Phosphorylation-dependent autoinhibition of myosin light chain phosphatase accounts for Ca2+ sensitization force of smooth muscle contraction.
The reversible regulation of myosin light chain phosphatase (MLCP) in response to agonist stimulation and cAMP/cGMP signals plays an important role in the regulation of smooth muscle (SM) tone. Here, we investigated the mechanism underlying the inhibition of MLCP induced by the phosphorylation of myosin phosphatase targeting subunit (MYPT1), a regulatory subunit of MLCP, at Thr-696 and Thr-853 using glutathione S-transferase (GST)-MYPT1 fragments having the inhibitory phosphorylation sites. GST-MYPT1 fragments, including only Thr-696 and only Thr-853, inhibited purified MLCP (IC(50) = 1.6 and 60 nm, respectively) when they were phosphorylated with RhoA-dependent kinase (ROCK). The activities of isolated catalytic subunits of type 1 and type 2A phosphatases (PP1 and PP2A) were insensitive to either fragment. Phospho-GST-MYPT1 fragments docked directly at the active site of MLCP, and this was blocked by a PP1/PP2A inhibitor microcystin (MC)-LR or by mutation of the active sites in PP1. GST-MYPT1 fragments induced a contraction of beta-escin-permeabilized ileum SM at constant pCa 6.3 (EC(50) = 2 microm), which was eliminated by Ala substitution of the fragment at Thr-696 or by ROCK inhibitors or 8Br-cGMP. GST-MYPT1-(697-880) was 5-times less potent than fragments including Thr-696. Relaxation induced by 8Br-cGMP was not affected by Ala substitution at Ser-695, a known phosphorylation site for protein kinase A/G. Thus, GST-MYPT1 fragments are phosphorylated by ROCK in permeabilized SM and mimic agonist-induced inhibition and cGMP-induced activation of MLCP. We propose a model in which MYPT1 phosphorylation at Thr-696 and Thr-853 causes an autoinhibition of MLCP that accounts for Ca(2+) sensitization of smooth muscle force.
Khromov
Alexander
A
Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.
Choudhury
Nandini
N
Stevenson
Andra S
AS
Somlyo
Avril V
AV
Eto
Masumi
M
D000409:Alanine / Q000737:chemistry; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D020134:Catalytic Domain; D004928:Escin / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D009119:Muscle Contraction; D009130:Muscle, Smooth / Q000378:metabolism*; D009154:Mutation; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D013912:Threonine / Q000737:chemistry
D013912:Threonine; D004928:Escin; D005982:Glutathione Transferase; D043263:Myosin-Light-Chain Phosphatase; D000409:Alanine; D002118:Calcium