Phosphorylation of a single head of smooth muscle myosin activates the whole molecule.
Regulatory light chain (RLC) phosphorylation activates smooth and non-muscle myosin II, but it has not been established if phosphorylation of one head turns on the whole molecule. Baculovirus expression and affinity chromatography were used to isolate heavy meromyosin (HMM) containing one phosphorylated and one dephosphorylated RLC (1-P HMM). Motility and steady-state ATPase assays indicated that 1-P HMM is nearly as active as HMM with two phosphorylated heads (2-P HMM). Single-turnover experiments further showed that both the dephosphorylated and phosphorylated heads of 1-P HMM can be activated by actin. Singly phosphorylated full-length myosin was also an active species with two cycling heads. Our results suggest that phosphorylation of one RLC abolishes the asymmetric inhibited state formed by dephosphorylated myosin [Liu, J., et al. (2003) J. Mol. Biol. 329, 963-972], allowing activation of both the phosphorylated and dephosphorylated heads. These findings help explain how smooth muscles are able to generate high levels of stress with low phosphorylation levels.
Rovner
Arthur S
AS
Department of Molecular Physiology and Biophysics, Health Sciences Research Facility, University of Vermont, Burlington, Vermont 05405-0068, USA.
Fagnant
Patricia M
PM
Trybus
Kathleen M
KM
D000199:Actins / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002645:Chickens; D009130:Muscle, Smooth / Q000378:metabolism*; D015879:Myosin Subfragments / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D024745:Smooth Muscle Myosins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018411:Spodoptera
D000199:Actins; D015879:Myosin Subfragments; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D024745:Smooth Muscle Myosins