Cyclic GMP specifically suppresses Type-Iα cGMP-dependent protein kinase expression by ubiquitination |
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Authors: | Nupur B. Dey Jennifer L. Busch Sharron H. Francis Jackie D. Corbin Thomas M. Lincoln |
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Affiliation: | 1. Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical University, 1838 North Guangzhou Ave, Guangzhou, Guangdong 510515, China;2. Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research, Affiliated Hospital of Hainan Medical University, Hainan Medical University, Haikou, Hainan 570102, China;3. The Institute of Biosciences and Technology, Texas A&M University System Health Science Center, Houston, TX 77030, USA;4. Texas Heart Institute, Houston, TX 77030, USA;5. Department of Biology and Biochemistry, University of Houston, Houston, TX 77204, USA |
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Abstract: | 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. |
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