Ca2+-independent phosphorylation of myosin in rat caudal artery and chicken gizzard myofilaments

Smooth muscle contraction is activated primarily by the Ca 2+ -calmodulin (CaM)-dependent phosphorylation of the 20 kDa light chains (LC 20 ) of myosin. Activation can also occur in some instances without a change in intracellular free [Ca 2+ ] or indeed in a Ca 2+ -independent manner. These signalling pathways often involve inhibition of myosin light chain phosphatase and unmasking of basal kinase activity leading to LC 20 phosphorylation and contraction. We have used demembranated rat caudal arterial smooth muscle strips and isolated chicken gizzard myofilaments in conjunction with the phosphatase inhibitor microcystin-LR to investigate the mechanism of Ca 2+ -independent phosphorylation of LC 20 and contraction. Treatment of Triton X-100-demembranated rat caudal arterial smooth muscle strips with microcystin at pCa 9 triggered a concentration-dependent contraction that was slower than that induced by pCa 4.5 or 6 but reached comparable steady-state levels of tension. This Ca 2+ -independent, microcystin-induced contraction correlated with phosphorylation of LC 20 at serine-19 and threonine-18. Whereas Ca 2+ -dependent LC 20 phosphorylation and contraction were inhibited by a synthetic peptide (AV25) based on the autoinhibitory domain of myosin light chain kinase (MLCK), Ca 2+ -independent, microcystin-induced LC 20 phosphorylation and contraction were resistant to AV25. Ca 2+ -independent LC 20 kinase activity was also detected in chicken gizzard smooth muscle myofilaments and catalysed phosphorylation of endogenous myosin LC 20 at serine-19 and/or threonine-18. This is in contrast to MLCK which phosphorylates threonine-18 only after prior phosphorylation of serine-19. Gizzard Ca 2+ -independent LC 20 kinase could be separated from MLCK by differential extraction from myofilaments and by CaM affinity chromatography. Its activity was resistant to AV25. We conclude that inhibition of smooth muscle myosin light chain phosphatase (MLCP) unmasks the activity of a Ca 2+ -independent LC 20 kinase associated with the myofilaments and distinct from MLCK. This kinase, therefore, probably plays a role in Ca 2+ sensitization and Ca 2+ -independent contraction of smooth muscle in response to stimuli that act via Ca 2+ -independent pathways, leading to inhibition of MLCP.