Abstract-Cyclic GMP modulates gene expression in vascular smooth muscle cells (SMCs) in part by stimulating cGMP-dependent protein kinase I (PKGI) and the phosphorylation of transcription factors. In some cells, cGMP increases nuclear translocation of PKGI and PKGI-dependent phosphorylation of transcription regulators; however, these observations have been variable, and the mechanisms mediating nuclear PKGI translocation are incompletely understood. We tested the hypothesis that proteolytic cleavage of PKGI is required for cGMP-stimulated nuclear compartmentation of PKGI and phosphorylation of transcription factors. We detected an NH 2 -terminal PKGI fragment with leucine zipper domain immunoreactivity in the cytosol and endoplasmic reticulum of SMCs, but only a COOH-terminal PKGI fragment containing the catalytic region (now termed PKGI␥) was observed in the Golgi apparatus (GA) and nucleoplasm. Posttranslational PKGI processing in the GA was critical for nuclear compartmentation of PKGI␥ because GA disruption with nocodazol or brefeldin A inhibited PKGI␥ nuclear localization. PKGI␥ immunoreactivity was particularly abundant in the nucleolus of interphase SMCs where its colocalization with the nucleolar dense fibrillar component protein fibrillarin closely matched the level of nucleolar assembly. Purified nucleolar PKGI␥ enzyme activity was insensitive to cGMP stimulation, which is consistent with its lack of the NH 2 -terminal autoinhibitory domain. Mutation of a putative proteolytic cleavage region in PKGI inhibited cGMPmediated phosphorylation of cAMP response element-binding protein, cAMP response element-dependent transcription, and nuclear localization of PKGI␥. These observations suggest that posttranslational modification of PKGI critically influences the nuclear translocation of PKGI and activities of cGMP in SMCs. Key Words: cGKI Ⅲ guanylate cyclase Ⅲ gene expression regulation Ⅲ signal transduction Ⅲ vascular disease C yclic GMP is a key regulator of vascular smooth muscle cell (SMC) cytoskeletal kinetics, proliferation, and differentiation, and abnormalities in cGMP signaling have been associated with pulmonary and peripheral vascular disease. 1 cGMP is synthesized by guanylyl cyclases, which are activated by nitric oxide, carbon monoxide, and natriuretic peptides, and is metabolized by phosphodiesterases. Through the interaction with cytoplasmic proteins, cGMP influences SMC shape and migration and decreases vascular tone. Recently, cGMP has also been observed to modulate the expression of genes that influence SMC phenotype and proliferation. 2 For example, cGMP has been noted to regulate SMC gene expression by increasing the phosphorylation of transcription factors, such as cAMP response element-binding protein (CREB) [3][4][5] and activating transcription factor-1 (ATF-1), 5 altering the expression of transcription regulators such as activator protein-1 (AP-1) 3 and the growth arrest-specific homeobox transcription factor (GAX), 6 and regulating the activity of other nuclear factors, such as se...
Objective: Pediatric pulmonary vascular disease (PVD) is associated with excessive VSMC proliferation. Because recent studies suggest that nitric oxide attenuates PVD and VSMC proliferation by stimulating PKGI activity, the mechanisms affecting PKGI's nuclear localization were investigated.Methods: PKGI was mapped to intracellular compartments using immunologic and biochemical methods and its catalytic activity was determined using a PKGI‐specific peptide substrate. The domain structure of nuclear PKGI was analyzed using antibodies and detailed using Edman‐based protein sequencing.Results: PKGI immunoreactivity was detected in the nuclei of VSMC, where its activity was cGMP‐independent. Immunoblotting revealed that the nuclear PKGI isoform (PKGIγ) is 18 kDa smaller than the cytosolic one and lacks a NH2‐terminal dimerization domain, which tethers it to cytosolic proteins. The PKGI dimerization domain was observed in the perinuclear region where it colocalized primarily with the endoplasmic reticulum. Sequencing of immunopurified endogenous nuclear PKGIγ confirmed the absence of a dimerization domain and revealed that its putative scissile amino acids are not encoded by DNA located near typical RNA splice donor‐acceptor guidance nucleotides.Conclusions: A novel constitutively active isoform of PKGI that appears to result from PKGI proteolytic processing is localized in the nuclei of VSMC.
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