Kinetics of Myosin Light Chain Kinase Activation of Smooth Muscle Myosin in an in Vitro Model System

详细信息    查看全文

• 作者：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. Baker
• 刊名：Biochemistry
• 出版年：2013
• 出版时间：November 26, 2013
• 年：2013
• 卷：52
• 期：47
• 页码：8489-8500
• 全文大小：497K
• 年卷期：v.52,no.47(November 26, 2013)
• ISSN：1520-4995

文摘

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鈥揝MM interactions. To determine the rate-limiting step underlying activation of SMM by MLCK, we measured the kinetics of calcium-calmodulin (Ca²⁺CaM)鈥揗LCK-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, k_p^o, of 1.17 heads s^鈥? MLCK^鈥?. 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^鈥?, which was similar to the k_p^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 K_d 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鈥?6 s^鈥?) would be much faster than with pSMM (<0.1鈥?.2 s^鈥?). This suggests that the probability of MLCK interacting with unphosphorylated versus phosphorylated SMM is 55鈥?60 times greater. This would avoid sequestering MLCK to unproductive interactions with previously phosphorylated SMM, potentially leading to faster rates of phosphorylation in muscle.