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

Abstract

Smooth muscle contraction is activated primarily by the Ca2+‐calmodulin (CaM)‐dependent phosphorylation of the 20 kDa light chains (LC20) of myosin. Activation can also occur in some instances without a change in intracellular free [Ca2+] or indeed in a Ca2+‐independent manner. These signalling pathways often involve inhibition of myosin light chain phosphatase and unmasking of basal kinase activity leading to LC20 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 Ca2+‐independent phosphorylation of LC20 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 Ca2+‐independent, microcystin‐induced contraction correlated with phosphorylation of LC20 at serine‐19 and threonine‐18. Whereas Ca2+‐dependent LC20 phosphorylation and contraction were inhibited by a synthetic peptide (AV25) based on the autoinhibitory domain of myosin light chain kinase (MLCK), Ca2+‐independent, microcystin‐induced LC20 phosphorylation and contraction were resistant to AV25. Ca2+‐independent LC20 kinase activity was also detected in chicken gizzard smooth muscle myofilaments and catalysed phosphorylation of endogenous myosin LC20 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 Ca2+‐independent LC20 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 Ca2+‐independent LC20 kinase associated with the myofilaments and distinct from MLCK. This kinase, therefore, probably plays a role in Ca2+ sensitization and Ca2+‐independent contraction of smooth muscle in response to stimuli that act via Ca2+‐independent pathways, leading to inhibition of MLCP.

Distinct kinases are involved in contraction of cat esophageal and lower esophageal sphincter smooth muscles

“…To ensure the specificity of MLCK inhibition, we used SM-1, a synthetic peptide corresponding to the autoinhibitory sequence of MLCK (residues 783-804 of chicken gizzard MLCK) (65), which is the most selective inhibitor of Ca 2ϩ / CaM-dependent MLCK (21,30,38). As observed with the other phosphatase inhibitors, contraction induced by anti-PP1c was inhibited by SM-1 in LES but not in ESO smooth muscle cells (Figs.…”

“…Nevertheless, several kinases are capable of phosphorylating intact myosin at Ser 19 of MLC: CaM-dependent PKII (16), MAP kinase-activated protein kinase-2 (39), ROK (2), ILK (13), and ZIP kinase (49). Of these candidates, ILK has been shown to associate with the contractile machinery of smooth muscle and therefore is likely to be a kinase responsible for Ca 2ϩ -independent myosin phosphorylation and contraction of rat tail arterial smooth muscle in response to the phosphatase inhibitor microcystin-LR (13,65). However, we cannot rule out the involvement of other candidate kinases.…”

“…ILK has been implicated in Ca 2ϩ -independent contraction of vascular smooth muscle elicited by phosphatase inhibition (14,65). We therefore examined the possibility that activation of ILK may play a role in phosphatase inhibitor/ PKC⑀-mediated contraction of ESO smooth muscle.…”

Role of alpha adrenergic receptors in erectile function

“…In addition to these primary mechanisms other mechanisms are proposed in which phosphorylation of MLC 20 takes place in the absence of increased intracellular Ca 2 -calmodulin. 62 The altered balance between the activities of Ca 2 -independent kinase and inhibition of phosphatase increases MLC 20 phosphorylation causing contraction in the absence of increased intracellular Ca 2 ( Figure 4). Other mechanisms of smooth muscle contraction have implicated G protein activating Ca 2 -sensitizing cascade via monomeric GTpase RhoA.…”

Differential signalling by muscarinic receptors in smooth muscle: m2-mediated inactivation of myosin light chain kinase via Gi3, Cdc42/Rac1 and p21-activated kinase 1 pathway, and m3-mediated MLC20 (20 kDa regulatory light chain of myosin II) phosphorylation via Rho-associated kinase/myosin phosphatase targeting subunit 1 and protein kinase C/CPI-17 pathway

“…However, the agonist-induced Ca 2+ transient is dissipated rapidly, and MLCK activity decreases to resting levels, whereas MLC 20 phosphorylation and contraction are maintained [3][4][5]. The G-protein-dependent pathway responsible for sustained MLC 20 phosphorylation and contraction involves inhibition of MLC phosphatase (MLCP, also known as smooth muscle myosin phosphatase 1) and activation of one or more Ca 2+ -independent MLC kinase(s) [6][7][8][9][10][11][12][13][14][15][16][17]. Studies of Ca 2+ sensitization, demonstrating G-protein-dependent augmentation Abbreviations used: ACh, acetylcholine; Cdc42, cell division cycle 42 kinase; PKC, protein kinase C; CPI-17, PKC-activated 17 kDa inhibitor protein of type 1 phosphatase; 4-DAMP, 4-diphenylacetoxy-N-methylpiperidine; DN, dominant negative; DTT, dithiothreitol; MLC 20 , 20 kDa regulatory light chain of myosin II; MLCK, myosin light chain kinase; MLCP, MLC phosphatase; MYPT1, myosin phosphatase targeting subunit; PAK1, p21-activated kinase 1; PEt, phosphatidylethanol; PI, phosphoinositide; PLC-β1, phospholipase C-β1; PLD, phospholipase D; PTx, pertussis toxin; Rho kinase, Rho-associated kinase; TCA, trichloroacetic acid.…”