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

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 extranuclear anchoring proteins. The inositol 1,4,5-triphosphate OPA receptor-associated cGMP kinase substrate (IRAG) binds to the N-terminus of PKG I beta, but not PKG lot, and in smooth muscle cells, IRAG and PKG I alpha are in a complex with the IP3 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 I beta in baby hamster kidney cells prevented cGMP-induced PKG I alpha translocation to the nucleus, and decreased cGMP/PKG I beta transactivation of a cAMP-response element-dependent reporter gene. These effects required the PKG I beta/IRAG association, as demonstrated by a binding-incompetent IRAG mutant, and were specific for PKG I beta, as nuclear translocation and reporter gene activation by PKG I alpha was not affected by IRAG. A phosphorylation-deficient IRAG mutant that is no longer functionally regulated by PKG phosphorylation suppressed cGMP/PKG I beta 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. (c) 2008 Elsevier Inc. All rights reserved.