chain kinase (MLCK) proposes that MLCK is bound tightly to actin but weakly to SMM. We found that MLCK and calmodulin (CaM) co-purify with unphosphorylated SMM from chicken gizzard, suggesting that they are tightly bound. Although the MLCK:SMM molar ratio in SMM preparations was well below stoichiometric (1:73+/-9), the ratio was approximately 23-37% of that in gizzard tissue. Fifteen to 30% of MLCK was associated with CaM at approximately 1 nM free [Ca(2+)]. There were two MLCK pools that bound unphosphorylated SMM with K(d) approximately 10 and 0.2 microM and phosphorylated SMM with K(d) approximately 20 and 0.2 microM. Using an in vitro motility assay to measure actin sliding velocities, we showed that the co-purifying MLCK-CaM was activated by Ca(2+) and phosphorylation of SMM occurred at a pCa(50) of 6.1 and at a Hill coefficient of 0.9. Similar properties were observed from reconstituted MLCK-CaM-SMM. Using motility assays, co-sedimentation assays, and on-coverslip enzyme-linked immunosorbent assays to quantify proteins on the motility assay coverslip, we provide strong evidence that most of the MLCK is bound directly to SMM through the telokin domain and some may also be bound to both SMM and to co-purifying actin through the N-terminal actin-binding domain. These results suggest that this MLCK may play a role in the initiation of contraction.