Myosin Regulatory Light Chain Diphosphorylation Slows Relaxation of Arterial Smooth Muscle

• Published in: The Journal of biological chemistry 2012-07, Vol.287 (29), p.24064-24076
• Main Authors: Sutherland, Cindy, Walsh, Michael P.
• Format: Article
• Language: English
• Subjects: Animals; Arteries - metabolism; Arteries - physiology; Calcium; In Vitro Techniques; Male; Muscle Relaxation - physiology; Muscle, Smooth, Vascular - metabolism; Muscle, Smooth, Vascular - physiology; Myosin; Myosin Light Chains - metabolism; Phosphorylation - physiology; Protein Kinases; Protein Phosphorylation; Rats; Rats, Sprague-Dawley; Signal Transduction; Smooth Muscle
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.371609

The principal signal to activate smooth muscle contraction is phosphorylation of the regulatory light chains of myosin (LC20) at Ser19 by Ca2+/calmodulin-dependent myosin light chain kinase. Inhibition of myosin light chain phosphatase leads to Ca2+-independent phosphorylation at both Ser19 and Thr18 by integrin-linked kinase and/or zipper-interacting protein kinase. The functional effects of phosphorylation at Thr18 on steady-state isometric force and relaxation rate were investigated in Triton-skinned rat caudal arterial smooth muscle strips. Sequential phosphorylation at Ser19 and Thr18 was achieved by treatment with adenosine 5′-O-(3-thiotriphosphate) in the presence of Ca2+, which induced stoichiometric thiophosphorylation at Ser19, followed by microcystin (phosphatase inhibitor) in the absence of Ca2+, which induced phosphorylation at Thr18. Phosphorylation at Thr18 had no effect on steady-state force induced by Ser19 thiophosphorylation. However, phosphorylation of Ser19 or both Ser19 and Thr18 to comparable stoichiometries (0.5 mol of Pi/mol of LC20) and similar levels of isometric force revealed differences in the rates of dephosphorylation and relaxation following removal of the stimulus: t½ values for dephosphorylation were 83.3 and 560 s, and for relaxation were 560 and 1293 s, for monophosphorylated (Ser19) and diphosphorylated LC20, respectively. We conclude that phosphorylation at Thr18 decreases the rates of LC20 dephosphorylation and smooth muscle relaxation compared with LC20 phosphorylated exclusively at Ser19. These effects of LC20 diphosphorylation, combined with increased Ser19 phosphorylation (Ca2+-independent), may underlie the hypercontractility that is observed in response to certain physiological contractile stimuli, and under pathological conditions such as cerebral and coronary arterial vasospasm, intimal hyperplasia, and hypertension. Background: The regulatory light chains of smooth muscle myosin are phosphorylated at Ser19 and Thr18. Results: Phosphorylation at Thr18 does not increase force elicited by Ser19 phosphorylation, but reduces the rate of relaxation. Conclusion: Diphosphorylation slows relaxation compared with monophosphorylation at Ser19. Significance: Knowledge of the functional effects of myosin diphosphorylation is important for understanding the underlying causes of hypercontractility.