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Kinetics of myosin light chain kinase activation of smooth muscle myosin in an in vitro model system.

ABSTRACT: During activation of smooth muscle contraction, one myosin light chain kinase (MLCK) molecule rapidly phosphorylates many smooth muscle myosin (SMM) molecules, suggesting that muscle activation rates are influenced by the kinetics of MLCK-SMM interactions. To determine the rate-limiting step underlying activation of SMM by MLCK, we measured the kinetics of calcium-calmodulin (Ca²?CaM)-MLCK-mediated SMM phosphorylation and the corresponding initiation of SMM-based F-actin motility in an in vitro system with SMM attached to a coverslip surface. Fitting the time course of SMM phosphorylation to a kinetic model gave an initial phosphorylation rate, kp(o), of ~1.17 heads s?¹ MLCK?¹. Also, we measured the dwell time of single streptavidin-coated quantum dot-labeled MLCK molecules interacting with surface-attached SMM and phosphorylated SMM using total internal reflection fluorescence microscopy. From these data, the dissociation rate constant from phosphorylated SMM was 0.80 s?¹, which was similar to the kp(o) mentioned above and with rates measured in solution. This dissociation rate was essentially independent of the phosphorylation state of SMM. From calculations using our measured dissociation rates and Kd values, and estimates of SMM and MLCK concentrations in muscle, we predict that the dissociation of MLCK from phosphorylated SMM is rate-limiting and that the rate of the phosphorylation step is faster than this dissociation rate. Also, association with SMM (11-46 s?¹) would be much faster than with pSMM (<0.1-0.2 s?¹). This suggests that the probability of MLCK interacting with unphosphorylated versus phosphorylated SMM is 55-460 times greater. This would avoid sequestering MLCK to unproductive interactions with previously phosphorylated SMM, potentially leading to faster rates of phosphorylation in muscle.

SUBMITTER: Hong F

PROVIDER: S-EPMC3886827 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Kinetics of myosin light chain kinase activation of smooth muscle myosin in an in vitro model system.

Hong Feng F Facemyer Kevin C KC Carter Michael S MS Jackson Del R del R Haldeman Brian D BD Ruana Nick N Sutherland Cindy C Walsh Michael P MP Cremo Christine R CR Baker Josh E JE

Biochemistry 20131111 47

During activation of smooth muscle contraction, one myosin light chain kinase (MLCK) molecule rapidly phosphorylates many smooth muscle myosin (SMM) molecules, suggesting that muscle activation rates are influenced by the kinetics of MLCK-SMM interactions. To determine the rate-limiting step underlying activation of SMM by MLCK, we measured the kinetics of calcium-calmodulin (Ca²⁺CaM)-MLCK-mediated SMM phosphorylation and the corresponding initiation of SMM-based F-actin motility in an in vitro ...[more]

PMID: 24144337

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Project description:Myosin light chain kinase (MLCK) phosphorylates S19 of the myosin regulatory light chain (RLC), which is required to activate myosin's ATPase activity and contraction. Smooth muscles are known to display plasticity in response to factors such as inflammation, developmental stage, or stress, which lead to differential expression of nonmuscle and smooth muscle isoforms. Here, we compare steady-state kinetics parameters for phosphorylation of different MLCK substrates: (1) nonmuscle RLC, (2) smooth muscle RLC, and heavy meromyosin subfragments of (3) nonmuscle myosin IIB, and (4) smooth muscle myosin II. We show that MLCK has a ~2-fold higher kcat for both smooth muscle myosin II substrates compared with nonmuscle myosin IIB substrates, whereas Km values were very similar. Myosin light chain kinase has a 1.6-fold and 1.5-fold higher specificity (kcat /Km ) for smooth versus nonmuscle-free RLC and heavy meromyosin, respectively, suggesting that differences in specificity are dictated by RLC sequences. Of the 10 non-identical RLC residues, we ruled out 7 as possible underlying causes of different MLCK kinetics. The remaining 3 residues were found to be surface exposed in the N-terminal half of the RLC, consistent with their importance in substrate recognition. These data are consistent with prior deletion/chimera studies and significantly add to understanding of MLCK myosin interactions.Significance of the studyPhosphorylation of nonmuscle and smooth muscle myosin by myosin light chain kinase (MLCK) is required for activation of myosin's ATPase activity. In smooth muscles, nonmuscle myosin coexists with smooth muscle myosin, but the two myosins have very different chemo-mechanical properties relating to their ability to maintain force. Differences in specificity of MLCK for different myosin isoforms had not been previously investigated. We show that the MLCK prefers smooth muscle myosin by a significant factor. These data suggest that nonmuscle myosin is phosphorylated more slowly than smooth muscle myosin during a contraction cycle.

Project description:Introduction: Phosphorylation of smooth muscle (SM) myosin regulatory light chain (RLC20) is a critical switch leading to SM contraction. The canonical view held that only the short isoform of myosin light chain kinase (MLCK1) catalyzed this reaction. It is now accepted that auxiliary kinases may contribute to vascular SM tone and contractility. We have previously reported that p90 ribosomal S6 kinase (RSK2) functions as such a kinase, in parallel with MLCK1, contributing ∼25% of the maximal myogenic force in resistance arteries. Thus, RSK2 may be instrumental in the regulation of basal vascular tone and blood pressure. Here, we take advantage of a MLCK1 null mouse (mylk1 -/-) to further test our hypothesis that RSK2 can function as an MLCK, playing a significant physiological role in SM contractility. Methods: Using fetal (E14.5-18.5) SM tissues, as embryos die at birth, we investigated the necessity of MLCK for contractility and fetal development and determined the ability of RSK2 kinase to compensate for the lack of MLCK and characterized its signaling pathway in SM. Results and Discussion: Agonists induced contraction and RLC20 phosphorylation in mylk1 -/- SM was attenuated by RSK2 inhibition. The pCa-tension relationships in permeabilized strips of bladder showed no difference in Ca2+ sensitivity in WT vs mylk1 -/- muscles, although the magnitude of force responses was considerably smaller in the absence of MLCK. The magnitude of contractile responses was similar upon addition of GTPγS to activate the RhoA/ROCK pathway or calyculinA to inhibit the myosin phosphatase. The Ca2+-dependent tyrosine kinase, Pyk2, contributed to RSK2-mediated contractility and RLC20 phosphorylation. Proximity-ligation and immunoprecipitation assays demonstrated an association of RSK2, PDK1 and ERK1/2 with MLCK and actin. RSK2, PDK1, ERK1/2 and MLCK formed a signaling complex on the actin filament, positioning them for interaction with adjacent myosin heads. The Ca2+-dependent component reflected the agonist mediated increases in Ca2+, which activated the Pyk2/PDK1/RSK2 signaling cascade. The Ca2+-independent component was through activation of Erk1/2/PDK1/RSK2 leading to direct phosphorylation of RLC20, to increase contraction. Overall, RSK2 signaling constitutes a new third signaling pathway, in addition to the established Ca2+/CaM/MLCK and RhoA/ROCK pathways to regulate SM contractility.

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Kinetics of myosin light chain kinase activation of smooth muscle myosin in an in vitro model system.

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Kinetics of myosin light chain kinase activation of smooth muscle myosin in an in vitro model system.

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