Purification of smooth-muscle myosin free of calmodulin and myosin light-chain kinase. Susceptibility to oxidation.
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ABSTRACT:
SUBMITTER: Ngai PK
PROVIDER: S-EPMC1148259 | biostudies-other | 1987 Aug
REPOSITORIES: biostudies-other
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Project description: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.
| S-EPMC3382066 | biostudies-literature
Project description:Specific phosphorylation of the human ventricular cardiac myosin regulatory light chain (MYL2) modifies the protein at S15. This modification affects MYL2 secondary structure and modulates the Ca(2+) sensitivity of contraction in cardiac tissue. Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated S15 in MYL2 in vitro. Specific modification of S15 was verified using the direct detection of the phospho group on S15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory light chain S15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (S20 in smooth muscle) but failed to phosphorylate the myosin regulatory light chain in rabbit skeletal myosin. Phosphorylation kinetics, measured using a novel fluorescence method eliminating the use of radioactive isotopes, indicates similar Michaelis-Menten V(max) and K(M) for regulatory light chain S15 phosphorylation rates in MYL2, porcine ventricular myosin, and chicken gizzard myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant kinase in cardiac tissue on the basis of its specificity, kinetics, and tissue expression.
| S-EPMC3242870 | biostudies-literature
Project description:Both smooth muscle (SM) and non-muscle (NM) myosin II are expressed in hollow organs such as the bladder and uterus, but their respective roles in contraction and corresponding physiological functions remain to be determined. In this report, we assessed their roles by analyzing mice deficient of Myl9, a gene encoding the SM myosin regulatory light chain (SM RLC). We find that global Myl9-deficient bladders contracted with an apparent sustained phase, despite no initial phase. This sustained contraction was mediated by NM myosin RLC (NM RLC) phosphorylation by myosin light chain kinase (MLCK). NM myosin II was expressed abundantly in the uterus and young mice bladders, of which the force was accordingly sensitive to NM myosin inhibition. Our findings reveal distinct roles of SM RLC and NM RLC in SM contraction.
| S-EPMC7793928 | biostudies-literature
Project description:We have performed complementary time-resolved fluorescence resonance energy transfer (TR-FRET) experiments and molecular dynamics (MD) simulations to elucidate structural changes in the phosphorylation domain (PD) of smooth muscle regulatory light chain (RLC) bound to myosin. PD is absent in crystal structures, leaving uncertainty about the mechanism of regulation. Donor-acceptor pairs of probes were attached to three site-directed di-Cys mutants of RLC, each having one Cys at position 129 in the C-terminal lobe and the other at position 2, 3, or 7 in the N-terminal PD. Labeled RLC was reconstituted onto myosin subfragment 1 (S1). TR-FRET resolved two simultaneously populated structural states of RLC, closed and open, in both unphosphorylated and phosphorylated biochemical states. All three FRET pairs show that phosphorylation shifts the equilibrium toward the open state, increasing its mol fraction by approximately 20%. MD simulations agree with experiments in remarkable detail, confirming the coexistence of two structural states, with phosphorylation shifting the system toward the more dynamic open structural state. This agreement between experiment and simulation validates the additional structural details provided by MD simulations: In the closed state, PD is bent onto the surface of the C-terminal lobe, stabilized by interdomain salt bridges. In the open state, PD is more helical and straight, resides farther from the C-terminal lobe, and is stabilized by an intradomain salt bridge. The result is a vivid atomic-resolution visualization of the first step in the molecular mechanism by which phosphorylation activates smooth muscle.
| S-EPMC2889560 | biostudies-literature
Project description:A 5.6-kilobase cDNA clone has been isolated which includes the entire coding region for the myosin light chain kinase from rabbit uterine tissue. This cDNA, expressed in COS cells, encodes a Ca2+/calmodulin-dependent protein kinase with catalytic properties similar to other purified smooth muscle myosin light chain kinases. A module (TLKPVGNIKPAE), repeated sequentially 15 times, has been identified near the N terminus of this smooth muscle kinase. It is not present in chicken gizzard or rabbit skeletal muscle myosin light chain kinases. This repeat module and a subrepeat (K P A/V) are similar in amino acid content to repeated motifs present in other proteins, some of which have been shown to associate with chromatin structures. Immunoblot analysis after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, used to compare myosin light chain kinase present in rabbit, bovine, and chicken smooth and nonmuscle tissues, showed that within each species both tissue types have myosin light chain kinases with indistinguishable molecular masses. These data suggest that myosin light chain kinases present in smooth and nonmuscle tissues are the same protein.
| S-EPMC2836767 | biostudies-literature
Project description:The molecular and biochemical properties of myosin light chain kinases from chicken skeletal and smooth muscle were investigated by recombinant DNA techniques. Deletion of the amino-terminal region of either the smooth or skeletal muscle myosin light chain kinase resulted in a decrease in Vmax with no significant change in Km values for light chain substrates. Skeletal/smooth muscle chimeric kinases were inactive when a 65-residue region amino-terminal of the catalytic core was exchanged between the two forms. Changing alanine 494 to glutamic acid within this region in the chicken skeletal muscle myosin light chain kinase increased the Km values for light chains 10-fold. These results are consistent with the hypothesis that the region amino-terminal of the catalytic core in myosin light chain kinases is involved in light chain recognition. A skeletal muscle kinase which contained the smooth muscle calmodulin binding domain remained regulated by Ca2+/calmodulin. Thus, the calmodulin binding domains of smooth and skeletal muscle myosin light chain kinases share structural elements necessary for regulation.
| S-EPMC2836765 | biostudies-literature
Project description:It has recently been shown that at relatively high molar ratios of myosin light-chain kinase (MLCKase) to calmodulin (CM) almost complete inhibition of the kinase activity occurs [Sobieszek (1991) J. Mol. Biol. 220, 947-957]. This inhibition resulted in a highly co-operative activation of MLCKase by CM, whereas the opposite activation (of CM by kinase) was hyperbolic, as expected (unco-operative). This difference in activation was observed only for kinase preparations preincubated with sub-stoichiometric amounts of CM, and only when micromolar concentrations of Ca2+ were present. The inhibitory effect was variable and depended not only on the concentration ratio of kinase to CM but also on the MLCKase preparation. For most of the preparations full inhibition required 5-15 min of preincubation at 25 degrees C and a 3-6-fold molar excess of kinase over CM. The inhibition was reversible, since full activity could be obtained after saturation of the kinase by additional CM. The inhibitory effect did not require ATP (excluding phosphorylation-type modifications of the kinase), and dephosphorylation of the kinase was not involved, since inhibition of an endogenous MLCK phosphatase by microcystin-LR did not decrease the inhibitory effect. Since the co-operative activation by CM was observed for cross-linked MLCKase preparations enriched in kinase dimers, but was absent for the analogous preparations enriched in the oligomers, we concluded that Ca(2+)-CM-dependent changes in the oligomeric state of the kinase were responsible for the modification observed. The exact nature of these modifications remains to be established.
| S-EPMC1134896 | biostudies-other
Project description:The effects of replacement of each of the individual Met in calmodulin (CaM) with Leu on the activation of two CaM target enzymes [smooth muscle myosin light chain kinase (smMLCK) and calcineurin (CN)] were investigated. The KD and Pmax (percentage maximal activation) values for activation of both enzymes by M76L-CaM were indistinguishable from wild-type (wt)-CaM, which is consistent with the location of Met-76 in the central linker that is not involved in target protein interaction. The other eight Met in CaM are exposed in the hydrophobic surfaces that are involved in target-enzymes binding, and in general equivalent effects are observed for substitutions of Leu for Met residues in homologous positions in the two CaM domains. However, the importance of the interaction of specific Met residues with the target enzyme depends on the particular enzyme. Leu substitution at Met-36 or Met-109 reduced the affinity of MLCK for the mutant and the maximal activation of CN. MLCK had a higher KD for M51L-CaM whereas M124L-CaM activated the kinase to only 68% of maximal activity induced by wt-CaM; these mutants were indistinguishable from wt-CaM in activation of CN. M71L- and M144L-CaMs behaved like wt-CaM in activation of MLCK, but activated the phosphatase to only about 80% of maximal activity induced by wt-CAM. M72L-CaM exhibited an increased affinity for MLCK compared to wt-CaM and slightly decreased maximal activation, whereas M145L-CaM exhibited maximal activation significantly greater than that due to wt-CaM; these mutants behaved like wt-CaM with respect to CN activation. Finally, a mutant CaM in which all four C-terminal Met were replaced by Leu (M4-CT-L4-CaM) had similar affinities for MLCK and CN as wt-CaM but maximal activation of these enzymes by this mutant was only 60-70% of that achieved with wt-CaM. These results imply that, in addition to removing the autoinhibitory domain from the active site of the target enzyme, CaM must induce a conformational change in the active site itself.
| S-EPMC1219332 | biostudies-other
Project description:The activity of smooth and non-muscle myosin II is regulated by phosphorylation of the regulatory light chain (RLC) at serine 19. The dephosphorylated state of full-length monomeric myosin is characterized by an asymmetric intramolecular head-head interaction that completely inhibits the ATPase activity, accompanied by a hairpin fold of the tail, which prevents filament assembly. Phosphorylation of serine 19 disrupts these head-head interactions by an unknown mechanism. Computational modeling (Tama et al., 2005. J. Mol. Biol. 345, 837-854) suggested that formation of the inhibited state is characterized by both torsional and bending motions about the myosin heavy chain (HC) at a location between the RLC and the essential light chain (ELC). Therefore, altering relative motions between the ELC and the RLC at this locus might disrupt the inhibited state. Based on this hypothesis we have derived an atomic model for the phosphorylated state of the smooth muscle myosin light chain domain (LCD). This model predicts a set of specific interactions between the N-terminal residues of the RLC with both the myosin HC and the ELC. Site directed mutagenesis was used to show that interactions between the phosphorylated N-terminus of the RLC and helix-A of the ELC are required for phosphorylation to activate smooth muscle myosin.
| S-EPMC4065857 | biostudies-literature
Project description:The mylk1 gene is a large gene spanning approximately 250 kb and comprising at least 31 exons. The mylk1 gene encodes at least four protein products: two isoforms of the 220-kDa myosin light chain kinase (MLCK), a 130-kDa MLCK, and telokin. Transcripts encoding these products are derived from four independent promoters within the mylk1 gene. The kinases expressed from the mylk1 gene have been extensively characterized and function to regulate the activity of nonmuscle and smooth muscle myosin II. Activation of these myosin motors by MLCK modulates a variety of contractile processes, including smooth muscle contraction, cell adhesion, migration, and proliferation. Dysregulation of these processes contributes to a number of diseases. The noncatalytic gene product telokin also has been shown to modulate contraction in smooth muscle cells through its ability to inhibit myosin light chain phosphatase. Given the crucial role of the products of the mylk1 gene in regulating numerous contractile processes, it seems intuitive that alterations in the transcriptional activity of the mylk1 gene also will have a significant impact on many physiological and pathological processes. In this review we highlight some of the recent studies that have described the transcriptional regulation of mylk1 gene products in smooth muscle tissues and discuss the implications of these findings for regulation of expression of other smooth muscle-specific genes.
| S-EPMC2836780 | biostudies-literature