Myosin Regulatory Light Chain Diphosphorylation Slows Relaxation of Arterial Smooth Muscle

The principal signal to activate smooth muscle contraction is phosphorylation of the regulatory light chains of myosin (LC 20 ) at Ser 19 by Ca 2+ /calmodulin-dependent myosin light chain kinase. Inhibition of myosin light chain phosphatase leads to Ca 2+ -independent phosphorylation at both Ser 19 and Thr 18 by integrin-linked kinase and/or zipper-interacting protein kinase. The functional effects of phosphorylation at Thr 18 on steady-state isometric force and relaxation rate were investigated in Triton-skinned rat caudal arterial smooth muscle strips. Sequential phosphorylation at Ser 19 and Thr 18 was achieved by treatment with adenosine 5′- O -(3-thiotriphosphate) in the presence of Ca 2+ , which induced stoichiometric thiophosphorylation at Ser 19 , followed by microcystin (phosphatase inhibitor) in the absence of Ca 2+ , which induced phosphorylation at Thr 18 . Phosphorylation at Thr 18 had no effect on steady-state force induced by Ser 19 thiophosphorylation. However, phosphorylation of Ser 19 or both Ser 19 and Thr 18 to comparable stoichiometries (0.5 mol of P i /mol of LC 20 ) and similar levels of isometric force revealed differences in the rates of dephosphorylation and relaxation following removal of the stimulus: t ½ values for dephosphorylation were 83.3 and 560 s, and for relaxation were 560 and 1293 s, for monophosphorylated (Ser 19 ) and diphosphorylated LC 20 , respectively. We conclude that phosphorylation at Thr 18 decreases the rates of LC 20 dephosphorylation and smooth muscle relaxation compared with LC 20 phosphorylated exclusively at Ser 19 . These effects of LC 20 diphosphorylation, combined with increased Ser 19 phosphorylation (Ca 2+ -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 Ser 19 and Thr 18 . Results: Phosphorylation at Thr 18 does not increase force elicited by Ser 19 phosphorylation, but reduces the rate of relaxation. Conclusion: Diphosphorylation slows relaxation compared with monophosphorylation at Ser 19 . Significance: Knowledge of the functional effects of myosin diphosphorylation is important for understanding the underlying causes of hypercontractility.