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CGMP-dependent protein kinase anchoring by IRAG regulates its nuclear translocation and transcriptional activity.

ABSTRACT: Type I cGMP-dependent protein kinases (PKGs) translocate to the nucleus to regulate gene expression in some, but not all cell types; we hypothesized that nuclear translocation of PKG may be regulated by extra-nuclear anchoring proteins. The inositol 1,4,5-triphosphate (IP(3)) receptor-associated cGMP kinase substrate (IRAG) binds to the N-terminus of PKG Ibeta, but not PKG Ialpha, and in smooth muscle cells, IRAG and PKG Ibeta are in a complex with the IP(3) receptor at endoplasmatic reticulum membranes, where the complex regulates calcium release [Schlossmann et al., Nature, 404 (2000) 197]. We found that co-expression of IRAG and PKG Ibeta in baby hamster kidney cells prevented cGMP-induced PKG Ibeta translocation to the nucleus, and decreased cGMP/PKG Ibeta transactivation of a cAMP-response element-dependent reporter gene. These effects required the PKG Ibeta/IRAG association, as demonstrated by a binding-incompetent IRAG mutant, and were specific for PKG Ibeta, as nuclear translocation and reporter gene activation by PKG Ialpha was not affected by IRAG. A phosphorylation-deficient IRAG mutant that is no longer functionally regulated by PKG phosphorylation suppressed cGMP/PKG Ibeta transcriptional activity, indicating that IRAG's effect was not explained by changes in intracellular calcium, and was not related to competition of IRAG with other PKG substrates. These results demonstrate that PKG anchoring to a specific binding protein is sufficient to dictate subcellular localization of the kinase and affect cGMP signaling in the nucleus, and may explain why nuclear translocation of PKG I does not occur in all cell types.

SUBMITTER: Casteel DE

PROVIDER: S-EPMC2477739 | biostudies-literature | 2008 Jul

REPOSITORIES: biostudies-literature

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cGMP-dependent protein kinase anchoring by IRAG regulates its nuclear translocation and transcriptional activity.

Casteel Darren E DE Zhang Tong T Zhuang Shunhui S Pilz Renate B RB

Cellular signalling 20080326 7

Type I cGMP-dependent protein kinases (PKGs) translocate to the nucleus to regulate gene expression in some, but not all cell types; we hypothesized that nuclear translocation of PKG may be regulated by extra-nuclear anchoring proteins. The inositol 1,4,5-triphosphate (IP(3)) receptor-associated cGMP kinase substrate (IRAG) binds to the N-terminus of PKG Ibeta, but not PKG Ialpha, and in smooth muscle cells, IRAG and PKG Ibeta are in a complex with the IP(3) receptor at endoplasmatic reticulum m ...[more]

PMID: 18450420

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Project description:A cultured porcine pulmonary artery (PA) model was used to examine the effects of prolonged nitric oxide (NO) treatment on the response to acutely applied NO, cGMP analog, or atrial natriuretic peptide (ANP). Twenty-four-hour treatment with the NO donor (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO) resulted in >10-fold decrease in the response to acutely applied DETA-NO. In parallel with this, the relaxant response to acutely applied cGMP analog, beta-phenyl-1,N(2)-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate, Sp isomer (Sp-8-Br-PET-cGMPS), and ANP decreased. The reduction in ANP responsiveness in PA was not associated with a reduction in cGMP levels evoked by 10(-6) M ANP. Twenty-four hours in culture and treatment with DETA-NO decreased total cGMP-dependent protein kinase (cGKI) mRNA level compared with that in freshly prepared PA (1.05 +/- 0.12, 0.42 +/- 0.08, and 0.11 +/- 0.01 amol/mug, respectively). Total cGKI protein levels were decreased to a lesser extent by 24 h in culture and further decreased by 24-h DETA-NO treatment compared with that in freshly prepared PA (361 +/- 33, 272 +/- 20, and 238 +/- 25 ng/mg total protein, respectively). Maximal cGMP-stimulated phosphotransferase activity was reduced in 24-h cultured and DETA-NO-treated PA (986 +/- 84, 815 +/- 81, and 549 +/- 78 pmol P(i).min(-1).mg soluble protein(-1)), but the cGMP concentration resulting in 50% of maximal phosphotransferase activity was not. cGKI specific activity (maximal cGMP-activated phosphotransferase activity/ng cGKI) was significantly reduced in PA treated with DETA-NO for 24 h compared with freshly prepared and 24-h cultured PA (1.95 +/- 0.22, 2.64 +/- 0.25, and 2.85 +/- 0.28 pmol P(i).min(-1).ng cGKI(-1), respectively). We conclude that prolonged NO treatment induces decreased acute NO responsiveness in PA in part by decreasing cGMP sensitivity. It does so by decreasing both cGKI expression and cGKI specific activity.

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CGMP-dependent protein kinase anchoring by IRAG regulates its nuclear translocation and transcriptional activity.

Publications

cGMP-dependent protein kinase anchoring by IRAG regulates its nuclear translocation and transcriptional activity.

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