Cyclic GMP specifically suppresses Type-Ialpha cGMP-dependent protein kinase expression by ubiquitination.
Type I cGMP-dependent protein kinase (PKG-I) mediates nitric oxide (NO) and hormone dependent smooth muscle relaxation and stimulates smooth muscle cell-specific gene expression. Expression of PKG-I in cultured smooth muscle cells depends on culture conditions and is inhibited by inflammatory cytokines such as interleukin-I and tumor necrosis factor-alpha, which are known to stimulate Type II NO synthase (iNOS) expression. We report here that the suppression of PKG-I protein levels in smooth muscle cells is triggered by the ubiquitin/26S proteasome pathway. Incubation of vascular smooth muscle cells with phosphodiesterase-resistant cyclic GMP analogs (e.g., 8-bromo-cGMP) decreases PKG-I protein level in a time- and concentration-dependent manner. To study this process, we tested the effects of 8-Br-cGMP on PKG-I protein level in Cos7 cells, which do not express endogenous type I PKG mRNA. 8-Br-cGMP induced the ubiquitination and down-regulation of PKG-Ialpha, but not PKG-Ibeta. Treatment of cells with the 26S proteasome inhibitor, MG-132, increased ubiquitination of PKG. Blocking PKG-I catalytic activity using the cell-permeant specific PKG-I inhibitor, DT-2, inhibited cGMP-induced PKG-I ubiquitination and down-regulation, suggesting that PKG catalytic activity and autophosphorylation were required for suppression of PKG-I level. Mutation of the known autophosphorylation sites of PKG-Ialpha to alanine uncovered a specific role for autophosphorylation of serine-64 in cGMP-dependent ubiquitination and suppression of PKG-I level. The results suggest that chronic elevation of cGMP, as seen in inflammatory conditions, triggers ubiquitination and degradation of PKG-Ialpha in smooth muscle.
Dey
Nupur B
NB
The Department of Physiology, College of Medicine, University of South Alabama, Mobile, AL 36688, USA.
Busch
Jennifer L
JL
Francis
Sharron H
SH
Corbin
Jackie D
JD
Lincoln
Thomas M
TM
D000818:Animals; D019556:COS Cells; D002417:Cattle; D002522:Chlorocebus aethiops; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology*; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015536:Down-Regulation / Q000187:drug effects; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000201:enzymology; D009569:Nitric Oxide / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection; D054875:Ubiquitination / Q000187:drug effects*
D007527:Isoenzymes; D010726:Phosphodiesterase Inhibitors; D010768:Phosphoserine; C016276:8-bromocyclic GMP; D009569:Nitric Oxide; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases; C569718:PRKG1 protein, human; C569720:Prkg1 protein, mouse; D006152:Cyclic GMP