Uncoupling of GPCR and RhoA-induced Ca2+-sensitization of chicken amnion smooth muscle lacking CPI-17.
Ca2+-sensitization of smooth muscle occurs through inhibition of myosin light chain phosphatase (MLCP) leading to an increase in the MLCK:MLCP activity ratio. MLCP is inhibited through phosphorylation of its regulatory subunit (MYPT-1) following activation of the RhoA/Rho kinase (ROK) pathway or through phosphorylation of the PP1c inhibitory protein, CPI-17, by PKC delta or ROK. Here, we explore the crosstalk between these two modes of MLCP inhibition in a smooth muscle of a natural CPI-17 knockout, chicken amnion. GTPgammaS elicited Ca2+-sensitized force which was relaxed by GDI or Y-27632, however, U46619, carbachol and phorbol ester failed to induce Ca2+-sensitized force, but were rescued by recombinant CPI-17, and were sensitive to Y-27632 inhibition. In the presence, but not absence, of CPI-17, U46619 also significantly increased GTP.RhoA. There was no affect on MYPT-1 phosphorylation at T695, however, T850 phosphorylation increased in response to GTPgammaS stimulation. Together, these data suggest a role for CPI-17 upstream of RhoA activation possibly through activation of another PP1 family member targeted by CPI-17.
Stevenson
Andrá S
AS
Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences, 1300 Jefferson Park Avenue, Charlottesville, VA 22908, USA.
Matthew
John D
JD
Eto
Masumi
M
Luo
Shizhen
S
Somlyo
Andrew P
AP
Somlyo
Avril V
AV
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid / Q000494:pharmacology; D017607:Aluminum Compounds / Q000494:pharmacology; D000650:Amnion / Q000033:anatomy & histology*; D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002645:Chickens / Q000033:anatomy & histology / Q000502:physiology; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005459:Fluorides / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism; D066298:In Vitro Techniques; D009119:Muscle Contraction / Q000502:physiology; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D009130:Muscle, Smooth / Q000187:drug effects / Q000502:physiology*; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D054645:Protein Phosphatase 1; D021122:Protein Subunits / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D014662:Vasoconstrictor Agents / Q000494:pharmacology; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D017607:Aluminum Compounds; D001427:Bacterial Toxins; D004791:Enzyme Inhibitors; D009124:Muscle Proteins; D010750:Phosphoproteins; D021122:Protein Subunits; D043562:Receptors, G-Protein-Coupled; D014662:Vasoconstrictor Agents; C050992:tetrafluoroaluminate; C016276:8-bromocyclic GMP; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D006160:Guanosine Triphosphate; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D043263:Myosin-Light-Chain Phosphatase; D020742:rhoA GTP-Binding Protein; D006152:Cyclic GMP; D005459:Fluorides; D002118:Calcium