Proteolytic processing of cGMP-dependent protein kinase I mediates nuclear cGMP signaling in vascular smooth muscle cells

Abstract

Cyclic GMP modulates gene expression in vascular smooth muscle cells (SMCs) in part by stimulating cGMP-dependent protein kinase I (PKGI) and the phosphorylation of transcription factors. In some cells, cGMP increases nuclear translocation of PKGI and PKGI-dependent phosphorylation of transcription regulators; however, these observations have been variable, and the mechanisms mediating nuclear PKGI translocation are incompletely understood. We tested the hypothesis that proteolytic cleavage of PKGI is required for cGMP-stimulated nuclear compartmentation of PKGI and phosphorylation of transcription factors. We detected an NH(2)-terminal PKGI fragment with leucine zipper domain immunoreactivity in the cytosol and endoplasmic reticulum of SMCs, but only a COOH-terminal PKGI fragment containing the catalytic region (now termed PKGIgamma) was observed in the Golgi apparatus (GA) and nucleoplasm. Posttranslational PKGI processing in the GA was critical for nuclear compartmentation of PKGIgamma because GA disruption with nocodazol or brefeldin A inhibited PKGIgamma nuclear localization. PKGIgamma immunoreactivity was particularly abundant in the nucleolus of interphase SMCs where its colocalization with the nucleolar dense fibrillar component protein fibrillarin closely matched the level of nucleolar assembly. Purified nucleolar PKGIgamma enzyme activity was insensitive to cGMP stimulation, which is consistent with its lack of the NH(2)-terminal autoinhibitory domain. Mutation of a putative proteolytic cleavage region in PKGI inhibited cGMP-mediated phosphorylation of cAMP response element-binding protein, cAMP response element-dependent transcription, and nuclear localization of PKGIgamma. These observations suggest that posttranslational modification of PKGI critically influences the nuclear translocation of PKGI and activities of cGMP in SMCs.

Figure 1

PKGI domain nuclear immunoreactivity in SMC. (A) PKGI has a functional leucine zipper (LZ) domain, cGMP-binding regulatory domain (REG), and catalytic region (CR), which were examined using the indicated antibodies. (B) In the nucleus of rat pulmonary artery smooth muscle cells (PASMC) and indicated SMC lines, PKGI_CR immunoreactivity was observed although PKGI_LZ reactivity was not detected. (C) Recombinant PKGI was also localized in SMC nuclei. RFL6 cells expressing PKGIβ-FLAG and GFP with a nuclear localization sequence (GFP_NLS) were treated with digitonin, which permits soluble cytosolic PKGI to diffuse from the cell. PKGIβ-FLAG was detected in the nucleus with an anti-FLAG antibody (αFLAG) and co-localized with the GFP_NLS epifluorescence.

Figure 2

PKGI proteolytic cleavage in SMC. (A) Hypotonic cell shock and isopycnic density centrifugation yielded nuclear (N) and cytoplasmic (C) SMC protein fractions with differential protein abundance and CREB and pyruvate dehydrogenase E2 subunit (E2) immunoreactivity. (B) Purified A7r5 cell nuclear proteins contained a ∼18-kDa protein fragment (arrowhead) with PKGI_LZ immunoreactivity and a 60-kDa protein (arrow) with PKGI_REG and PKGI_CR immunoreactivity. Although these PKGI fragments were not identified in the cytosolic fraction, full-length ∼78-kDa PKGI in the cytosol exhibited all of these immunoreactivities. (C) PKGIβ LZ domain immunoreactivity was identified in perinuclear regions in Z-dimension optical sections of A7r5 nuclei. (D) and (E) PKGIα was also fragmented in intact cells. NH₂-terminal (arrowheads) and COOH-terminal fragments (arrow) of PKGIα and PKGIβ and full-length PKGI isoforms (*) were identified in lysates of BHK cells expressing FLAG-PKGIα and FLAG-PKGIβ. The apparent masses of the NH₂-terminal fragments of the PKGI isoforms are consistent with the different sizes of the their LZ domains. Mass spectroscopy identified peptide portions (red letters) of the LZ domain of PKGIα and the precipitating SBP2 epitope (box).

Figure 3

cGMP increased PKGIγ generation and nuclear localization. (A) 8-Br-cGMP increased PKGIγ levels in the lysates of cells transfected with the indicated amounts of a plasmid encoding PKGIβ-FLAG. A non-specific biotin-binding protein was also detected in RFL-6 (*) that did not have anti-PKGI_CR antibody reactivity. (B) With increased SMC density, PKGIγ levels in nuclei increased and became independent of cGMP treatment. A7r5 cells seeded at the indicated density were exposed to 8-CPT-cGMP and PKGIγ was detected in lysed purified nuclei using an anti-PKGI_CR antibody. (C) and (D) cGMP increased detection of PKGIγ in the GA and nucleoplasm and the NH₂-terminal PKGIβ fragment in the ER. RFL-6 cells were treated with 8-Br-cGMP, exposed to digitonin, and fixed; PKGIγ was detected with an anti-PKGI_CR antibody, GA α-D-galactoside was visualized using lectin, and ER protein disulfide isomerase with an anti-antibody.

Figure 4

An intact GA was important for PKGIγ nuclear localization. (A) Nz and BFA caused GA disassociation and decreased PKGIγ nuclear localization in 8-Br-cGMP-treated RFL-6 cells. (B) cGMP-induced nuclear PKGIγ localization was abolished by Nz exposure and inhibited by BFA treatment. Nuclear colocalization of PKGIβ-FLAG and GFP_NLS, shown in the inset, was objectively quantified in digitonin-treated RFL-6 cells as detailed in the text. Results are expressed as means±SD, n=6 per group, and typical of three independent experiments. * and †P<0.05, indicated treatment vs. the other treatment groups.

Figure 5

PKGIγ immunoreactivity and enzyme activity were localized in SMC nucleoli. (A) PKGIγ immunofluorescence co-localized with A7r5 cell nucleoli, which were identified by their characteristic interaction with RNA-binding dyes and exclusion of DAPI. (B) PKGIγ identified by PKGI_CR immunoreactivity also associated with purified A7r5 cell nucleoli, which exhibited Azure C and RNA-binding dye staining. (C) In contrast with whole cell lysates from COS7 cells, lysates of A7r5 cells and their nucleoli had abundant PKGI enzyme activity and immunoreactivity. Results are expressed as means±SD; n=3 each group, and typical of three independent experiments. * and †P<0.05, indicated groups vs. other treatment groups. (D) DRB caused nucleolar disassembly and disrupted nucleolar PKGI_CR immunoreactivity. PKGIγ was primarily observed in the beaded structures of the DRB-treated nucleoli detected by Azure C staining (arrow). (E) Following removal of DRB, PKGIγ localized in the reassembling nucleolus in a similar manner as fibrillarin, a nucleolar dense fibrillar component protein. The time in hours after DRB removal is indicated.

Figure 6

Mutation of the putative proteolytic cleavage area in PKGI inhibited cGMP-mediated gene expression and nuclear localization of PKGIγ. (A) PCR generated plasmids encoding mutant PKGIβ-FLAG (Mut1—5) with alanines (box) in a putative proteolysis area suggested by NH₂-terminal amino acid sequence analysis of a fragment of immuno-purified A7r5 cell PKGIγ. (B) Mutation of the putative PKGI cleavage site inhibits cGMP-mediated CREB phosphorylation. Immunoblotting with anti-phospho-CREB antibodies revealed a decrease in phospho-CREB in lysates of cGMP-treated BHK cells expressing mutant PKGIβ-FLAG compared to those expressing wild type PKGI-FLAG. The cells had equivalent cGMP-stimulated cytosolic PKGI activity levels as reflected by phospho-VASP immunoreactivity. (C) PKGI cleavage site mutation inhibits cGMP-dependent CRE-activated gene transactivation. BHK cells transfected with a CRE-luciferase reporter plasmid and expressing Mut3 PKGI-FLAG did not have increased luciferase activity when exposed to 8-Br-cGMP. Results are expressed as means±SD, n=6 each group, and typical of three independent experiments. *P<0.05, indicated treatment vs. the other treatment groups. (D) PKGI cleavage site mutation inhibited PKGIγ nuclear localization. BHK cells expressing Mut3 or wild type PKGI-FLAG were treated with 8-Br-cGMP and examined for FLAG immunoreactivity. 8-Br-cGMP did not increase nuclear PKGIγ immunoreactivity in cells expressing Mut3. Results are expressed as means±SD, n=6 each group, and typical of three independent experiments. *P<0.05, indicated treatment vs. the other treatment groups.