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, kpo, of ~1.17 heads s−1·MLCK−1. Also we measured the dwell time of single QD-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 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 Kds, and estimates of [SMM] and [MLCK] 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 to SMM (11-46 s−1) would be much faster than to pSMM (<0.1-0.2 s−1). This suggests that the probability of MLCK interacting with unphosphorylated versus pSMM 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.

Diffusion of myosin light chain kinase on actin: A mechanism to enhance myosin phosphorylation rates in smooth muscle

“…Previously we showed, by observing single molecules in vitro, that the MLCK-myosin detachment rate is likely to be rate limiting for phosphorylation at 1.2 heads s 1 MLCK 1 (Hong et al, 2013), a value that is in reasonable agreement with the measured ratios of MLCK to myosin and typical phosphorylation rates in muscle. Therefore, diffusion time will be much shorter than phosphorylation time, ensuring that diffusion is not rate limiting for phosphorylation.…”

“…The ratio of active MLCK to SMM in smooth muscle is relatively low (Injeti et al, 2008), and thus one MLCK must phosphorylate many SMMs within 1-2 s to account for phosphorylation rates (see references in Hong et al, 2013). The time, t, it takes a single MLCK molecule to diffuse a distance, d, can be estimated by the first-passage time for 1-D diffusion, t = d 2 /2D (Berg, 1993;Howard, 2001).…”

“…Therefore, by examining MLCK motion on stress fibers, we were able to explore the idea that the state of myosin, specifically its affinity for actin and its phosphorylation state, could affect the motion of MLCK and N75. MLCK is a long molecule that, when extended, is likely to be long enough to simultaneously bind both actin and myosin (Mabuchi et al, 2010), and importantly, the MBD binds tightly to unphosphorylated but weakly to phosphorylated SMM (Sellers and Pato, 1984;Hong et al, 2011Hong et al, , 2013. Because N75 lacks the MBD, comparison of MLCK with N75 will reveal the extent to which MBDmyosin interactions of MLCK control its motion.…”

Diffusion of myosin light chain kinase on actin: A mechanism to enhance myosin phosphorylation rates in smooth muscle

“…Therefore, by examining MLCK motion on stress fibers, we were able to explore the idea that the state of myosin, specifically its affinity for actin and its phosphorylation state, could affect the motion of MLCK and N75. MLCK is a long molecule that, when extended, is likely to be long enough to simultaneously bind both actin and myosin ( Mabuchi et al, 2010 ), and importantly, the MBD binds tightly to unphosphorylated but weakly to phosphorylated SMM ( Sellers and Pato, 1984 ; Hong et al, 2011 , 2013 ). Because N75 lacks the MBD, comparison of MLCK with N75 will reveal the extent to which MBD–myosin interactions of MLCK control its motion.…”

“…Diffusion of MLCK on actin may be an important mechanism for enhancing rates of SMM phosphorylation in smooth muscle. The ratio of active MLCK to SMM in smooth muscle is relatively low ( Injeti et al, 2008 ), and thus one MLCK must phosphorylate many SMMs within 1–2 s to account for phosphorylation rates (see references in Hong et al, 2013 ). The time, t , it takes a single MLCK molecule to diffuse a distance, d , can be estimated by the first-passage time for 1-D diffusion, t = d 2 /2 D ( Berg, 1993 ; Howard, 2001 ).…”