Published online before print June 5, 2008, doi: 10.1161/CIRCRESAHA.108.176321
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© 2008 American Heart Association, Inc.
Molecular Medicine |
Proteolytic Processing of cGMP-Dependent Protein Kinase I Mediates Nuclear cGMP Signaling in Vascular Smooth Muscle Cells
From the Cardiovascular Research Center, Departments of Anesthesia (T.S., H.N., J.R.), Pediatrics (J.R.), and Medicine (J.R.), Massachusetts General Hospital, Boston; and Harvard Medical School.
Correspondence to Jesse Roberts Jr, MD, MGH-East, CVRC 4th Floor, 149 13th Street, Charlestown MA 02129. E-mail roberts{at}cvrc.mgh.harvard.edu
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Abstract |
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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 NH2-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 PKGI
) was observed in the Golgi apparatus (GA) and nucleoplasm. Posttranslational PKGI processing in the GA was critical for nuclear compartmentation of PKGI because GA disruption with nocodazol or brefeldin A inhibited PKGI nuclear localization. PKGI 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 PKGI enzyme activity was insensitive to cGMP stimulation, which is consistent with its lack of the NH2-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 PKGI. These observations suggest that posttranslational modification of PKGI critically influences the nuclear translocation of PKGI and activities of cGMP in SMCs.
Key Words: cGKI • guanylate cyclase • gene expression regulation • signal transduction • vascular disease
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Introduction |
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Cyclic GMP is a key regulator of vascular smooth muscle cell (SMC) cytoskeletal kinetics, proliferation, and differentiation, and abnormalities in cGMP signaling have been associated with pulmonary and peripheral vascular disease.1 cGMP is synthesized by guanylyl cyclases, which are activated by nitric oxide, carbon monoxide, and natriuretic peptides, and is metabolized by phosphodiesterases. Through the interaction with cytoplasmic proteins, cGMP influences SMC shape and migration and decreases vascular tone. Recently, cGMP has also been observed to modulate the expression of genes that influence SMC phenotype and proliferation.2 For example, cGMP has been noted to regulate SMC gene expression by increasing the phosphorylation of transcription factors, such as cAMP response element-binding protein (CREB)3–5 and activating transcription factor-1 (ATF-1),5 altering the expression of transcription regulators such as activator protein-1 (AP-1)3 and the growth arrest-specific homeobox transcription factor (GAX),6 and regulating the activity of other nuclear factors, such as serum response factor (SRF).7
cGMP-dependent protein kinase I (PKGI) appears to modulate many of the nuclear activities of cGMP. On activation by cGMP, PKGI increases the phosphorylation of several nuclear transcription factors. PKGI is a threonine/serine kinase present in 2 isoforms in SMCs as a result of alternate mRNA splicing. These isoforms, PKGI
and PKGIβ, have distinct NH2-terminal leucine zipper (LZ) domains.1,8 Through these LZ domains, the PKGI isoforms homodimerize and also interact with heterologous proteins, which anchor them in the cytosol and in proximity to their phosphorylation targets.9 The conserved COOH-terminal portion of PKGI contains 2 cGMP-binding domains and a catalytic region containing Mg2+/ATP-binding, kinase, and substrate recognition domains. The importance of PKGI in modulating transcription factor phosphorylation is supported by the observation that in cells lacking PKGI, such as baby hamster kidney (BHK cells), cGMP does not phosphorylate CREB or ATF-1. In contrast, in cells that express PKGI, such as some low passage SMCs, 3T3 fibroblasts, and BHK cells transfected with PKGI-encoding plasmids, cGMP stimulates phosphorylation of CREB and ATF-1.3,5
Several studies suggest that nuclear translocation of PKGI is important for cGMP-mediated regulation of transcription factors in SMCs. PKGI has been detected in nuclei in some SMC lines.3,10 Moreover, cGMP induces nuclear localization of recombinant PKGI in BHK cells, which do not express endogenous PKGI, where it mediates cGMP-stimulated phosphorylation of CREB.3 Importantly, this activity of cGMP in BHK cells is dependent on the activity of a putative monomeric nuclear localization sequence in PKGI that resides within the Mg2+/ATP-binding domain.10 In addition, the nuclear activities of cGMP have been observed to occur independently of the Ca2+, PKA, and MAPK signaling pathways.3 However, there is variability in the PKGI nuclear localization and activities reported in several studies. In some investigations, PKGI was not observed in the nucleus of primary SMCs or SMC lines11–13 and did not modulate cGMP-dependent gene expression.11 One study reported that in cells that lack cGMP-modulated PKGI translocation, cGMP does not modulate CREB phosphorylation or cAMP response element (CRE)-dependent gene expression.11 This variability in cGMP-mediated nuclear PKGI localization and nuclear function suggests that additional mechanisms might influence PKGI-dependent cGMP nuclear signaling. In this study, we investigated the role of PKGI proteolysis in PKGI function and localization and established its importance in mediating cGMP-induced nuclear events in SMCs.
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Materials and Methods |
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Detailed information is provided in the supplemental materials (available online at http://circres.ahajournals.org).
Antibodies and Reagents
The anti-PKGI regulatory domain (PKGIREG) antibody was kindly provided by S. Janssens (University of Leuven, Belgium),14 and the human antipyruvate dehydrogenase complex E2 subunit autoantibodies were provided by D. Bloch (Mass General Hospital, Boston).15 The other antibodies and reagents were obtained from commercial sources.
Cell Culture and Transfection
Pulmonary artery smooth muscle cells (PASMCs) were isolated from rats16; all other cells were obtained from American Type Culture Collection. PASMCs and RFL-6 cells were maintained in RPMI 1640 and the others in DMEM medium. PASMC medium was supplemented with 10% Nuserum, and all other media contained 10% fetal bovine serum. Cells were transfected using a cationic lipid-based reagent. Luciferase activities were measured using a commercially available kit and a luminometer and normalized to the average luciferase activity observed in cGMP-treated wild type PKGIβ expressing cells.
Subcellular Fractionation
SMC nuclei and nucleoli were purified as described in the Supplemental Information in the presence of protease inhibitors. Nuclear purity was confirmed using phase contrast microscopy; nucleolar purity was confirmed using phase contrast microscopy and Azure C and fluorescent RNA-binding dyes.
Plasmid Construction and Mutagenesis
The plasmids encoding wild-type and mutant PKGI were constructed using standard techniques,17 and a plasmid encoding murine PKGIβ (provided by M. Uhler, University of Michigan, Ann Arbor).11 FLAG-PKGIβ and PKGIβ-FLAG represent NH2-terminal and COOH-terminal FLAG epitope-tagged PKGIβ, respectively.
Purification and Analysis of PKGI Fragments
The purified NH2-terminal fragment of PKGI was identified after trypsic digestion using LC-MS/MS and database interrogation. To assess the NH2-terminal end of PKGI, nuclei of 8-Br-cGMP–treated A7r5 cells were purified and homogenized in lysis buffer and PKGI was collected using immobilized anti-PKGCR antibody. After SDS-PAGE, purified PKGI was subjected to protein microsequencing using Edman degradation.
Immunodetection of Proteins
Cells and purified nuclei and nucleoli were fixed with 4% paraformaldehyde, exposed to 100% methanol, and blocked with 1% serum in PBS. After incubation with primary antibody or preimmune serum, biotin-conjugated secondary antibodies and fluorochrome-linked strepavidin or fluorescent probe-labeled secondary antibodies were used and subsequently detected using epifluorescent microscopy. For Western blotting, protein fractions were resolved using SDS-PAGE, transferred to polyvinylidene fluoride membranes, exposed to indicated primary antibodies, and detected using enzyme-conjugated secondary antibodies and chemiluminescence.
PKGI Enzyme Activity
PKG enzyme activity was measured using a PKGI-specific peptide substrate as previously described.16
Quantification of PKGI Nuclear Localization
Cells transfected with plasmids encoding wild-type and mutant PKGIβ-FLAG and green fluorescent protein with a nuclear localization sequence (GFPNLS) were treated with 8-Br-cGMP, exposed to digitonin in PBS containing protease inhibitors, and then fixed as described above. PKGIβ-FLAG, reacted with biotinylated anti-FLAG antibody and Alexa Fluor 610-conjugated streptavidin, and GFPNLS were detected using epifluorescence. Identically registered epifluorescent images of 6 randomly-oriented, nonoverlapping 1.0-mm2 fields were obtained and analyzed using ImageJ.18
Statistical Analysis
Data are represented as mean±SD. Data were analyzed using a randomized complete block design and, because no differences were found between the groups, the results were analyzed using one-way ANOVA. When significant differences were detected, a Scheffé test was used post hoc. Significance was determined at P<0.05.
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Results |
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Nuclear PKGI Lacks NH2-Terminal LZ Domain Immunoreactivity
We reasoned that the reported variability in nuclear localization of PKGI could be attributable to a posttranslational modification of PKGI that modifies its immunologic detection. Therefore, several polyclonal antibodies (PKGI epitopes are schematically defined in Figure 1A) were used to localize PKGI functional domains to SMC compartments. Although the nuclei of many pulmonary artery SMCs and cells of SMC and myofibroblast cell lines were observed to harbor PKGICR immunoreactivity, none exhibited PKGIβ LZ domain reactivity, although it was detected in the cytosol (Figure 1B). However, nuclear PKGICR staining was not detected in all SMCs. For example, some later SMCs and A7r5 cell passages did not exhibit nuclear PKGICR immunoreactivity despite the detection of PKGI LZ domain and COOH-terminal immunoreactivity in the cytosol. Moreover, in individual cultures that exhibited nuclear PKGICR immunoreactivity, expression was nonhomogeneous. For example, in areas where SMCs were confluent, abundant cytosolic and nuclear PKGICR immunoreactivity was often detected. In contrast, in subconfluent areas of the same culture, less cytosolic anti-PKGICR reactivity and little or no nuclear PKGICR immunoreactivity were evident. These results suggest the importance of using an objective sampling method when quantifying nuclear compartmentation of PKGI in cultured cells.
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Although PKGI expressed from transfected plasmids has been detected in nuclei of some cells,10,11 it is not known whether their functional domains are differentially localized. We examined whether the COOH-terminal portion of PKGI overexpressed in RFL6 cells localizes to the nucleus as the endogenous protein does. To improve detection of nuclear PKGI, after RFL6 cells were transiently transfected with plasmids encoding PKGI-FLAG, digitonin was used to permeabilize the plasma membrane and permit the egress of soluble cytosolic PKGI while nuclear PKGI was retained. To aid in nuclear PKGI-FLAG localization, cells were cotransfected with a plasmid encoding green fluorescent protein with a nuclear localization sequence. Immunohistochemistry with anti-FLAG revealed the COOH-terminal portion of PKGIβ colocalized with GFP in the nucleus (Figure 1C) but no nuclear PKGIβ LZ domain immunoreactivity (data not shown).
PKGI is Cleaved in Vascular SMCs
PKGI cleavage might account for the differential compartmentation of PKGI epitopes. We therefore evaluated purified nuclear proteins for evidence of PKGI fragmentation. Plasma membrane disruption and isopycnic density centrifugation permitted the purification of SMC nuclei and effective separation of nuclear and cytosolic proteins (Figure 2A). Immunoblotting using equal amounts of nuclear and cytosolic proteins revealed that SMC nuclear protein fractions did not contain full-length PKGI with NH2-terminal and COOH-terminal epitope immunoreactivity. However, an 
18-kDa PKGI fragment with LZ domain immunoreactivity (Figure 2B, arrowhead), and a 60-kDa fragment (PKGI) with PKGIREG and PKGICR immunoreactivity were detected in nuclear proteins. Because we previously found no nuclear immunoreactivity for the PKGIβ LZ domain in intact SMCs (Figure 1B), we performed immunofluorescence studies using purified nuclei to clarify the PKGI LZ domain localization. PKGILZ immunoreactivity mapped to asymmetrical perinuclear structures in purified SMC nuclei (Figure 2C), colocalizing with molecules restricted to the Golgi apparatus (supplemental Figure I). These data suggested that PKGI fragmentation occurs outside the nuclear envelope and that only the COOH-terminal fragment of PKGI enters the nucleus.
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We assessed whether PKGI is similarly cleaved and compartmented in SMCs. Because no antibody detecting the PKGI LZ domain was available, BHK cells were transfected with a plasmid that encodes NH2-terminal FLAG-tagged PKGI. The NH2-terminal fragment of PKGI was detected with anti-FLAG antibody and the COOH-terminal PKGI fragment with anti-PKGILZ antibody. Immunoblotting revealed NH2-terminal fragments (Figure 2D, arrowheads) consistent with the lengths of PKGI and PKGIβ LZ domains and a PKGI fragment (arrow). Mass spectroscopy confirmed the identity of the purified NH2-terminal fragment (Figure 2E). The observed cleavage of both PKGI isoforms, the generation of PKGI of an identical size on fragmentation of either PKGI or PKGIβ (Figure 2D, arrow), and the 60-kDa size of the PKGI fragment suggested that the PKGI cleavage site resides in a conserved cGMP-binding domain of the PKGI isoforms.
cGMP Increases PKGI Nuclear Localization
Previous studies revealed that cGMP increased PKGI immunoreactivity in the nucleus of BHK and some SMC lines.10 Because only PKGI was identified in SMC nuclei, we examined the effect of cGMP on nuclear PKGI levels. cGMP was found to increase nuclear PKGI abundance in BHK and RFL-6 cells expressing PKGIβ-FLAG (Figure 3A). Moreover, because PKGI expression is increased in postconfluent SMCs,19 the influence of SMC density on PKGI levels in purified nuclei was examined. cGMP increased nuclear PKGI levels in sparsely plated A7r5 cells, but as SMC density increased, so did the level of nuclear PKGI immunoreactivity in the absence of cGMP stimulation (Figure 3B). This suggests that as SMCs become more confluent and PKGI expression increases, PKGI cleavage and nuclear PKGI translocation increases independent of cGMP exposure.
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PKGI fragments were detected in the Golgi apparatus (GA; Figure 2C and supplemental Figure I) and cGMP appeared to increase PKGI fractionation, thus we investigated whether cGMP increases localization of PKGI to the GA. Exposure of RFL-6 cells to cGMP increased PKGI immunoreactivity in the GA and endoplasmic reticulum (ER; Figure 3C and 3D). PKGICR domain immunoreactivity colocalized with the GA whereas PKGILZ immunoreactivity also colocalized with the ER. These results suggest that cGMP increases PKGI mobilization to the ER and GA, where cleavage might increase PKGI accumulation in the GA and its transfer to the nucleoplasm.
Intact Golgi Apparatus Is Required for Nuclear Trafficking of PKGI
The GA contains endopeptidases that process some signaling proteins and thereby regulate their nuclear trafficking.20 We examined the role of the GA in regulating PKGI nuclear compartmentation. The effects of both Nz and BFA on PKGI nuclear localization were tested because they dissociate the GA through different mechanisms: whereas Nz causes microtubular depolymerization and relocation of GA fragments into cytosolic islands,21,22 BFA inhibits protein transport from the ER to the GA by inhibiting GTP-exchange factors23 and disassembles the cis/middle- and trans-Golgi complexes, causing them to fuse with the ER.24 PKGI nuclear localization was inhibited in cGMP-treated RFL-6 cells exposed to either Nz or BFA. After cGMP exposure, PKGICR immunoreactivity continued to be associated with the Nz- and BFA-disrupted GA, however PKGI nuclear transport decreased (Figure 4A). Objective quantitative analysis revealed that GA disruption with Nz completely blocked cGMP-stimulated PKGI nuclear localization while BFA partially inhibited it (Figure 4B). GA disruption did not inhibit PKGI fractionation: immunoblotting revealed abundant PKGI in cGMP-exposed RFL-6 cells treated with these compounds (data not shown), indicating that the effect of GA disruption appears to be on the nuclear translocation of PKGI, and not on proteolysis of PKGI.
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P<0.05, indicated treatment vs the other treatment groups.
PKGI Is Associated With the Nucleoli of Interphase SMCs
We further explored the apparent association between PKGI and SMC nucleoli (Figures 1B and 3
C). PKGICR immunoreactivity colocalized with SMC nucleoli in situ and with SMC nucleoli purified by ultracentrifugation (Figure 5A and 5B). Moreover, isolated SMC nucleoli exhibited cGMP-independent PKGI enzyme activity at a level similar to that detected in cGMP-treated A7r5 whole cell lysates (Figure 5C). This suggests that removal of the NH2-terminal autoinhibitory pseudosubstrate site of PKGI through proteolytic cleavage releases inhibition of kinase activity, revealing high constitutive PKGI activity. This observation is consistent with in vitro studies where the NH2-terminal region of PKGI was removed with purified proteases.25,26
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Transcription and processing of precursor rRNA and packaging of rRNA into ribosomal particles occur within structurally distinct areas of nucleoli. Specifically, the fibrillar centers are thought to harbor rDNA, the dense fibrillar component (DFC) is where precursor rRNA is transcribed and matured and preribosome formation commences, and the granular compartment is where the preribosome is assembled.27 DRB is an adenosine analogue that inhibits rRNA production and permits evaluation of nucleolar assembly and activity.28,29 DRB exposure causes the nucleoli to reversely unravel into intranuclear structures resembling a string of beads, which comprised RNA polymerase I and are thought to be single units of rDNA transcription, and a strand of nontranscribed DNA spacer regions.28–30 To investigate whether PKGI actively associates with the nucleolar subcomponents, SMCs were treated with DRB and the nucleolar association of PKGI was assessed. DRB disassembled A7r5 cell nucleoli into beaded strands that retained PKGI immunoreactivity (Figure 5D). During DRB-mediated nucleolar disassembly and reassembly, PKGI localized in the nucleolus in a similar manner as fibrillarin (Figure 5E), a DFC protein that processes precursor rRNA,31 indicating that PKGI has a dynamic relationship with SMC nucleoli.
Regulation of Gene Expression by cGMP Requires Nuclear PKGI
Previous studies indicate that nuclear PKGI modulates gene expression in part by phosphorylating CREB.2–4 Because cGMP induces nuclear localization that appears dependent on PKGI cleavage, we examined whether mutation of a putative cleavage site inhibits cGMP-dependent CREB phosphorylation and activity and PKGI nuclear localization. To identify the PKGI cleavage site, the NH2-terminal end of immunopurified PKGI was detailed using Edman-based amino acid sequencing. Accounting for a protein fragment contributed by bovine serum albumin, peptides commencing with serine138 and glutamic acid153 of PKGI were identified (detailed in supplemental Figure III). We generated plasmids with alanine substitutions in this region (mapped in Figure 6A and supplemental Figure III) and assessed their ability to express catalytically active PKGI and support cGMP-dependent CREB phosphorylation and CRE-dependent gene expression in BHK cells. Transfection conditions in these experiments were optimized so that wild-type and mutant PKGI had equivalent immunoreactivity levels. The wild-type and mutant PKGI forms exhibited similar cGMP-stimulated cytosolic kinase activity, as shown by the ability of cGMP to stimulate PKGI-dependent VASP serine239 phosphorylation (Figure 6B). However, Mut3 and Mut4 exhibited decreased cGMP-dependent CREB phosphorylation. Of interest, these 2 mutants had amino acid substitutions associated with the amino acid sequence KVEVTK, which has similarity to a putative proprotein convertase motif.32 Moreover, the amino acids in this area have a high level of species homology in PKGI (supplemental Figure III). Previous studies found that nuclear localization of PKGI critical for cGMP-dependent stimulation of CRE-mediated gene expression.10 We examined the ability of PKGI Mut3 to confer cGMP-dependent CRE-promoter activity. cGMP increased CRE-dependent promoter activity in BHK cells expressing wild-type PKGIβ but not in cells expressing PKGI Mut3 (Figure 6C). Additional studies revealed that this PKGI mutant exhibited less cGMP-stimulated PKGI nuclear localization (Figure 6D). These studies suggest that proteolytic cleavage of PKGI within the cGMP-binding domain is critical for the nuclear activities of cGMP.
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Discussion |
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The principal objective of this investigation was to examine mechanisms that regulate the nuclear localization of PKGI and modulation of gene expression by cGMP. In SMCs, cGMP exposure facilitated the cleavage of the NH2-terminal LZ domain from PKGI and the nuclear localization of PKGI
, a constitutively active kinase fragment of PKGI. The Golgi apparatus appeared to have an important role in modulating the nuclear translocation of PKGI; Golgi apparatus disruption with either nocodazol or brefeldin A inhibited the cGMP-stimulated nuclear localization of PKGI. Additionally, PKGI nuclear localization appeared to be required for the regulation of gene expression by cGMP. Mutation of PKGI in the putative proteolysis site inhibited cGMP-mediated CREB-phosphorylation, CRE-dependent gene expression, and the nuclear localization of PKGI. cGMP regulates the expression of genes that modulate SMC proliferation and differentiation, therefore these results support a central role for PKGI in mediating the influence of cGMP on cell phenotype. Moreover, they provide additional evidence that proteolytic pathways regulate some canonical signaling systems. Protein cleavage is also a critical step in the nuclear translocation and activities of sterol regulatory element binding protein (SREBP) and Notch.20 In these pathways, proteolytic removal of a membrane-binding domain releases a protein fragment that can enter the nucleus and modulate gene expression. Similarly, NH2-terminal cleavage of PKGI removes an LZ domain that tethers PKGI to cytosolic proteins. The resulting active kinase PKGI fragment enters the SMC nucleus and phosphorylates proteins that regulate gene expression.
Although the functional domains of PKGI and PKA are arranged similarly, the requirement for proteolytic processing to enable cyclic nucleotide-dependent nuclear signaling distinguishes PKGI from PKA. The PKA heterodimer consists of separate regulatory and catalytic subunits. On cAMP binding, the catalytic subunit is released from the regulatory unit which anchors it in the cytosol and enters the nucleus where it modulates gene expression.33 In PKGI, the anchoring (LZ) and regulatory and catalytic (PKGI) domains are on a single protein molecule that appear to require cleavage to allow PKGI nuclear translocation. Another difference between PKA and PKGI is the requirement for a nuclear localization sequence (NLS) for PKGI nuclear localization. Unlike the PKA catalytic subunit, which is small enough to passively diffuse into the nucleus,34 PKGI is too large to enter the nucleus without an NLS to facilitate docking to nuclear pore complexes.35 Gudi and coworkers identified a monomeric NLS in PKGI that facilitated nuclear PKGI localization in BHK cells on cGMP treatment.3,10 This NLS is in the Mg2+/ATP binding domain of PKGI and is present in PKGI.
Studies suggest that cGMP-induced conformational changes in PKGI may expose a cryptic proteolytic site to endopeptidases, regulating PKGI cleavage. For example, in vitro studies indicate that cGMP-binding unfolds PKGI and increases the sensitivity of PKGI to endopeptidase-induced fractionation.36,37 In these experiments, PKGI was not cleaved in the cGMP-binding region, as we report here. This suggests that SMCs contain an endopeptidase that cleaves amino acid residues in the cGMP-binding region and is distinct from those investigated in the in vitro studies. It might also indicate that additional PKGI posttranslational modifications or protein interactions are required to permit PKGI proteolysis within the cGMP-binding region.
The association of PKGI with the nucleolus in SMCs is a novel finding. The PKGI nucleolar compartmentation we report, particularly during nucleolar disassociation and reassembly, and presence of PKGI enzyme activity within purified nucleoli support the notion that PKGI is actively integrated within the nucleolus of SMCs. These data suggest that cGMP signaling might influence nucleolar function. Recent proteomic screens indicate that the nucleolus contains not only proteins required for ribosomal biogenesis, but also kinases such as protein kinase C and PKA, which likely transduce cytoplasmic signals.38
The identification of PKGI proteolysis in SMCs has important implications for vascular disease mechanisms and the development of novel therapies. Indirect evidence suggests that abnormalities in PKGI processing contributes to neointima formation in injured vessels.13,14,16,39 For example, Monks and coworkers observed that carotid artery injury in rats increases PKGI expression in neointimal SMCs.39 However, because the increased PKGI was detected in the perinuclear region of SMCs and not within the nucleus, PKGI proteolysis likely was diminished in the injured vessels.39 Decreased nuclear PKGI activity might contribute to SMC proliferation in injured carotid arteries. In early passaged SMCs, in which we detected PKGI proteolysis and nuclear PKGI, PKGI decreases cell proliferation.16 In contrast, in murine SMCs, in which PKGI does not localize in the nucleus,13 PKGI increases cell proliferation.40 Such an attenuation of PKGI proteolysis might inhibit the cytostatic effect of PKGI-based therapies. For example, Sinnaeve and coworkers found that although overexpression of PKGI does not decrease neointimal formation in the injured porcine aorta, transduction of an LZ domain-deficient mutant PKGI, which could passively diffuse into SMC nuclei, is protective.14 Thus, although PKGI expression is upregulated in some injured vessels, deficient proteolysis could prevent PKGI generation and nuclear translocation, blocking cGMP-induced protection against neointimal cell proliferation. Our data indicate that PKGI-like molecules may be able to protect against vascular diseases in which PKGI proteolytic mechanisms are diminished.
In summary, we identified a novel mechanism that regulates cGMP nuclear signaling. The observation that PKGI proteolytic cleavage critically regulates this signaling pathway has important implications for understanding how cyclic nucleotides regulate gene expression in health and disease.
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Acknowledgments |
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We thank Michael Uhler (Univ. Michigan), Stefan Janssens (Univ. Leuven), Donald Bloch (Mass General Hospital) for generously providing reagents and Ashok Khatri (Mass General Hospital Protein Core Facility) for assisting in the amino acid microsequencing.
Sources of Funding
The National Institutes of Health grant HL080316 supported this work.
Disclosures
None.
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Footnotes |
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Original received March 25, 2008; revision received May 8, 2008; accepted May 28, 2008.
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