Cyclic GMP specifically suppresses Type-Iα cGMP-dependent protein kinase expression by ubiquitination

• Published in: Cellular signalling 2009-06, Vol.21 (6), p.859-866
• Main Authors: Dey, Nupur B., Busch, Jennifer L., Francis, Sharron H., Corbin, Jackie D., Lincoln, Thomas M.
• Format: Article
• Language: English
• Subjects: cGMP; Gene expression; Nitric oxide; Phenotype; Phosphorylation; PKG; Smooth muscle; Ubiquitin
• ISSN: 0898-6568; 1873-3913
• DOI: 10.1016/j.cellsig.2009.01.014

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-α, 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-Iα, but not PKG-Iβ. 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-Iα 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-Iα in smooth muscle.