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Ca 2+ ‐independent phosphorylation of myosin in rat caudal artery and chicken gizzard myofilaments
Author(s) -
Weber L. P.,
Lierop J. E.,
Walsh M. P.
Publication year - 1999
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1111/j.1469-7793.1999.0805u.x
Subject(s) - myosin light chain kinase , phosphorylation , myofilament , myosin , threonine , phosphatase , biochemistry , dephosphorylation , muscle contraction , chemistry , serine , calmodulin , contraction (grammar) , biophysics , biology , endocrinology , enzyme
1 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. 2 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. 3 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. 4 This Ca 2+ ‐independent, microcystin‐induced contraction correlated with phosphorylation of LC 20 at serine‐19 and threonine‐18. 5 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. 6 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. 7 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. 8 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.