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Kinetics of Myosin Light Chain Kinase Activation of Smooth Muscle Myosin in an in Vitro Model System
journal contribution
posted on 2013-11-26, 00:00 authored by Feng Hong, Kevin
C. Facemyer, Michael S. Carter, Del R. Jackson, Brian D. Haldeman, Nick Ruana, Cindy Sutherland, Michael P. Walsh, Christine R. Cremo, Josh E. BakerDuring
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, kpo, 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 kpo 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.