Elsevier

Cellular Signalling

Volume 21, Issue 6, June 2009, Pages 859-866
Cellular Signalling

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

https://doi.org/10.1016/j.cellsig.2009.01.014Get rights and content

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.

Introduction

Cyclic GMP-dependent protein kinase (PKG) is a widely expressed serine/threonine protein kinase and is an important mediator of nitric oxide (NO) and hormone signaling in smooth muscle cells (SMC), neurons, and platelets [1], [2], [3], [4], [5], [6]. PKG is expressed as two gene products in eukaryotic cells: PKG-I and PKG-II. PKG-I, in turn, is expressed as two alternatively spliced variants wherein the first or second exon is spliced into the initial coding sequences to yield PKG-Iα and PKG-Iβ, respectively [7], [8], [9]. The two isoforms of PKG-I (Mr = 80 kDa) are identical except for the N-terminal ∼ 100 amino acids, which comprise the autoinhibitory, autophosphorylation and dimerization subdomains.

In SMC, only PKG-I is expressed, and both α and β isoforms of PKG-I are believed to mediate relaxation through a variety of mechanisms. These include phosphorylation of proteins that regulate intracellular calcium homeostasis or calcium sensitivity [10], [11], [12], and stimulation of the expression of several SMC-specific gene products, as demonstrated in cell culture [13], [14], [15]. PKG-I null mice predictably exhibit disruptions in smooth muscle relaxation and are hypertensive, and the viable null animals die within 5 weeks due to gastrointestinal obstruction [16]. Conditional smooth muscle-specific PKG-I null animals have also been described, but these animals do not demonstrate abnormal cardiovascular or gastrointestinal phenotypes [17], [18].

The level of PKG-I protein expression in vascular SMC is variable [15], [19], [20], [21], [22], [23]. For example, rat aortic SMC express high levels of PKG-I in primary and early-passaged cultures, but with continuing passage the levels of PKG-I decline. Furthermore, PKG-I mRNA levels in SMC and other tissues are highly variable, often do not correspond with protein expression, and are difficult to detect using standard northern blot procedures [24], [25]. Recently, we reported that inflammatory cytokines such as IL-1β and TNF-α inhibit PKG-I mRNA and protein expression in primary cultures of bovine aortic SMC using real time PCR and western blotting, respectively [25]. The reduction in expression of PKG-I in vascular SMC under these inflammatory conditions may contribute to phenotypic modulation of the SMC from differentiated, contractile cells to more proliferative, secretory cells that are characteristic of inflammatory vascular lesions including those seen in atherosclerosis [13], [14], [26]. In other studies, NO-cGMP signaling has been shown to inhibit vascular SMC proliferation and/or stimulate vascular SMC apoptosis [19], [26], [27], [28], [29], [30]. Obviously, a better understanding of the mechanisms regulating PKG-I expression in SMC would be of great importance in defining the role of the NO-cGMP signaling pathway in inflammatory vascular disorders.

One of the principle actions of inflammatory cytokines (e.g., IL-1β and TNF-α) on cells is the induction of Type II NO synthase (iNOS) [30], [31], [32], [33], [34], [35]. Cytokine-induced iNOS expression leads to activation of NO-sensitive guanylyl cyclase, commonly known as soluble guanylyl cyclase (sGC), marked elevations in cellular cGMP in SMC [31], [32], [33], [34], [35], [36], and as mentioned above, down-regulation of PKG-I expression [25]. Studies in our laboratory demonstrated that cGMP analogs decreased PKG-I both mRNA and protein levels [24]. There have been only a few studies directed towards understanding the molecular mechanisms regulating PKG-I expression in cells, including vascular SMC. Our laboratory has examined the proximal human PKG-I promoter and has determined that the principle transcription factors that regulate PKG-I mRNA expression are members of the Sp family and the Upstream Stimulatory Factor (USF) family [37], [38]. Likewise, Pilz's laboratory has suggested that Kruppel-like transcription factor-4 (KLF-4) interacts with the Sp1/Sp3 sites on the PKG-I promoter and appears to be necessary for gene transcription [23]. More recent studies have reported that a reciprocal relationship may exist between the expression of sGC α and β subunits and the expression of PKG-I protein in various SMC culture systems [39].

One of the major mechanisms regulating protein turnover in eukaryotic cells is the ubiquitin–26S proteasome system. With regard to cellular protein kinases, it has been shown that the binding of phorbol myristate acetate (PMA) to protein kinase C (PKC) leads to activation of the enzyme in the short term and down-regulation of PKC expression upon longer exposures due to ubiquitination of the enzyme [40], [41], [42]. Because the ligand for PKG (i.e., cGMP or its analogs) down-regulates PKG-I expression, we hypothesize that cGMP leads to down-regulation of PKG-I via the ubiquitin-proteasome pathway. The findings presented here may have major implications not only for the regulation of the NO-cGMP signaling pathway in cells, but also for strategies to use PDE-5 inhibitors and other drugs that affect the pathway.

Section snippets

Materials

Cell culture sera (fetal bovine and calf) and Dulbecco's Modified Minimal Essential Medium (DMEM) were purchased from Invitrogen-Gibco (Grand Island, NY). 8-para-cholorophenylthio-cGMP (8-pCPT-cGMP) and 8-bromo-cyclic GMP (8-Br-cGMP) were purchased from Biolog (Bremen, Germany), and DETA-NONOate was obtained from Alexis Biochemicals (San Diego, CA). Polyclonal antibody to PKG-I was acquired from StressGen Biotechnology, Inc. (Victoria, British Columbia, Canada), monoclonal antibodies for

Effects of cGMP analogs on PKG-I protein expression in VSMC

As previously reported by this laboratory, NO donors and inflammatory cytokines that elevate cellular cGMP suppressed PKG-I mRNA expression and inhibited PKG-I promoter activity in vascular SMC [24], [25]. To determine whether or not cGMP itself regulated PKG-I expression, mouse aortic SMC were incubated with 8-Br-cGMP (1 mM) for various times. Protein was extracted and PKG-I levels were analyzed by western blotting. As shown in Fig. 1, 8-Br-cGMP decreased PKG-I levels at the earliest time

Discussion

Activation of PKG-I in SMC mediates NO-dependent and hormone-induced relaxation, and more recent studies have shown that PKG-I is an important regulator of vascular SMC gene expression [13], [14], [15]. Restoration of PKG-I expression to PKG-deficient vascular SMC or overexpression of PKG-I in vascular SMC increases the expression of various SMC-specific marker proteins such as smooth muscle myosin heavy chain and calponin, and decreases the expression of extracellular matrix proteins.

Acknowledgements

This work was supported by NIH grants HL66164 (T.M.L.) and DK40029 (J.D.C.).

References (60)

  • S.M. Lohmann et al.

    Trends Biochem. Sci.

    (1997)
  • T.M. Lincoln et al.

    J. Biol. Chem.

    (1988)
  • S. Orstavik et al.

    Genomics

    (1997)
  • R.B. Rosenberg et al.

    Biochem. Med. Metab. Biol.

    (1994)
  • H. Sellak et al.

    J. Biol. Chem.

    (2005)
  • N.C. Browner et al.

    J. Biol. Chem.

    (2004)
  • H.W. Lee et al.

    J. Biol. Chem.

    (1996)
  • J.L. Busch et al.

    J. Biol. Chem.

    (2002)
  • W.L. Kuo et al.

    J. Biol. Chem.

    (2004)
  • J.A. Smith et al.

    J. Biol. Chem.

    (1996)
  • T.M. Lincoln et al.

    J. Biol. Chem.

    (1978)
  • H. Liu et al.

    J. Biol. Chem.

    (1997)
  • T.M. Lincoln et al.

    FASEB J.

    (1993)
  • X. Wang et al.

    J. Neurochem.

    (1997)
  • T.M. Lincoln et al.

    J. Appl. Physiol.

    (2001)
  • F. Hofmann et al.

    J. Cell Sci.

    (2000)
  • F. Hofmann et al.

    Physiol. Rev.

    (2006)
  • S.H. Francis et al.

    Second Messengers Phosphoprot.

    (1988)
  • M. Sausbier et al.

    Circ. Res.

    (2000)
  • J. Schlossmann et al.

    Nature

    (2000)
  • H.K. Surks et al.

    Science

    (1999)
  • N.J. Boerth et al.

    J. Vasc. Res.

    (1997)
  • N.B. Dey et al.

    Circ. Res.

    (1998)
  • T. Zhang, S. Zhuang, D.E. Casteel, D.J. Looney, G.R. Boss, and R.B. Pilz, J. Biol. Chem. 282 (2007)...
  • A. Pfeifer et al.

    EMBO J.

    (1998)
  • W. Wolfsgruber et al.

    Proc. Natl. Acad. Sci. U. S. A.

    (2003)
  • R. Feil et al.

    Circ. Res.

    (2003)
  • J.D. Chiche et al.

    J. Biol. Chem.

    (1998)
  • T.L. Cornwell et al.

    Am. J. Physiol.

    (1994)
  • T.A. Wyatt et al.

    Am. J. Physiol.

    (1998)
  • Cited by (19)

    View all citing articles on Scopus
    1

    Permanent address: Department of Biology, Wheaton College, Wheaton, IL 60187, USA.

    View full text