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Address correspondence to Lonny R. Levin, Dept. of Pharmacology, Joan and Sanford I. Weill Medical College and Graduate School of Medical Sciences of Cornell University, 1300 York Ave., New York, NY 10021. Tel.: (212) 746-6752. Fax: (212) 747-6241. email: llevin{at}med.cornell.edu
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Abstract |
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Key Words: CREB; PKA; gene expression; compartmentalization; signal transduction
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Introduction |
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cAMP elicits its cellular effects by activation of three known classes of effector proteins: exchange proteins activated by cAMP (EPAC), cyclic nucleotide gated ion channels, and protein kinase A (PKA). A subset of these targets resides at the plasma membrane, where they exist in macromolecular signaling complexes that also include a G protein coupled receptor, its transducing G protein, and the source of cAMP, a tmAC isoform (Davare et al., 2001). The cAMP generated by tmACs acts locally (Rich et al., 2000, 2001; Zaccolo and Pozzan, 2002), most likely restricted by phosphodiesterase "firewalls" (Zaccolo and Pozzan, 2002), which define the limits of these cAMP signaling microdomains. However, targets of cAMP do not solely reside at the plasma membrane. EPAC is localized to the nuclear membrane and mitochondria (Qiao et al., 2002), and PKA is tethered throughout the cell by a class of proteins called AKAP (A-kinaseanchoring proteins; Michel and Scott, 2002). The observation that cAMP does not diffuse far from tmACs (Bacskai et al., 1993; Zaccolo and Pozzan, 2002) reveals that there must be another source of cAMP modulating the activity of these distally localized targets.
sAC (Buck et al., 1999) is widely expressed in mammalian cells (Sinclair et al., 2000). Unlike tmACs, sAC is G protein insensitive (Buck et al., 1999), and among mammalian cyclases, it is uniquely responsive to intracellular levels of bicarbonate (Chen et al., 2000). The ubiquitous presence of carbonic anhydrases ensures that the intracellular bicarbonate concentration (and sAC activity) will reflect changes in pH (Pastor-Soler et al., 2003) and/or CO2. Because CO2 is the end product of energy-producing metabolic processes, sAC is poised to function as a cell's intrinsic sensor of metabolic activity (Zippin et al., 2001). sAC possesses no transmembrane spanning domains (Buck et al., 1999) and is distributed to subcellular compartments containing cAMP targets (Zippin et al., 2003) that are distant from the plasma membrane. sAC was also found localized inside the mammalian cell nucleus (Zippin et al., 2003).
To evaluate how sAC-generated cAMP might differ from the second messenger generated by tmACs, we explored a prototypical cAMP-dependent pathway, PKA-dependent phosphorylation of cAMP response element binding protein (CREB; De Cesare and Sassone-Corsi, 2000). In a widely accepted signal transduction paradigm, extracellular signals (i.e., hormones and neurotransmitters) affect CREB family phosphorylation by stimulation of plasma membranebound tmACs. The generated cAMP activates nearby PKA, and the liberated catalytic subunit then appears to translocate through the cytoplasm to phosphorylate and activate CREB proteins residing inside the nucleus (Riabowol et al., 1988b; Hagiwara et al., 1993). Intracellular signals, such as metabolic activity, also modulate CREB phosphorylation in a cAMP-dependent manner (Daniel et al., 1998; Singh et al., 2001; Trumper et al., 2002), but the mechanism has yet to be established. Localization of sAC inside the nucleus, in close proximity to the CREB family proteins, and its regulation by calcium and bicarbonate suggested that sAC might be responsible for modulating CREB activity in response to intracellular signals.
In this paper, we demonstrate the existence of a nuclear cAMP signaling microdomain that mediates bicarbonate-dependent activation of the transcription factor CREB. Bicarbonate activation of CREB represents an example of a mammalian cAMP-dependent pathway solely modulated by intrinsic cellular signals. This nuclear cAMP signaling cascade functions independently from the classically defined mechanisms leading to CREB activation, demonstrating that cAMP is a locally acting second messenger that can work autonomously in different compartments within a single cell.
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Results |
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Isolated nuclei contain components of a cAMP signaling microdomain
Bicarbonate treatment of whole cells leads to rapid induction of CREB phosphorylation (Fig. 2). To test whether the nuclear localized sAC and PKA were responsible for this bicarbonate-induced CREB activation, we prepared isolated nuclei from suspension HeLa cells, a cell line with well-established protocols for the isolation and enrichment of nuclei. Cells were lysed using digitonin, and nuclear preparations were purified by density centrifugation through an OptiPrep gradient. Western analyses of the same cell equivalents from each fraction using cellular markers for different subcellular compartments (histone H1, NaK ATPase 1 subunit, cytochrome c oxidase subunit III [COX], and ß-tubulin) confirmed that the nuclear fractions (P2) were positive for nuclear markers (histone) with undetectable levels of plasma membrane (NaK ATPase), mitochondrial (COX), or cytoplasmic (tubulin) contamination (Fig. 5 A). To confirm that the P2 fraction did not contain any detectable mitochondria, a possible source of both sAC and PKA contamination, we overloaded the P2 fraction, but COX antigen was still not detected (unpublished data). Visual inspection and DAPI fluorescence confirmed that the final preparation was enriched for intact nuclei (Fig. 5, B and C), and, as expected, isolated nuclei contained both CREB and sAC proteins by immunocytochemistry (Fig. 5 B) and Western blotting (Fig. 5 D).
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sAC represents the only source of cAMP detectable in isolated nuclei
We previously demonstrated that sAC activity was present in COS7 cell nuclei (Zippin et al., 2003). We now show that bicarbonate-responsive sAC is the only source of cAMP in nuclei isolated from suspension HeLa cells. Whereas forskolin potently stimulates cAMP production in whole cell lysates (Fig. 6 A), there was no significant increase in cAMP elicited by forskolin in isolated nuclei (Fig. 6 B). There was a significant level of basal adenylyl cyclase activity in isolated nuclei, which was stimulated by bicarbonate addition (Fig. 6 B). Both the bicarbonate-stimulated and basal activities were inhibited by sAC-selective inhibitors (Fig. 6, C and D). We have identified several sAC inhibitors (Fig. 6 C), inert toward tmACs (Fig. 6 D), in a screen of a combinatorial chemical library (unpublished data). In the presence of two representative, structurally unrelated inhibitors (KH1 and KH2 each display an IC50 10 µM toward recombinant human sAC protein), the cAMP generated in the presence of bicarbonate in P2 nuclei was reduced to a level below that of basal. These results indicate that in addition to mediating the bicarbonate-induced increase in cAMP in isolated nuclei, sAC is also responsible for the observed basal adenylyl cyclase activity.
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Next, we confirmed that the effects of bicarbonate on CREB phosphorylation were mediated by nuclear sAC and PKA. CREB phosphorylation induced by bicarbonate was substantially reduced by the PKA inhibitors, H89 (50%) and RpcAMPs (70%; Fig. 7 C), revealing the involvement of cAMP-responsive PKA holoenzyme. The chemical inhibitors effective at blocking sAC-generated cAMP accumulation (Fig. 6 B, KH1 and KH2) were also effective in preventing bicarbonate-induced CREB phosphorylation (Fig. 7 D), demonstrating, once again, that sAC is responsible for the bicarbonate-stimulated cAMP-dependent phosphorylation of CREB in the mammalian cell nucleus.
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Discussion |
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The nuclear cAMP signaling cascade induced by bicarbonate produced a rapid activation of CREB family members in both whole cells and nuclei, whereas PGE2 and forskolin, tmAC-specific activators, produced a delayed response exclusively in whole cells. Therefore, cAMP-mediated activation of CREB family members by tmACs and sAC proceed via independent pathways. CREB activation by hormones or neurotransmitters via tmACs apparently requires time for movement of PKA catalytic subunit from the plasma membrane into the nucleus (Riabowol et al., 1988a; Hagiwara et al., 1993). This delayed activation is consistent with hormonal control of gene expression providing a long-term response to predominantly sustained extracellular signals (Bailey et al., 1996). In contrast, the newly described nuclear sAC activation pathway proceeds rapidly without requiring the translocation of any constituent. In this regard, the sAC nuclear microdomain is capable of responding quickly to subtle fluctuations in intrinsic signals, such as local intracellular concentrations of bicarbonate and calcium.
In tissues, sAC is not present within the nucleus of every cell. In liver, sAC appears to be predominantly extranuclear but enriched in a subset of the nuclei (Fig. 4, A and B, A arrows). PKA holoenzyme appears to be enriched within the same subset of nuclei (Fig. 4 A, A arrows), and interestingly, these are the nuclei that are also positive for CREB phosphorylation (Fig. 4 B, A arrows). The presence of both positive and negative nuclei for sAC, PKA, and CREB phosphorylation in the same tissue suggests that there may be coordinated regulation of the presence of this newly described nuclear signaling microdomain.
The demonstration that bicarbonate treatment of whole cells leads to activation of the CREB family of transcription factors reveals that bicarbonate itself induces a signal transduction cascade. Cellular bicarbonate levels reflect intracellular pH as well as CO2 generation (Bevensee et al., 2000); therefore, bicarbonate signaling pathways would respond to a wide variety of cellular transitions. Immunostaining revealed that sAC is present at mitochondria, centrioles, mitotic spindles, and mid-bodies (Zippin et al., 2003), suggesting the existence of multiple cAMP signaling microdomains within a single cell. A remaining challenge will be to determine whether sAC molecules in these different microdomains are subject to independent and unique modes of regulation, permitting a variety of distinct responses independently mediated by the same second messenger.
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Materials and methods |
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Immunocytochemistry
Cells or nuclei were washed in PBS and fixed for either 30 min in 4% PFA and permeabilized in 0.1% Triton X-100 or fixed for 15 min in 2% PFA and permeabilized in 0.05% Triton X-100. Liver from adult rat was rapidly excised, placed between two thinly sliced pieces of bovine liver, and snap frozen in isopentane cooled with liquid nitrogen. 6-µM-thick cryosections were collected on superfrost slides (Fisher Scientific) and stained within 1 d of sectioning. Tissue was fixed for 30 min in 4% PFA and permeabilized in 0.1% Triton X-100 for 15 min. All samples were blocked in 2% BSA for at least 1 h. Cells or tissues were stained with anti-sAC R41 or R52 biotinylated mAbs or R21 mAb (1:100) generated against human 48-kD isoform of sAC (sACt) antigen as described previously (Zippin et al., 2003), anti-PKA regulatory subunit (RI and RII
) polyclonal antisera (1:100; Chemicon and Cedarlane Laboratories Limited) or mAbs (Becton Dickinson), and anti-CREB or antiphospho-CREB polyclonal antisera (1:500; Cell Signaling Technologies) overnight in 2% BSA, 0.01% Triton X-100; washed three times for 10 min each in 2% BSA, 0.01% Triton X-100; stained for 1 h at RT with goat antirabbit Alexa Fluor 488, goat antimouse Alexa Fluor 568, or goat antimouse Alexa Fluor 594 (Molecular Probes); treated with DAPI for 5 min or To-Pro 3 (1:500; Molecular Probes) for 15 min; and washed and mounted with gelvatol/DABCO (Sigma-Aldrich).
For phospho-CREB immunolocalization, cells or nuclei were fixed in 4% PFA for 30 min, permeabilized in 0.1% Triton X-100 for 15 min, blocked for at least 1 h in 3% BSA, and immunostained using phospho-CREB polyclonal antisera (1:500; Cell Signaling Technologies) overnight at 4°C. Staining was visualized by incubation with goat antirabbit Alexa Fluor 488 (Molecular Probes) for 1 h at RT, treated with DAPI for 5 min, and washed and mounted with gelvatol/DABCO (Sigma-Aldrich). Fluorescent images were recorded by a digital camera (Hamamatsu) connected to an inverted epifluorescent microscope (Nikon). Images were taken at the same exposure time and gain, and all photographic manipulations were performed equally. Phospho-CREBpositive nuclei were quantified in multiple fields from each stained slide by a blinded experimenter.
Confocal images were acquired with a confocal system (model LSM 510; Carl Zeiss MicroImaging, Inc.). Goat antirabbit Alexa Fluor 488 was excited with a 488-nM Kr/Ar laser, goat antimouse Alexa Fluor 568 was excited with a 568-nm Kr/Ar laser, and To-Pro 3 was excited with a 633-nm Kr/Ar laser.
Isolation of nuclei
Nuclei were isolated by cellular lysis followed by differential centrifugation (Spector et al., 1998) through OptiPrep (Axis-Shield). HeLa cells grown in suspension were lysed by detergent treatment in TM-2 buffer (0.01 M Tris-HCl, pH 7.4, 1.5 mM MgCl2, 150 mM NaCl, 0.5 mM PMSF, 10 µg/ml apoprotin, and 10 µg/ml leupeptin) containing 100 µg/ml of digitonin followed by a 1,000-g spin. Supernatant (S1) was removed and the pellet was resuspended in 0.25 M sucrose, 25 mM KCl, 30 mM MgCl2, and 20 mM Tris-HCl, pH 7.8. The resuspended pellet and 60% OptiPrep iodixanol were mixed (30% OptiPrep final) and centrifuged at 10,000 g for 20 min. The supernatant was removed and the nuclei-enriched pellet (P2) was resuspended in TM-2 buffer without detergent.
CREB phosphorylation and adenylyl cyclase assays
Equal aliquots of nuclei-enriched P2 preparations were incubated in 50 µl of the final volume of 100 mM Tris, pH 7.2, 10 mM MgCl2, and 5 mM ATP for CREB phosphorylation and 100 mM Tris, pH 7.2, 10 mM MgCl2, 5 mM ATP, and 0.5 mM IBMX for adenylyl cyclase assay with the indicated additions for 10 min (CREB phosphorylation) or 15 min (adenylyl cyclase) at 37°C. Reactions were stopped by the addition of 20 µl of SDS sample buffer (CREB phosphorylation) or by being placed into a 100°C heat block for 3 min (adenylyl cyclase).
For whole cell and isolated nuclei CREB phosphorylation assays, equal cell or nuclear equivalents were separated under reducing conditions using a 10% SDS-PAGE, transferred to a PVDF membrane, and probed for CREB (rabbit polyclonal antiserum; Upstate Biotechnology) and phosphorylated CREB (rabbit polyclonal antiserum; Upstate Biotechnology). HRP-conjugated secondary antibodies were used, and bands were visualized using ECL. Image analysis software (model Fluorchem 8800; Alpha Innotech) was used to quantitate Western results. Intensities of phospho-CREB bands were normalized to total CREB.
cAMP produced in the cyclase assays was detected using a competition-based assay with [3H]cAMP (Amersham Biosciences) and compared with a cAMP standard curve for quantitation.
Inhibitor profiles were determined by adenylyl cyclase assay (Assay Designs, Inc.) using purified sAC protein (Litvin et al., 2003) in the presence of 10 mM NaHCO3, 0.5 mM CaCl2, 10 mM MgCl2, and 10 mM ATP or a mixture of purified catalytic domains, C1 and C2, from Type VII tmAC (Yan and Tang, 2002) in the presence of 5 mM MgCl2 and 1 mM ATP as previously described.
Quantitation of isolated nuclei (P2 fraction) immunocytochemistry
Nuclei were treated with Mg2+-ATP alone or in combination with bicarbonate, forskolin, or 8-Br-cAMP for 10 min, spread on a chilled slide, stored at -20°C, and immunostained using phospho-CREBspecific antisera as described. Nuclei were also treated with DAPI to differentiate intact nuclei from membrane ghosts. DAPI-positive nuclei were scored for phosho-CREB immunofluorescence. Nuclei with detectable staining (Fig. 7 A, NaHCO3) were considered positive for CREB phosphorylation, whereas nuclei with no detectable staining (Fig. 7 A, Basal) were counted as negative. Multiple microscopic fields were photographed for each condition, and data was combined from three to five separate experiments.
Western analysis
Equal cell equivalents, unless otherwise noted, were separated under reducing conditions using a 10% SDS-PAGE, transferred to PVDF membrane, and blocked in 5% milk. The blots were probed with antibodies against either NaK ATPase (monoclonal, 1:50; Santa Cruz Biotechnology, Inc.), histone H1 (monoclonal, 1:100; Santa Cruz Biotechnology, Inc.), cytochrome oxidase subunit III (monoclonal, 2 µg/ml; Molecular Probes), ß-tubulin (monoclonal, 1:1000; Sigma-Aldrich), sAC (R21 mAb, 1:500), monoclonal RI or RII
antibodies (1:250; Becton Dickinson), or polyclonal RI
or RII
antisera (1:5000; Chemicon) overnight. HRP-conjugated secondary antibodies were used and bands were visualized using ECL.
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Acknowledgments |
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This work was supported by grants from the National Institutes of Health (AR32147 to D.A. Fischman, GM62328 and HD42060 to J. Buck, HD38722 to L.R. Levin, and MSTP-GM07739 to J.H. Zippin), the Ellison Medical Foundation (to J. Buck), the American Diabetes Association (to J.H. Zippin), and the Barbara and Stephen Friedman Fellowship Endowment (to J.H. Zippin).
Submitted: 21 November 2003
Accepted: 2 January 2004
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