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

Abstract

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, PKGIγ, 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 PKGIγ 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 α1-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 PKGIγ 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.

Keywords: guanosine 3′,5′-cyclic monophosphate-dependent protein kinase I; nitric oxide and guanosine 3′,5′-cyclic monophosphate signaling; proprotein convertase.

Fig. 1.

Structural characterization of a putative proprotein convertase (PC) cleavage site in cGMP-dependent protein kinase I (PKGI). A: PKGIα and PKGIβ isoforms and cleaved PKGIγ share COOH-terminal functional domains that are defined in the text. PKGIγ-fragment amino acid sequencing suggested that PKGI is cleaved following KVEVTK, after residue 152 in PKGIα and 167 in PKGIβ. This sequence corresponds to a typical PC recognition site: (K/R)-Xn-(K/R), where n = 0, 2, 4, or 6 and X is not a cysteine. The putative PC recognition site in PKGI, shown in the box, resides in the cGMP-binding domain area and is conserved in many species. Cleavage at this site would result in an ∼60-kDa PKGI fragment. LZ, leucine zipper-like; AI, autoinhibitory. B: polarity map in PKGI in the putative cleavage and PC recognition site, which is in bold, supports the possibility that this site might bind to charged amino acid residues in the active cleft of PCs. The polarity scale was defined by Grantham (25).

Fig. 2.

Furin overexpression increases PKGI proteolysis. Separate cultures of human embryonic kidney (HEK) 293 cells were transfected with 1 μg of plasmids that encode murine PKGIβ with a COOH-terminal FLAG tag and either human furin with a COOH-terminal V5 tag or a control plasmid, pcDNA3. The next day, soluble protein fractions were obtained, and steady-state PKGIβ proteolysis was detected using an anti-FLAG antibody and immunoblotting. Anti-V5 antibody revealed furin·V5 expression; α-tubulin immunoreactivity was used as a loading control. A and B: furin overexpression was associated with increased PKGIβ proteolysis. Cells overexpressing furin had increased amounts of an ∼60-kDa fragment with FLAG immunoreactivity, similar to previously described PKGIγ. B: densitometry revealed that furin overexpression increased PKGIβ proteolysis nearly 50%; n = 3 each group, *P < 0.05.

Fig. 3.

PC5 also proteolyzes PKGI. A: HEK293 cells were transfected with the indicated amounts of plasmids that encode PC5A or PC7 and with 1 μg of pcDNA3·PKGIβ·FLAG. The total amount of transfected DNA was normalized using control plasmids. PKGI proteolysis was detected using an anti-FLAG antibody and immunoblotting. Whereas exposure to increased amounts of a PC5A-encoding plasmid increased the levels of the immunoreactive PKGIγ·FLAG fragment (arrow), transfection with the PC7-encoding plasmid did not increase the detected amount of this PKGI cleavage product. B: to confirm that active PC7 was expressed following transfection, PC enzyme activity was determined using an internally quenched PC-substrate fluorogenic probe and lysates of HEK293 cells transfected with 1.5 μg of the indicated plasmids. Transfection with the PC7-encoding plasmid used in the experiment detailed in A increased PC enzyme activity in the cell lysates; n = 6 each group, *P < 0.05 vs. each other. C: HEK293 cells were transfected with 1.5 μg of pcDNA3 or pcDNA3·furin·V5, and, the following day, proteins were precipitated from the cell media, resolved using SDS-PAGE, and electroblotted to a membrane, and shed furin·V5 was detected using an anti-V5 antibody. Cells transfected with furin-encoding plasmid secreted cleaved furin into the cell media.

Fig. 4.

Definition of the PC proteolysis site in PKGI. HEK293 cells were transfected with 1 μg of plasmids that encode PKGIβ·FLAG with and without an alanine-substitution mutation in the putative PC recognition domain, e.g., δKVEVTK; control plasmids were used to balance the amount of transfected DNA. Steady-state PKGI proteolysis was assessed using an anti-FLAG antibody and immunoblotting. Although PKGIβ·FLAG proteolysis was increased in cells with furin overexpression, the proteolysis of PKGIβ·FLAG with a mutation in the putative PC recognition domain was not increased in those overexpressing furin.

Fig. 5.

Furin overexpression increases PKGIβ proteolysis in cells with dysfunctional furin. LoVo cells were infected with 200 multiplicity of infection of an adenovirus that encodes PKGIβ·FLAG and then transfected with or without 4.5 μg of a plasmid that encodes furin (pSVL·furin) or empty vector (pSVL). PKGI proteolysis was detected (arrow) in soluble cell lysates using an anti-FLAG antibody and immunoblotting. Overexpression of furin increased PKGIβ·FLAG proteolysis in cells with deficient endogenous furin.

Fig. 6.

PC inhibition decreases PKGI proteolysis in cells. Cells were transfected with plasmids that encode α₁-PDX, an inhibitor of PC activity, PKGI isoforms with a COOH-terminal FLAG tag, or control plasmids. Later, soluble proteins in cell lysates were denatured and resolved using gel electrophoresis and transferred to a charged membrane for immunoblotting. In some experiments, furin and a furin-α₁-PDX complex were detected with an anti-furin antibody. In others, PKGI proteolysis was detected using an anti-FLAG antibody. A: HEK293 cells express profurin (*) and mature furin (open arrow), which results from profurin autoproteolysis, and associated N-glycosylated species. Transfection with pSVL·furin increased the level of the encoded mature ∼85-kDa furin protein. Coexpression of α₁-PDX and furin caused the formation of a higher-molecular-weight anti-furin antibody-reactive band (closed arrow) that is indicative of the formation of an α₁-PDX-furin complex and PC inhibition. Similar α-tubulin immunoreactivity showed equal protein transfer. B: in HEK293 cells overexpressing PKGIβ·FLAG, transfection with the α₁-PDX-encoding plasmid caused inhibition of PKGIβ transgene proteolysis and PKGIγ generation.

Fig. 7.

PC inhibition decreases PC activity and proteolysis-dependent nuclear PKGIγ localization in vascular SMC. A: rat aortic SMC were serum starved and then treated in media containing 10% FBS with and without 50 μm decanoyl-Arg-Val-Lys-Arg-chloromethylketone (dec-RVKR-CMK), a membrane-permeable PC inhibitor. Subsequently, COOH-terminal insulin-like growth factor I receptor (IGF-IR) immunoreactivity was detected in cell lysates using a specific antibody and immunoblotting. Whereas the endoplasmic reticulum (ER) form of pro-IGF-IR and cleaved IGF-IRβ was detected in all cell lysates, the latter being increased with serum stimulation, the Golgi apparatus (GA) form of uncleaved pro-IGF-IR was detected in cells treated with dec-RVKR-CMK. B: rat aortic smooth muscle cells were pretreated with and without 50 μM dec-RVKR-CMK and then with and without the inhibitor and 200 μM 8-(p-chlorophenylthio)-cGMP (8-pCPT-cMP), a membrane-permeable cGMP analog. Endogenous PKGI immunoreactivity was detected using an antibody that binds to the COOH-terminal PKGI domain (PKG_CR), a fluorescently tagged secondary antibody, and laser-scanning confocal microscopy with optical sections through the cell nucleus. Proteolysis-dependent nuclear PKGIγ translocation stimulated by 8-pCPT-cGMP was inhibited by treatment with dec-RVKR-CMK.