A novel constitutively active isoform of cGMP‐dependent protein kinase I (PKGI) is localized in the nucleus of vascular smooth muscle cells (VSMC)

Abstract

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.