The Golgi apparatus regulates cGMP‐dependent protein kinase I (PKGI) proteolytic cleavage in vascular smooth muscle cells (SMC)

Abstract

cGMP regulates vascular tone and structure through stimulating PKGI. Although encoded by a single gene product, two PKGI isoforms, PKGIα and PKGIβ, form through alternate mRNA splicing. They differ in their NH2‐terminal dimerization domains. Proteolysis of the PKGI isoforms and the nuclear localization of their conserved COOH‐terminal kinase fragment (PKGIγ) play a critical role in regulating SMC gene expression and differentiation. Although PKGI has been detected in the perinuclear region of SMC, and proprotein convertases (PCs) that are resident in the Golgi apparatus (GA) have been found to stimulate PKGI proteolysis, the role of the GA in PKGI processing is not understood. Using pulmonary artery SMC and rat fetal lung fibroblast (RFL‐6) cells, which express endogenous PKGI, we determined that PKGI immunoreactivity co‐localizes with index proteins residing in the endoplasmic reticulum (ER), ER‐Golgi intermediate complex, GA cisterna, and the trans‐Golgi network, including furin a PKGI‐cleaving PC. Moreover, over‐expression of Sar1 [T39N], a GDP‐bound trans‐dominant Sar1 mutant that inhibits ER exit site budding, was found to inhibit PKGI localization to GA cisterna. Also, IP3R‐associated cGMP kinase substrate (IRAG) was found not only to tether overexpressed PKGIβ to the ER and decrease its GA localization, but also to decrease PKGI proteolysis and PKGIγ nuclear translocation. Monensin, which inhibits intra‐GA protein transport, was also found to decrease PKGI cleavage. Together these studies detail for the first time a role for ER‐GA transport in PKGI post‐translational modifications that regulate nuclear signaling. NIH R01HL096779 supported this work.