Proprotein convertases play an important role in regulating PKGI endoproteolytic cleavage and nuclear transport

Abstract

Kato S, Zhang R, Roberts, Jr. JD. Proprotein convertases play an important role in regulating PKGI endoproteolytic cleavage and nuclear transport. Am J Physiol Lung Cell Mol Physiol 305: L130 -L140, 2013. First published May 17, 2013 doi:10.1152/ajplung.00391.2012.-Nitric oxide and cGMP modulate vascular smooth muscle cell (SMC) phenotype by regulating cell differentiation and proliferation. Recent studies suggest that cGMP-dependent protein kinase I (PKGI) cleavage and the nuclear translocation of a constitutively active kinase fragment, PKGI␥, are required for nuclear cGMP signaling in SMC. However, the mechanisms that control PKGI proteolysis are unknown. Inspection of the amino acid sequence of a PKGI cleavage site that yields PKGI␥ and a protease database revealed a putative minimum consensus sequence for proprotein convertases (PCs). Therefore we investigated the role of PCs in regulating PKGI proteolysis. We observed that overexpression of PCs, furin and PC5, but not PC7, which are all expressed in SMC, increase PKGI cleavage in a dose-dependent manner in human embryonic kidney (HEK) 293 cells. Moreover, furin-induced proteolysis of mutant PKGI, in which alanines were substituted into the putative PC consensus sequence, was decreased in these cells. In addition, overexpression of furin increased PKGI proteolysis in LoVo cells, which is an adenocarcinoma cell line expressing defective furin without PC activity. Also, expression of ␣1-PDX, an engineered serpin-like PC inhibitor, reduced PC activity and decreased PKGI proteolysis in HEK293 cells. Last, treatment of low-passage rat aortic SMC with membrane-permeable PC inhibitor peptides decreased cGMP-stimulated nuclear PKGI␥ translocation. These data indicate for the first time that PCs have a role in regulating PKGI proteolysis and the nuclear localization of its active cleavage product, which are important for cGMP-mediated SMC phenotype. guanosine 3=,5=-cyclic monophosphate-dependent protein kinase I; proprotein convertase; nitric oxide and guanosine 3=,5=-cyclic monophosphate signaling NITRIC OXIDE (NO) and cGMP play an important role in regulating vascular function and structure. In several disease models, decreased NO and cGMP signaling are associated with dedifferentiation and excessive proliferation of vascular smooth muscle cells (SMC) (9, 60), which can contribute to vasomotor instability and hypertension. In models of newborn lung disease, for example, pulmonary injury often causes decreased NO and cGMP signaling, pulmonary SMC hyperplasia, pulmonary hypertension, right heart failure, and reduced somatic growth. In such cases, selective pulmonary NO treatment has been observed to decrease abnormal pulmonary artery SMC proliferation (66, 67), improve alveolar development (4,8,45,52), and prevent pulmonary hypertension (66,67).NO and cGMP regulate vascular SMC phenotype primarily by stimulating cGMP-dependent protein kinase I (PKGI) (10, 15, 47, 95) through mechanisms that are incompletely understood. PKGI is expressed in vascular SMC as two isofo...

Soluble guanylate cyclase modulates alveolarization in the newborn lung

“…For example, previously, we reported that NO and cGMP regulate pulmonary artery SMC differentiation through stimulating PKGI (21). Nuclear cGMP signaling in vascular SMC appears to require PKGI proteolysis, in part mediated by proprotein convertases, and the nuclear translocation of an active kinase fragment that transactivates gene expression (38,70). Moreover, others have observed decreased pulmonary vascular development in newborn (10) and increased muscularization of peripheral lung arteries in adult mice with NOS deficiency (69).…”

“…26 and 60). Although the precise mechanisms through which cGMP regulates cell phenotype are incompletely understood, recently, cGMP-stimulated PKGI has been observed to undergo proteolysis, in part through the activity of proprotein convertases (38), and a constitutively active PKGI kinase fragment has been observed to localize in the nucleus of cells (70). This nuclear PKGI has been observed in some studies to be required for the transactivation of gene expression by cGMP (29,70).…”

cGMP-dependent protein kinase I in vascular smooth muscle cells improves ischemic stroke outcome in mice

“…5,83,87 Upon cGMP stimulation, smooth muscle cell cGKI has been observed to localize to the nucleus and phosphorylate transcription factors. In these cells, cGMP stimulates cGKI proteolysis, caused in part by proprotein convertases residing within the endomembrane system, 53,88 releasing a COOH-terminal, constitutively active kinase fragment. 86 This cGKI fragment migrates into the nucleus, 86 through classical nuclear, pore-regulated mechanisms, 6 and transactivates gene expression and regulates cell phenotype.…”

Transforming growth factor-β downregulates sGC subunit expression in pulmonary artery smooth muscle cells via MEK and ERK signaling

“…Upon cGMP stimulation, PKGI can localize to the nucleus and phosphorylate transcription regulators (13,31,32). In SMC, cGMP stimulates the proteolysis of PKGI, which is caused in part by proprotein convertases residing within the endomembrane system, releasing a COOH-terminal portion of the molecule (PKGI␥) (41,42,86). This constitutively active PKGI␥ fragment migrates into the nucleus via mechanisms requiring importins, transactivates gene expression, and regulates cell phenotype (15).…”