AMP-activated protein kinase phosphorylates and desensitizes smooth muscle myosin light chain kinase.
Smooth muscle contraction is initiated by a rise in intracellular calcium, leading to activation of smooth muscle myosin light chain kinase (MLCK) via calcium/calmodulin (CaM). Activated MLCK then phosphorylates the regulatory myosin light chains, triggering cross-bridge cycling and contraction. Here, we show that MLCK is a substrate of AMP-activated protein kinase (AMPK). The phosphorylation site in chicken MLCK was identified by mass spectrometry to be located in the CaM-binding domain at Ser(815). Phosphorylation by AMPK desensitized MLCK by increasing the concentration of CaM required for half-maximal activation. In primary cultures of rat aortic smooth muscle cells, vasoconstrictors activated AMPK in a calcium-dependent manner via CaM-dependent protein kinase kinase-beta, a known upstream kinase of AMPK. Indeed, vasoconstrictor-induced AMPK activation was abrogated by the STO-609 CaM-dependent protein kinase kinase-beta inhibitor. Myosin light chain phosphorylation was increased under these conditions, suggesting that contraction would be potentiated by ablation of AMPK. Indeed, in aortic rings from mice in which alpha1, the major catalytic subunit isoform in arterial smooth muscle, had been deleted, KCl- or phenylephrine-induced contraction was increased. The findings suggest that AMPK attenuates contraction by phosphorylating and inactivating MLCK. This might contribute to reduced ATP turnover in the tonic phase of smooth muscle contraction.
Horman
Sandrine
S
Hormone and Metabolic Research Unit, de Duve Institute, Université Catholique de Louvain, B-1200 Brussels.
Morel
Nicole
N
Vertommen
Didier
D
Hussain
Nusrat
N
Neumann
Dietbert
D
Beauloye
Christophe
C
El Najjar
Nicole
N
Forcet
Christelle
C
Viollet
Benoit
B
Walsh
Michael P
MP
Hue
Louis
L
Rider
Mark H
MH
D055372:AMP-Activated Protein Kinases; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001011:Aorta / Q000737:chemistry / Q000201:enzymology*; D001562:Benzimidazoles / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D002645:Chickens; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology*; D009129:Muscle Tonus / Q000187:drug effects / Q000502:physiology; D009130:Muscle, Smooth / Q000737:chemistry / Q000201:enzymology*; D032389:Myocytes, Smooth Muscle / Q000737:chemistry / Q000201:enzymology*; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053644:Naphthalimides / Q000494:pharmacology; D010656:Phenylephrine / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011189:Potassium Chloride / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D014661:Vasoconstriction / Q000187:drug effects / Q000502:physiology; D014662:Vasoconstrictor Agents / Q000494:pharmacology
D001562:Benzimidazoles; D002147:Calmodulin; D009097:Multienzyme Complexes; D053644:Naphthalimides; C458525:STO 609; D014662:Vasoconstrictor Agents; D010656:Phenylephrine; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D017346:Protein Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D009219:Myosin-Light-Chain Kinase; D055372:AMP-Activated Protein Kinases; D002118:Calcium