CGMP‐dependent protein kinase I (PKGI) undergoes post‐translational modifications in the Golgi apparatus (GA)

Abstract

PKGI mediates how nitric oxide and cGMP regulate vascular sooth muscle cell phenotype and pulmonary artery structure through incompletely understood mechanisms. Recently we determined that trans‐Golgi network (TGN)‐resident proprotein convertases, such as furin, stimulate PKGI proteolysis and regulate the nuclear localization of its kinase domain, PKGIγ. The current project tested whether PKGI undergoes proteolysis and glycosylation in the GA.Using specific antibodies and confocal microscopy, we observed that PKGI is colocalized with furin and TGN38, TGN‐resident proteins, in the peri‐nuclear compartment of BHK cells. Moreover, using this method and also immunoblotting, we noted that IP3 receptor‐associated cGMP kinase substrate (IRAG), overexpressed in RFL‐6 cells tethers PKGIβ to the endoplasmic reticulum and decreases PKGI proteolysis and PKGIγ nuclear translocation. Also, immunoblotting showed that brefeldin A and monensin, which decrease pre‐and intra‐GA protein transport respectively, inhibit PKGI proteolysis. Lastly, the resistance to endoglycosidase H and maintenance of PNGase F digestion of PKGIγ immunopurified from BHK cells indicated that PKGIγ is N‐glycosylated in the GA.These data demonstrate for the first time that the Golgi apparatus plays an important role in the regulation of PKGI post‐translational modifications.These studies were supported by NIH grant R01HL096779.