Inhibition of Nuclear Import of Calcineurin Prevents Myocardial Hypertrophy
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Matthias Hallhuber, Natalie Burkard, Ron
参见附件。
the Department of Medicine I (M.H., N.B., R.W., O.R.)
Institute of Physiology (K.S.)
Rudolf-Virchow-Center (S.E.), DFG-Research Center for Experimental Biomedicine, University of Wuerzburg, Germany
University Department of Medicine (M.H.B., L.N.), Manchester Royal Infirmary, UK
the Institute of Experimental and Clinical Pharmacology and Toxicology (L.H.), University of Freiburg, Germany.
Abstract
The time that transcription factors remain nuclear is a major determinant for transcriptional activity. It has recently been demonstrated that the phosphatase calcineurin is translocated to the nucleus with the transcription factor nuclear factor of activated T cells (NF-AT). This study identifies a nuclear localization sequence (NLS) and a nuclear export signal (NES) in the sequence of calcineurin. Furthermore we identified the nuclear cargo protein importin1 to be responsible for nuclear translocation of calcineurin. Inhibition of the calcineurin/importin interaction by a competitive peptide (KQECKIKYSERV), which mimicked the calcineurin NLS, prevented nuclear entry of calcineurin. A noninhibitory control peptide did not interfere with the calcineurin/importin binding. Using this approach, we were able to prevent the development of myocardial hypertrophy. In angiotensin II-stimulated cardiomyocytes, [3H]-leucine incorporation (159%±9 versus 111%±11; P<0.01) and cell size were suppressed significantly by the NLS peptide compared with a control peptide. The NLS peptide inhibited calcineurin/NF-AT transcriptional activity (227%±11 versus 133%±8; P<0.01), whereas calcineurin phosphatase activity was unaffected (298%±9 versus 270%±11; P=NS). We conclude that calcineurin is not only capable of dephosphorylating NF-AT, thus enabling its nuclear import, but the presence of calcineurin in the nucleus is also important for full NF-AT transcriptional activity.
Key Words: angiotensin II calcineurin gene regulation hypertrophy NF-AT nuclear-localizing signals
Introduction
The calcineurin/nuclear factor of activated T cells (NF-AT) signaling cascade is a crucial transducer of cellular function. It has recently emerged that in addition to the transcription factor NF-AT, the phosphatase calcineurin is also translocated to the nucleus.1–4 Our traditional understanding of calcineurin activation via sustained high Ca2+ levels5,6 was also advanced by recent findings from our laboratory that showed that calcineurin is activated by proteolysis of the C-terminal autoinhibitory domain.1 This leads to the constitutive activation and nuclear translocation of calcineurin. Calcineurin is therefore not only responsible for dephosphorylating NF-AT in the cytosol, thus enabling its nuclear import, but its presence in the nucleus is also significant in ensuring the full transcriptional activity of NF-AT.7
The formation of complexes between transcription factors and DNA regulates the transcriptional process. Therefore, the time that transcription factors remain nuclear is a major determinant of transcriptional activity. The movement of proteins more than 40 kDa into and out of the nucleus is governed by the nuclear pore complex (NPC), a multisubunit structure embedded in the nuclear envelope.8 Transcription factors and enzymes that regulate the activity of these proteins are shuttled across the nuclear envelope by proteins that recognize nuclear localization signals (NLS) and nuclear export signals (NESs) within these transcription factors. The positively charged NLSs are bound by importins and/or (also called karyopherins), which tether cargo to the cytosolic face of the NPC and facilitate translocation of proteins into the nucleus. Likewise, the Crm1 protein, also referred to as exportin, mediates the transfer of proteins out of the nucleus.9 The ability of the nuclear import and export machinery to access a NLS or NES is often dictated by signaling events that expose or mask these regulatory sequences.10
In this study, we investigated the precise mechanisms of calcineurin nuclear import and export.
Materials and Methods
Expression Constructs
Epitope tagged derivatives of calcineurin A, containing N-terminal enhanced green fluorescent protein (EGFP), were generated using the mammalian expression vector pEGFP-C3 (BD Biosciences/Clontech). The following C-terminal truncated mutants were amplified by PCR and cloned into the XbaI and XhoI sites of the pEGFP-C3 plasmid: CnA(1 to 525), CnA(1–485), CnA(1 to 465), CnA(1 to 445), CnA(1 to 425), CnA(1 to 415), CnA(171 to 190), and CnA(420 to 434). The generation of the FLAG-tagged calcineurin has been described previously.11
NLS Peptide and Control Peptide
The NLS peptide (sense) and the control peptide (nonsense) were synthesized by Genosphere Biotechnologies (Paris, France). To improve the import into the cells, a hydrophobic membrane permeable sequence (MPS)12 was attached to the N terminus. The NLS peptide mimicked the amino acid (aa) sequence of calcineurin A from aa 172 to 183. In the control peptide, the positive charged amino acids (position 172, 176, 178, and 182) were replaced by uncharged alanine and tyrosine.
Myocardial Infarction and Aortic Banding in Mice
For a detailed description, see the online data supplement, available at http://circres.ahajournals.org.
Preparation of Neonatal Rat Cardiomyocytes and Cell Culture Experiments
Neonatal rat cardiomyocytes of Wistar rats (Harlan-Winkelmann, Borchen, Germany) were isolated as described previously.13 Cells were resuspended in minimum essential medium (MEM) with 1% FCS (MEM/1). HeLa cells were cultured in DMEM with 10% FCS and 100 U/mL penicillin and 100 μg/mL streptomycin. All supplements were obtained from Sigma-Aldrich.
Transfection of Cell Cultures and Treatment of Cell Cultures
Neonatal rat cardiomyocytes were transfected with Lipofectamine (Invitrogen Life Technologies), 48 hours after preparation, on 6-well plates at a density of 1x106 cells per well or chamber slides at a density of 700 000 cells per cavity. Transfections were performed as described by the manufacturer. Cells were treated according to the respective experiments with the following chemicals: angiotensin II (Ang II) (10 μmol/L), phenylephrine (PE) (10 μmol/L), calpeptin (10 μmol/L), leptomycin B (LMB) (1 μmol/L), and NLS or control peptides (1 μmol/L). HeLa cells were transfected 24 hours after trypsination with GenePorter2 (Gene Therapy Systems) on 100-mm dishes, 6-well plates, or chamber slides at a confluence of 70% to 80%.
Calcineurin Enzymatic Activity and Protein Synthesis
Calcineurin-dependent NF-AT activity was determined using a luciferase assay according to the protocol of the manufacture (Promega). The NF-AT reporter plasmid pNP3-luci was used, which contains the Il-2 promoter in front of luciferase, whereas the promoter in the control plasmid pNP1-luci is in the reverse direction. To determine the CnA phosphatase activity, a commercial kit (CnA kit assay AK-816; Biomol, Hamburg, Germany) was used as described previously, with minor modifications.14 The RII-phosphopeptide (Biomol) was used as a specific substrate for calcineurin (PP2B). For measurement of cellular protein synthesis, the amount of incorporated [3H]-marked leucine was measured using a -counter in counts per milliliter per minute (cpm). The change in protein synthesis is expressed as a percentage of the cpm:DNA concentration ratio in unstimulated cells, which was taken as 100%. A detailed description is found in the online data supplement.
Western Blotting, Coimmunoprecipitation, and Immunostaining
Proteins were visualized with the ECL kit (GE Healthcare) according to the instructions of the manufacture. To analyze brain natriuretic peptide (BNP) expression an anti-BNP antibody was used (1:200; Biotrend, 1505-0639). For coimmunoprecipitation (Co-IP) experiments, HeLa cells were used according to a standard protocol (Immunoprecipitation Starter Pack, GE Healthcare). Protein/antibody complexes were precipitated with a mixture of 25 μL of protein A and protein G Sepharose beads for 1 hour at 4°C. To detect the CnA fragments, an anti-GFP (1:500; ab5450, Abcam) antibody was used. The subcellular distribution of calcineurin was determined by immunostaining (Figure IVB in the online data supplement). Antibodies used were anti-CnA antibody (StressGen, SPA-610), troponin I-specific antibody (Santa Cruz Biotechnology, sc15368), anti-FLAG antibody (Acris, DP3002), and Cy3/Cy2-labeled goat anti-rabbit IgG (The Jackson Laboratory (each 1:500).
Statistics
All data are presented as mean±SEM. Statistical analyses were performed using Student t test, significance was assigned a value of P<0.05 () and P<0.01 (). Nonsignificant differences are expressed as NS.
Results
In Vivo Nuclear Translocation of Calcineurin
We recently identified that posttranslational modification, specifically proteolysis of the autoinhibitory domain (AID), leads to activation of calcineurin and its strong nuclear translocation.1 The calpain-mediated cleavage of the C-terminal AID and the causative link to myocardial hypertrophy were demonstrated in human myocardial tissue. Here we demonstrate the nuclear translocation of CnA in different animal models of diseased myocardium (Figure 1). In wild-type mice (sham), there was a predominant cytosolic distribution of CnA, whereas in mice that underwent aortic banding or myocardial infarction, we observed a strong nuclear localization of CnA in the hypertrophied myocardium after 4 weeks similar changes could be demonstrated already after 2 days (supplemental Figure III). Nuclear accumulation of calcineurin was observed in 82±13% (P<0.01) of cardiomyocytes in pathological myocardial hypertrophy. In contrast, nuclear calcineurin was not observed in normal myocardium. For evaluation, >100 cells of 6 animals per group were counted.
Time Course
To assess whether CnA import into the nucleus is a chronic phenomenon or an acute response to a myocardial insult, we investigated the time course of CnA shuttling. A plasmid encoding EGFP-tagged full-length CnA was transfected into neonatal rat cardiomyocytes. Cells were stimulated with Ang II (10 μmol/L). Confocal microscopy revealed onset of nuclear translocation of calcineurin after 2 hours. After 4 hours of Ang II stimulation, CnA was predominantly nuclear (Figure 2A). After 6 hours, maximum of intensity of the EGFP-calcineurin signal was seen in the nucleus. We observed nuclear accumulation of calcineurin in >90% of the transfected cells (supplemental Figure I). Similarly, 2 hours after removal of Ang II from the medium, CnA was homogenously distributed in the cytosol and the nucleus and after 4 hours, CnA was localized in the perinuclear region. Six hours after removal of the stimulus, CnA was localized completely in the cytosol again (Figure 2B). To protect CnA from calpain-mediated proteolysis, which would cause constitutive activation of CnA and therefore persistent nuclear translocation, all experiments were performed in the presence of a membrane-permeable calpain inhibitor.1
Identification of a Nuclear Localization Sequence and the Corresponding Importin
To define the regions of calcineurin that are required for nuclear import different EGFP- or FLAG-tagged calcineurin deletion mutants (Figure 3A) were screened to assess for those that entered the nucleus and those that remained cytosolic. In general, deletion of the autoinhibitory domain led to nuclear translocation and deletion of the region starting with aa 173 (within the putative NLS) prevented calcineurin from entering the nucleus (Figure 3B). Sequence comparisons with known NLSs of other proteins enabled further delineation of the putative NLS region to the sequence from aa 171 to 190 of CnA. Fusion of this aa 171 to 190 fragment to the EGFP backbone resulted in translocation of the EGFP/NLS fusion protein into the nucleus, whereas the pure EGFP backbone remained cytosolic. Although full-length CnA resided in the cytosol, it was translocated into the nucleus after Ang II stimulation attributable to uncovering of the catalytic subunit and probably of the putative NLS. In contrast, deletion mutants aa 2 to 173 and aa 3 to 143, both lacking the putative NLS, remained exclusively cytosolic despite Ang II stimulation (Figure 3C).
Importin1 has been shown to bind the "nonclassical" NLS of different cargo proteins.15 Interactions between the CnA mutants and importin1 were therefore assessed to determine whether the functionally defined NLS physically interacts with importin1. As demonstrated by Co-IP, importin1 binds to full-length calcineurin (CnA[1 to 525]) and also to the deletion mutants CnA(1 to 415) and CnA(1 to 44) (Figure 4A).
To demonstrate further that the identified NLS in CnA is essential for the nuclear import of calcineurin, a peptide competition assay was used to prevent importin1/CnA binding. A peptide containing the putative NLS sequence of calcineurin (AAVALLPAVLAALAAKQECKIKYSERV) was synthesized and added to the medium. (Small capital letters give N-terminal extension to increase membrane permeability16; NLS sequence is underlined.) In control experiments, a nonsense peptide (control peptide) (AAVALLPAVLAALAAAQECAIAYSEYV) was used. Addition of the synthetic NLS peptide (1 μmol/L) saturated the binding domain of importin1 for CnA and, therefore, prevented CnA binding to importin1.
Specifically, the interaction domain was mapped to the region aa 171 to 190 as evidenced by the ability of the NLS peptide to abolish the interaction between importin1 and CnA completely. These data indicate that the NLS identified by functional analyses also mediates physical interactions between importin1 and calcineurin (Figure 4A).
Inhibition of this interaction suppressed nuclear import of a constitutively active calcineurin mutant (CnA[1 to 415]). The noninhibitory control peptide (1 μmol/L) did not interfere with the calcineurin/importin binding; accordingly, nuclear translocation of CnA was not inhibited (Figure 4B). The results were identical in cells treated with Ang II (10 μmol/L) and with the -adrenergic receptor agonist phenylephrine (10 μmol/L).
Detection of a Nuclear Export Signal
To screen the calcineurin sequence for nuclear export signals we used the NetNES 1.1 server (http://www.cbs.dtu.dk/services/NetNES). This program predicts leucine-rich NESs in eukaryotic proteins. Our input was the C terminus downstream of aa 410 of CnA. In this region, a typical NES was predicted between aa 420 and 434 (Figure 3A). To exactly identify the sequence in CnA that controls nuclear export, serial carboxy-terminally truncated CnA mutants with an N-terminal EGFP tag were generated and examined by confocal microscopy (Figures 3A and 5A). Experiments were performed in the presence of a calpain inhibitor to prevent calpain induced cleavage of the AID and to ensure functional integrity of calcineurin. Cells were stimulated with Ang II for 12 hours to achieve nuclear entry of CnA, followed by removal of the stimulus to promote nuclear export. Full-length CnA(1 to 525) was relocalized exclusively to the cytosol of transfected cardiomyocytes after removal of the stimulus. An extended deletion variant (1 to 415) was not able to leave the nucleus any more (Figure 5A).
These results suggest that sequences in the region downstream of aa 415 regulate nuclear export. Consistent with these findings and sequence comparisons with known NES sites, a CnA mutant lacking aa 420 to 434 remained exclusively nuclear after removal of the stimulus. Inhibition of calpain did not influence this result as the calpain cleavage site (at aa 424) was deleted in this mutation variant (Figure 5A).
To address whether CnA nuclear export is mediated by the export protein Crm1, experiments using the Crm1-specific inhibitor, LMB, were performed. Agonist-dependent nuclear import of full-length CnA was achieved by Ang II stimulation. Calpeptin was added to prevent proteolysis of CnA. The addition of LMB to prevent Crm1-mediated export suppressed nuclear export of CnA. Interestingly, LMB alone resulted in nuclear accumulation of calcineurin after 48 hours. There is, of course, a continuous shuttling of calcineurin across the nuclear membrane even under basal conditions. This supports the hypothesis that nucleocytoplasmic shuttling of CnA is coupled to an NES localized within the region containing aa 423 and 434 and is mediated by Crm1 (Figure 5B).
In vivo studies of pathological myocardial hypertrophy show that proteolysis of the calcineurin autoinhibitory domain at aa 424 results in a constitutively active calcineurin mutant lacking both the AID (aa 468 to 490) and the NES (aa 423 to 433). To determine whether loss of the AID or disruption of the NES is responsible for strong nuclear accumulation of CnA, the nuclear import and export qualities of an EGFP-tagged CnA mutant with the deletion of the NES, CnA(420 to 434), was investigated. In this case calcineurin resided in the cytosol. Stimulation of the transfected cells with Ang II resulted in subsequent translocation of CnA into the nucleus. Based on these results, we conclude that the AID not only blocks the catalytic activity of CnA but also masks the NLS. Removal of the AID via a conformational change in calcineurin following Ca2+ activation or by proteolysis of the AID leads to exposure of the NLS and resultant nuclear translocation of CnA. Subsequent removal of the stimulating Ang II agent from the medium resulted in a nuclear localization of the CnA(420 to 434) mutant, as the lack of the NES made it impossible for Crm1 to interact with CnA and to transport it back to the cytosol (Figure 6). Loss of the C-terminal part of CnA would, therefore, appear to regulate nuclear shuttling of CnA at the level of both nuclear import and export. Deprivation of the AID promotes import via importin1, and loss of the NES hinders nuclear export via Crm1 mediated mechanisms.
Inhibition of Myocardial Hypertrophy by a NLS Corresponding Peptide
We examined phosphatase activity, transcriptional activity, protein synthesis, cell size, and markers of myocardial hypertrophy in response to the peptide-related inhibition of CnA nuclear import. Phosphatase activity was assessed using a specific substrate (RII) for CnA.14 Cardiomyocytes were stimulated with Ang II (10 μmol/L), and CnA phosphatase activity was measured in the presence of the NLS peptide (1 μmol/L) or a nonsense control peptide (1 μmol/L). Total CnA phosphatase activity was not affected by inhibition of the access of importin1 to the CnA NLS (298±9% versus 270±11%; n=8; P=NS). Additionally, we assessed NF-ATc2 phosphorylation status because NF-AT is the physiological substrate for calcineurin. In cells that were stimulated with Ang II, there was an increase in dephosphorylated NF-ATc2 (120 kDa) compared with control cells. Addition of the NLS peptide had no significant effect on NF-ATc2 dephosphorylation. This indicates that the NLS peptide had no impact on phosphatase activity of calcineurin (supplemental Figure II). In contrast, transcriptional activity of the CnA/NF-AT signaling pathway was decreased significantly by the NLS peptide in cardiomyocytes stimulated with Ang II (10 μmol/L) (227±11% versus 133±8%; n=8; P<0.05) or with PE (10 μmol/L) (189±10% versus 91±7%; n=8; P<0.05) (Figure 7A). Similarly, myocardial hypertrophy, as evidenced by protein synthesis (159±9% versus 111±11%; n=8; P<0.05) and cell size (1180±91 μm2 versus 744±65 μm2; n=8; P<0.05) (Figure 7B), was suppressed by the NLS peptide. To further investigate the inhibitory effect of the NLS peptide, the expression of brain natriuretic peptide (BNP) as a molecular marker of myocardial hypertrophy was measured. In cardiomyocytes stimulated with Ang II to induce myocardial hypertrophy, the NLS peptide significantly reduced the expression of BNP (163±11% versus 88±8%; n=8; P<0.05) (Figure 7C). Transcriptional activity detected by an NF-AT luciferase reporter plasmid was decreased when nuclear import of CnA was blocked by the NLS peptide in a dose-dependent manner (Figure 7D).
These data indicate that despite full CnA phosphatase activity, CnA was unable to form effective transcriptional complexes. Full transcriptional activity of CnA/NF-AT is achieved only in the presence of nuclear calcineurin. Thus it is clear that calcineurin nuclear translocation is a prerequisite to the formation of effective NF-AT transcriptional complexes (Figure 8).
Discussion
The calcineurin/NF-AT signaling cascade is crucial for T-cell activation and for the development of myocardial hypertrophy. After activation, NF-AT nuclear localization is directly induced by calcineurin-mediated dephosphorylation of multiple conserved serine residues in the N terminus of these proteins, revealing a nuclear localization signal.16 Once dephosphorylated, NF-AT translocates into the nucleus and the transcriptional process begins.
The biological activity of transcription factors is in part regulated by their intracellular localization. In the case of the calcineurin/NF-AT signaling cascade this means inactive (hyperphosphorylated) NF-AT resides in the cytosol and activated (dephosphorylated) NF-AT resides in the nucleus. However, it has also been demonstrated by our group and others that full transcriptional activity of the calcineurin/NF-AT pathway is achieved only when calcineurin is also translocated into the nucleus. The nuclear half-life of NF-AT alone is very short. In the absence of active calcineurin, it is rapidly transported back into the cytoplasm within minutes.4 In this study, we investigated the mechanisms leading to nuclear import and export of calcineurin.
The active transport of proteins into the nucleus requires an array of proteins including nuclear cargo or carrier proteins (called importins or karyopherins, respectively), which in many instances make the primary contact with the classical NLSs of the imported protein.17 Classical NLSs consist of 5 to 11 amino acids. When importin binds to the target protein that contains the classical NLS, the complex interacts with accessory proteins such as importin and the small GTP-binding protein Ran. This complex binds to the nuclear pore and is then transported through it in an energy-dependent manner. Nonclassical NLSs can bind directly to importin , initiating nuclear transport through the nuclear pore complex.18 Similarly, NESs are responsible for binding to export proteins, so-called exportins. Exportins transport their target proteins across the nuclear envelop back into the cytosol. A number of proteins that shuttle across the nuclear membrane have been identified using Crm1 as the export shuttle (eg, NF-AT). Here, we have identified a NLS and a NES in the calcineurin sequence. We have also identified the respective carrier proteins for calcineurin shuttling across the nuclear membrane. Importin1 is responsible for the nuclear import, whereas the export protein Crm1 is required for nuclear export of calcineurin. These findings identify a potentially novel therapeutic strategy to inhibit myocardial hypertrophy. Inhibition of the calcineurin/importin1 interaction would prevent nuclear translocation of calcineurin and subsequently inhibit the full transcriptional activity of the calcineurin/NF-AT signaling pathway. Similar approaches, such as inhibition of the nuclear factor B/importin interaction19–20 and calcineurin/NF-AT interaction21 by competitive peptides, have already been successfully proven. Using this strategy, we synthesized a peptide comprising 12 amino acids that mimicked the NLS sequence of calcineurin and an N-terminal peptide extension of additional 15 amino acids to increase membrane permeability. This peptide was able to suppress calcineurin/importin1 interaction, which subsequently prevented calcineurin nuclear import. The physiological result was blunting of NF-AT transcriptional activity and inhibition of the development of myocardial hypertrophy. In contrast, calcineurin phosphatase activity was unaffected, although assessment of calcineurin phosphatase activity in vivo is often imprecise. As a surrogate the NLS peptide had no impact of NF-AT dephosphorylation.
These results demonstrate that inhibition of the calcineurin/importin interaction by interfering peptides is an effective tool to suppress calcineurin signaling. These results however raise the following question: What is the precise role of calcineurin in the nucleus The transcriptional effector of the CnA/NF-AT system is NF-AT through its DNA-binding domain. NF-AT factors share an imperfect Rel homology domain that is only capable of weak DNA binding in the monomeric or dimeric state. To strengthen NF-AT/DNA interactions, these factors prefer to interact cooperatively with other nuclear transcription factors such as AP-1 (c-Jun/c-Fos), GATA-4, and MEF-2.22 Therefore, calcineurin may act as a transcriptional coactivator. However, competition by calcineurin with the glycogen synthase kinase 3 (GSK3) to ensure further dephosphorylation of NF-AT in the nucleus or at least to prevent rephosphorylation is unlikely to be the major task of nuclear calcineurin, as CnA mutants devoid of phosphatase function also increase transcriptional activity of the CnA/NF-AT signaling pathway when translocated to the nucleus.7 Also, multiple other kinases beside glycogen synthase kinase 3 (GSK-3) such as c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases, casein kinase I (CK1), protein kinase A (PKA), and mitogen-activated protein kinase kinase 1 (MEKK1) (indirectly), all promote rephosphorylation of the serine-rich N terminus of NF-AT factors, enabling Crm1-mediated nuclear export.23–26
Another model of competition between CnA and Crm1 for the nuclear export sequence of NF-AT has also been proposed. It has previously been demonstrated that the nuclear export protein Crm1 is capable of transporting NF-AT out of the nucleus. A constitutive nuclear calcineurin will shift Crm1 off the NES of NF-AT and leave the CnA/NF-AT complex nuclear, thereby enhancing transcriptional output.7,27 Our data show that Crm1 not only exports NF-AT but also calcineurin from the nucleus. To interrupt transcriptional activity of the calcineurin/NF-AT signaling cascade, Crm1 is first required to export calcineurin, so that in a second round Crm1 can access the NESs of NF-AT and subsequently proceed with its nuclear export. This mechanism may be prevented in myocardial hypertrophy by the proteolysis of calcineurin by calpain at aa 424, resulting in a loss of the autoinhibitory domain including the NES. In this scenario, calcineurin remains nuclear because it is inaccessible to the export protein Crm1. These observations suggest that calcineurin function in the nucleus is largely driven via its anti-Crm1 as opposed to anti-GSK3 effects.
As import always precedes export, the inhibition of CnA nuclear import by peptide competition for the binding of the nuclear import protein importin1 presents a sophisticated approach to abolishing the deleterious effects of exaggerated NF-AT transcriptional activity. Nevertheless, assessment of the specific action of the NLS peptide on the calcineurin/NF-AT interaction must be performed before further experiments can be undertaken in vivo.
Acknowledgments
We thank J. M. Redondo (Universidad Autónoma de Madrid, Spain) for providing parts of the plasmid constructs, T. Renné (University of Wuerzburg, Germany) for advice on peptide synthesis, and C. Gebhardt for excellent technical support.
Sources of Funding
This work was supported by grants from the German Research Society (Ri 1085/3-1) and the Interdisziplinares Zentrum für Klinische Forschung Wuerzburg (E-25 to O.R. and S.E.).
Disclosures
None.
Footnotes
Original received March 10, 2006; revision received August 11, 2006; accepted August 16, 2006.
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the Department of Medicine I (M.H., N.B., R.W., O.R.)
Institute of Physiology (K.S.)
Rudolf-Virchow-Center (S.E.), DFG-Research Center for Experimental Biomedicine, University of Wuerzburg, Germany
University Department of Medicine (M.H.B., L.N.), Manchester Royal Infirmary, UK
the Institute of Experimental and Clinical Pharmacology and Toxicology (L.H.), University of Freiburg, Germany.
Abstract
The time that transcription factors remain nuclear is a major determinant for transcriptional activity. It has recently been demonstrated that the phosphatase calcineurin is translocated to the nucleus with the transcription factor nuclear factor of activated T cells (NF-AT). This study identifies a nuclear localization sequence (NLS) and a nuclear export signal (NES) in the sequence of calcineurin. Furthermore we identified the nuclear cargo protein importin1 to be responsible for nuclear translocation of calcineurin. Inhibition of the calcineurin/importin interaction by a competitive peptide (KQECKIKYSERV), which mimicked the calcineurin NLS, prevented nuclear entry of calcineurin. A noninhibitory control peptide did not interfere with the calcineurin/importin binding. Using this approach, we were able to prevent the development of myocardial hypertrophy. In angiotensin II-stimulated cardiomyocytes, [3H]-leucine incorporation (159%±9 versus 111%±11; P<0.01) and cell size were suppressed significantly by the NLS peptide compared with a control peptide. The NLS peptide inhibited calcineurin/NF-AT transcriptional activity (227%±11 versus 133%±8; P<0.01), whereas calcineurin phosphatase activity was unaffected (298%±9 versus 270%±11; P=NS). We conclude that calcineurin is not only capable of dephosphorylating NF-AT, thus enabling its nuclear import, but the presence of calcineurin in the nucleus is also important for full NF-AT transcriptional activity.
Key Words: angiotensin II calcineurin gene regulation hypertrophy NF-AT nuclear-localizing signals
Introduction
The calcineurin/nuclear factor of activated T cells (NF-AT) signaling cascade is a crucial transducer of cellular function. It has recently emerged that in addition to the transcription factor NF-AT, the phosphatase calcineurin is also translocated to the nucleus.1–4 Our traditional understanding of calcineurin activation via sustained high Ca2+ levels5,6 was also advanced by recent findings from our laboratory that showed that calcineurin is activated by proteolysis of the C-terminal autoinhibitory domain.1 This leads to the constitutive activation and nuclear translocation of calcineurin. Calcineurin is therefore not only responsible for dephosphorylating NF-AT in the cytosol, thus enabling its nuclear import, but its presence in the nucleus is also significant in ensuring the full transcriptional activity of NF-AT.7
The formation of complexes between transcription factors and DNA regulates the transcriptional process. Therefore, the time that transcription factors remain nuclear is a major determinant of transcriptional activity. The movement of proteins more than 40 kDa into and out of the nucleus is governed by the nuclear pore complex (NPC), a multisubunit structure embedded in the nuclear envelope.8 Transcription factors and enzymes that regulate the activity of these proteins are shuttled across the nuclear envelope by proteins that recognize nuclear localization signals (NLS) and nuclear export signals (NESs) within these transcription factors. The positively charged NLSs are bound by importins and/or (also called karyopherins), which tether cargo to the cytosolic face of the NPC and facilitate translocation of proteins into the nucleus. Likewise, the Crm1 protein, also referred to as exportin, mediates the transfer of proteins out of the nucleus.9 The ability of the nuclear import and export machinery to access a NLS or NES is often dictated by signaling events that expose or mask these regulatory sequences.10
In this study, we investigated the precise mechanisms of calcineurin nuclear import and export.
Materials and Methods
Expression Constructs
Epitope tagged derivatives of calcineurin A, containing N-terminal enhanced green fluorescent protein (EGFP), were generated using the mammalian expression vector pEGFP-C3 (BD Biosciences/Clontech). The following C-terminal truncated mutants were amplified by PCR and cloned into the XbaI and XhoI sites of the pEGFP-C3 plasmid: CnA(1 to 525), CnA(1–485), CnA(1 to 465), CnA(1 to 445), CnA(1 to 425), CnA(1 to 415), CnA(171 to 190), and CnA(420 to 434). The generation of the FLAG-tagged calcineurin has been described previously.11
NLS Peptide and Control Peptide
The NLS peptide (sense) and the control peptide (nonsense) were synthesized by Genosphere Biotechnologies (Paris, France). To improve the import into the cells, a hydrophobic membrane permeable sequence (MPS)12 was attached to the N terminus. The NLS peptide mimicked the amino acid (aa) sequence of calcineurin A from aa 172 to 183. In the control peptide, the positive charged amino acids (position 172, 176, 178, and 182) were replaced by uncharged alanine and tyrosine.
Myocardial Infarction and Aortic Banding in Mice
For a detailed description, see the online data supplement, available at http://circres.ahajournals.org.
Preparation of Neonatal Rat Cardiomyocytes and Cell Culture Experiments
Neonatal rat cardiomyocytes of Wistar rats (Harlan-Winkelmann, Borchen, Germany) were isolated as described previously.13 Cells were resuspended in minimum essential medium (MEM) with 1% FCS (MEM/1). HeLa cells were cultured in DMEM with 10% FCS and 100 U/mL penicillin and 100 μg/mL streptomycin. All supplements were obtained from Sigma-Aldrich.
Transfection of Cell Cultures and Treatment of Cell Cultures
Neonatal rat cardiomyocytes were transfected with Lipofectamine (Invitrogen Life Technologies), 48 hours after preparation, on 6-well plates at a density of 1x106 cells per well or chamber slides at a density of 700 000 cells per cavity. Transfections were performed as described by the manufacturer. Cells were treated according to the respective experiments with the following chemicals: angiotensin II (Ang II) (10 μmol/L), phenylephrine (PE) (10 μmol/L), calpeptin (10 μmol/L), leptomycin B (LMB) (1 μmol/L), and NLS or control peptides (1 μmol/L). HeLa cells were transfected 24 hours after trypsination with GenePorter2 (Gene Therapy Systems) on 100-mm dishes, 6-well plates, or chamber slides at a confluence of 70% to 80%.
Calcineurin Enzymatic Activity and Protein Synthesis
Calcineurin-dependent NF-AT activity was determined using a luciferase assay according to the protocol of the manufacture (Promega). The NF-AT reporter plasmid pNP3-luci was used, which contains the Il-2 promoter in front of luciferase, whereas the promoter in the control plasmid pNP1-luci is in the reverse direction. To determine the CnA phosphatase activity, a commercial kit (CnA kit assay AK-816; Biomol, Hamburg, Germany) was used as described previously, with minor modifications.14 The RII-phosphopeptide (Biomol) was used as a specific substrate for calcineurin (PP2B). For measurement of cellular protein synthesis, the amount of incorporated [3H]-marked leucine was measured using a -counter in counts per milliliter per minute (cpm). The change in protein synthesis is expressed as a percentage of the cpm:DNA concentration ratio in unstimulated cells, which was taken as 100%. A detailed description is found in the online data supplement.
Western Blotting, Coimmunoprecipitation, and Immunostaining
Proteins were visualized with the ECL kit (GE Healthcare) according to the instructions of the manufacture. To analyze brain natriuretic peptide (BNP) expression an anti-BNP antibody was used (1:200; Biotrend, 1505-0639). For coimmunoprecipitation (Co-IP) experiments, HeLa cells were used according to a standard protocol (Immunoprecipitation Starter Pack, GE Healthcare). Protein/antibody complexes were precipitated with a mixture of 25 μL of protein A and protein G Sepharose beads for 1 hour at 4°C. To detect the CnA fragments, an anti-GFP (1:500; ab5450, Abcam) antibody was used. The subcellular distribution of calcineurin was determined by immunostaining (Figure IVB in the online data supplement). Antibodies used were anti-CnA antibody (StressGen, SPA-610), troponin I-specific antibody (Santa Cruz Biotechnology, sc15368), anti-FLAG antibody (Acris, DP3002), and Cy3/Cy2-labeled goat anti-rabbit IgG (The Jackson Laboratory (each 1:500).
Statistics
All data are presented as mean±SEM. Statistical analyses were performed using Student t test, significance was assigned a value of P<0.05 () and P<0.01 (). Nonsignificant differences are expressed as NS.
Results
In Vivo Nuclear Translocation of Calcineurin
We recently identified that posttranslational modification, specifically proteolysis of the autoinhibitory domain (AID), leads to activation of calcineurin and its strong nuclear translocation.1 The calpain-mediated cleavage of the C-terminal AID and the causative link to myocardial hypertrophy were demonstrated in human myocardial tissue. Here we demonstrate the nuclear translocation of CnA in different animal models of diseased myocardium (Figure 1). In wild-type mice (sham), there was a predominant cytosolic distribution of CnA, whereas in mice that underwent aortic banding or myocardial infarction, we observed a strong nuclear localization of CnA in the hypertrophied myocardium after 4 weeks similar changes could be demonstrated already after 2 days (supplemental Figure III). Nuclear accumulation of calcineurin was observed in 82±13% (P<0.01) of cardiomyocytes in pathological myocardial hypertrophy. In contrast, nuclear calcineurin was not observed in normal myocardium. For evaluation, >100 cells of 6 animals per group were counted.
Time Course
To assess whether CnA import into the nucleus is a chronic phenomenon or an acute response to a myocardial insult, we investigated the time course of CnA shuttling. A plasmid encoding EGFP-tagged full-length CnA was transfected into neonatal rat cardiomyocytes. Cells were stimulated with Ang II (10 μmol/L). Confocal microscopy revealed onset of nuclear translocation of calcineurin after 2 hours. After 4 hours of Ang II stimulation, CnA was predominantly nuclear (Figure 2A). After 6 hours, maximum of intensity of the EGFP-calcineurin signal was seen in the nucleus. We observed nuclear accumulation of calcineurin in >90% of the transfected cells (supplemental Figure I). Similarly, 2 hours after removal of Ang II from the medium, CnA was homogenously distributed in the cytosol and the nucleus and after 4 hours, CnA was localized in the perinuclear region. Six hours after removal of the stimulus, CnA was localized completely in the cytosol again (Figure 2B). To protect CnA from calpain-mediated proteolysis, which would cause constitutive activation of CnA and therefore persistent nuclear translocation, all experiments were performed in the presence of a membrane-permeable calpain inhibitor.1
Identification of a Nuclear Localization Sequence and the Corresponding Importin
To define the regions of calcineurin that are required for nuclear import different EGFP- or FLAG-tagged calcineurin deletion mutants (Figure 3A) were screened to assess for those that entered the nucleus and those that remained cytosolic. In general, deletion of the autoinhibitory domain led to nuclear translocation and deletion of the region starting with aa 173 (within the putative NLS) prevented calcineurin from entering the nucleus (Figure 3B). Sequence comparisons with known NLSs of other proteins enabled further delineation of the putative NLS region to the sequence from aa 171 to 190 of CnA. Fusion of this aa 171 to 190 fragment to the EGFP backbone resulted in translocation of the EGFP/NLS fusion protein into the nucleus, whereas the pure EGFP backbone remained cytosolic. Although full-length CnA resided in the cytosol, it was translocated into the nucleus after Ang II stimulation attributable to uncovering of the catalytic subunit and probably of the putative NLS. In contrast, deletion mutants aa 2 to 173 and aa 3 to 143, both lacking the putative NLS, remained exclusively cytosolic despite Ang II stimulation (Figure 3C).
Importin1 has been shown to bind the "nonclassical" NLS of different cargo proteins.15 Interactions between the CnA mutants and importin1 were therefore assessed to determine whether the functionally defined NLS physically interacts with importin1. As demonstrated by Co-IP, importin1 binds to full-length calcineurin (CnA[1 to 525]) and also to the deletion mutants CnA(1 to 415) and CnA(1 to 44) (Figure 4A).
To demonstrate further that the identified NLS in CnA is essential for the nuclear import of calcineurin, a peptide competition assay was used to prevent importin1/CnA binding. A peptide containing the putative NLS sequence of calcineurin (AAVALLPAVLAALAAKQECKIKYSERV) was synthesized and added to the medium. (Small capital letters give N-terminal extension to increase membrane permeability16; NLS sequence is underlined.) In control experiments, a nonsense peptide (control peptide) (AAVALLPAVLAALAAAQECAIAYSEYV) was used. Addition of the synthetic NLS peptide (1 μmol/L) saturated the binding domain of importin1 for CnA and, therefore, prevented CnA binding to importin1.
Specifically, the interaction domain was mapped to the region aa 171 to 190 as evidenced by the ability of the NLS peptide to abolish the interaction between importin1 and CnA completely. These data indicate that the NLS identified by functional analyses also mediates physical interactions between importin1 and calcineurin (Figure 4A).
Inhibition of this interaction suppressed nuclear import of a constitutively active calcineurin mutant (CnA[1 to 415]). The noninhibitory control peptide (1 μmol/L) did not interfere with the calcineurin/importin binding; accordingly, nuclear translocation of CnA was not inhibited (Figure 4B). The results were identical in cells treated with Ang II (10 μmol/L) and with the -adrenergic receptor agonist phenylephrine (10 μmol/L).
Detection of a Nuclear Export Signal
To screen the calcineurin sequence for nuclear export signals we used the NetNES 1.1 server (http://www.cbs.dtu.dk/services/NetNES). This program predicts leucine-rich NESs in eukaryotic proteins. Our input was the C terminus downstream of aa 410 of CnA. In this region, a typical NES was predicted between aa 420 and 434 (Figure 3A). To exactly identify the sequence in CnA that controls nuclear export, serial carboxy-terminally truncated CnA mutants with an N-terminal EGFP tag were generated and examined by confocal microscopy (Figures 3A and 5A). Experiments were performed in the presence of a calpain inhibitor to prevent calpain induced cleavage of the AID and to ensure functional integrity of calcineurin. Cells were stimulated with Ang II for 12 hours to achieve nuclear entry of CnA, followed by removal of the stimulus to promote nuclear export. Full-length CnA(1 to 525) was relocalized exclusively to the cytosol of transfected cardiomyocytes after removal of the stimulus. An extended deletion variant (1 to 415) was not able to leave the nucleus any more (Figure 5A).
These results suggest that sequences in the region downstream of aa 415 regulate nuclear export. Consistent with these findings and sequence comparisons with known NES sites, a CnA mutant lacking aa 420 to 434 remained exclusively nuclear after removal of the stimulus. Inhibition of calpain did not influence this result as the calpain cleavage site (at aa 424) was deleted in this mutation variant (Figure 5A).
To address whether CnA nuclear export is mediated by the export protein Crm1, experiments using the Crm1-specific inhibitor, LMB, were performed. Agonist-dependent nuclear import of full-length CnA was achieved by Ang II stimulation. Calpeptin was added to prevent proteolysis of CnA. The addition of LMB to prevent Crm1-mediated export suppressed nuclear export of CnA. Interestingly, LMB alone resulted in nuclear accumulation of calcineurin after 48 hours. There is, of course, a continuous shuttling of calcineurin across the nuclear membrane even under basal conditions. This supports the hypothesis that nucleocytoplasmic shuttling of CnA is coupled to an NES localized within the region containing aa 423 and 434 and is mediated by Crm1 (Figure 5B).
In vivo studies of pathological myocardial hypertrophy show that proteolysis of the calcineurin autoinhibitory domain at aa 424 results in a constitutively active calcineurin mutant lacking both the AID (aa 468 to 490) and the NES (aa 423 to 433). To determine whether loss of the AID or disruption of the NES is responsible for strong nuclear accumulation of CnA, the nuclear import and export qualities of an EGFP-tagged CnA mutant with the deletion of the NES, CnA(420 to 434), was investigated. In this case calcineurin resided in the cytosol. Stimulation of the transfected cells with Ang II resulted in subsequent translocation of CnA into the nucleus. Based on these results, we conclude that the AID not only blocks the catalytic activity of CnA but also masks the NLS. Removal of the AID via a conformational change in calcineurin following Ca2+ activation or by proteolysis of the AID leads to exposure of the NLS and resultant nuclear translocation of CnA. Subsequent removal of the stimulating Ang II agent from the medium resulted in a nuclear localization of the CnA(420 to 434) mutant, as the lack of the NES made it impossible for Crm1 to interact with CnA and to transport it back to the cytosol (Figure 6). Loss of the C-terminal part of CnA would, therefore, appear to regulate nuclear shuttling of CnA at the level of both nuclear import and export. Deprivation of the AID promotes import via importin1, and loss of the NES hinders nuclear export via Crm1 mediated mechanisms.
Inhibition of Myocardial Hypertrophy by a NLS Corresponding Peptide
We examined phosphatase activity, transcriptional activity, protein synthesis, cell size, and markers of myocardial hypertrophy in response to the peptide-related inhibition of CnA nuclear import. Phosphatase activity was assessed using a specific substrate (RII) for CnA.14 Cardiomyocytes were stimulated with Ang II (10 μmol/L), and CnA phosphatase activity was measured in the presence of the NLS peptide (1 μmol/L) or a nonsense control peptide (1 μmol/L). Total CnA phosphatase activity was not affected by inhibition of the access of importin1 to the CnA NLS (298±9% versus 270±11%; n=8; P=NS). Additionally, we assessed NF-ATc2 phosphorylation status because NF-AT is the physiological substrate for calcineurin. In cells that were stimulated with Ang II, there was an increase in dephosphorylated NF-ATc2 (120 kDa) compared with control cells. Addition of the NLS peptide had no significant effect on NF-ATc2 dephosphorylation. This indicates that the NLS peptide had no impact on phosphatase activity of calcineurin (supplemental Figure II). In contrast, transcriptional activity of the CnA/NF-AT signaling pathway was decreased significantly by the NLS peptide in cardiomyocytes stimulated with Ang II (10 μmol/L) (227±11% versus 133±8%; n=8; P<0.05) or with PE (10 μmol/L) (189±10% versus 91±7%; n=8; P<0.05) (Figure 7A). Similarly, myocardial hypertrophy, as evidenced by protein synthesis (159±9% versus 111±11%; n=8; P<0.05) and cell size (1180±91 μm2 versus 744±65 μm2; n=8; P<0.05) (Figure 7B), was suppressed by the NLS peptide. To further investigate the inhibitory effect of the NLS peptide, the expression of brain natriuretic peptide (BNP) as a molecular marker of myocardial hypertrophy was measured. In cardiomyocytes stimulated with Ang II to induce myocardial hypertrophy, the NLS peptide significantly reduced the expression of BNP (163±11% versus 88±8%; n=8; P<0.05) (Figure 7C). Transcriptional activity detected by an NF-AT luciferase reporter plasmid was decreased when nuclear import of CnA was blocked by the NLS peptide in a dose-dependent manner (Figure 7D).
These data indicate that despite full CnA phosphatase activity, CnA was unable to form effective transcriptional complexes. Full transcriptional activity of CnA/NF-AT is achieved only in the presence of nuclear calcineurin. Thus it is clear that calcineurin nuclear translocation is a prerequisite to the formation of effective NF-AT transcriptional complexes (Figure 8).
Discussion
The calcineurin/NF-AT signaling cascade is crucial for T-cell activation and for the development of myocardial hypertrophy. After activation, NF-AT nuclear localization is directly induced by calcineurin-mediated dephosphorylation of multiple conserved serine residues in the N terminus of these proteins, revealing a nuclear localization signal.16 Once dephosphorylated, NF-AT translocates into the nucleus and the transcriptional process begins.
The biological activity of transcription factors is in part regulated by their intracellular localization. In the case of the calcineurin/NF-AT signaling cascade this means inactive (hyperphosphorylated) NF-AT resides in the cytosol and activated (dephosphorylated) NF-AT resides in the nucleus. However, it has also been demonstrated by our group and others that full transcriptional activity of the calcineurin/NF-AT pathway is achieved only when calcineurin is also translocated into the nucleus. The nuclear half-life of NF-AT alone is very short. In the absence of active calcineurin, it is rapidly transported back into the cytoplasm within minutes.4 In this study, we investigated the mechanisms leading to nuclear import and export of calcineurin.
The active transport of proteins into the nucleus requires an array of proteins including nuclear cargo or carrier proteins (called importins or karyopherins, respectively), which in many instances make the primary contact with the classical NLSs of the imported protein.17 Classical NLSs consist of 5 to 11 amino acids. When importin binds to the target protein that contains the classical NLS, the complex interacts with accessory proteins such as importin and the small GTP-binding protein Ran. This complex binds to the nuclear pore and is then transported through it in an energy-dependent manner. Nonclassical NLSs can bind directly to importin , initiating nuclear transport through the nuclear pore complex.18 Similarly, NESs are responsible for binding to export proteins, so-called exportins. Exportins transport their target proteins across the nuclear envelop back into the cytosol. A number of proteins that shuttle across the nuclear membrane have been identified using Crm1 as the export shuttle (eg, NF-AT). Here, we have identified a NLS and a NES in the calcineurin sequence. We have also identified the respective carrier proteins for calcineurin shuttling across the nuclear membrane. Importin1 is responsible for the nuclear import, whereas the export protein Crm1 is required for nuclear export of calcineurin. These findings identify a potentially novel therapeutic strategy to inhibit myocardial hypertrophy. Inhibition of the calcineurin/importin1 interaction would prevent nuclear translocation of calcineurin and subsequently inhibit the full transcriptional activity of the calcineurin/NF-AT signaling pathway. Similar approaches, such as inhibition of the nuclear factor B/importin interaction19–20 and calcineurin/NF-AT interaction21 by competitive peptides, have already been successfully proven. Using this strategy, we synthesized a peptide comprising 12 amino acids that mimicked the NLS sequence of calcineurin and an N-terminal peptide extension of additional 15 amino acids to increase membrane permeability. This peptide was able to suppress calcineurin/importin1 interaction, which subsequently prevented calcineurin nuclear import. The physiological result was blunting of NF-AT transcriptional activity and inhibition of the development of myocardial hypertrophy. In contrast, calcineurin phosphatase activity was unaffected, although assessment of calcineurin phosphatase activity in vivo is often imprecise. As a surrogate the NLS peptide had no impact of NF-AT dephosphorylation.
These results demonstrate that inhibition of the calcineurin/importin interaction by interfering peptides is an effective tool to suppress calcineurin signaling. These results however raise the following question: What is the precise role of calcineurin in the nucleus The transcriptional effector of the CnA/NF-AT system is NF-AT through its DNA-binding domain. NF-AT factors share an imperfect Rel homology domain that is only capable of weak DNA binding in the monomeric or dimeric state. To strengthen NF-AT/DNA interactions, these factors prefer to interact cooperatively with other nuclear transcription factors such as AP-1 (c-Jun/c-Fos), GATA-4, and MEF-2.22 Therefore, calcineurin may act as a transcriptional coactivator. However, competition by calcineurin with the glycogen synthase kinase 3 (GSK3) to ensure further dephosphorylation of NF-AT in the nucleus or at least to prevent rephosphorylation is unlikely to be the major task of nuclear calcineurin, as CnA mutants devoid of phosphatase function also increase transcriptional activity of the CnA/NF-AT signaling pathway when translocated to the nucleus.7 Also, multiple other kinases beside glycogen synthase kinase 3 (GSK-3) such as c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases, casein kinase I (CK1), protein kinase A (PKA), and mitogen-activated protein kinase kinase 1 (MEKK1) (indirectly), all promote rephosphorylation of the serine-rich N terminus of NF-AT factors, enabling Crm1-mediated nuclear export.23–26
Another model of competition between CnA and Crm1 for the nuclear export sequence of NF-AT has also been proposed. It has previously been demonstrated that the nuclear export protein Crm1 is capable of transporting NF-AT out of the nucleus. A constitutive nuclear calcineurin will shift Crm1 off the NES of NF-AT and leave the CnA/NF-AT complex nuclear, thereby enhancing transcriptional output.7,27 Our data show that Crm1 not only exports NF-AT but also calcineurin from the nucleus. To interrupt transcriptional activity of the calcineurin/NF-AT signaling cascade, Crm1 is first required to export calcineurin, so that in a second round Crm1 can access the NESs of NF-AT and subsequently proceed with its nuclear export. This mechanism may be prevented in myocardial hypertrophy by the proteolysis of calcineurin by calpain at aa 424, resulting in a loss of the autoinhibitory domain including the NES. In this scenario, calcineurin remains nuclear because it is inaccessible to the export protein Crm1. These observations suggest that calcineurin function in the nucleus is largely driven via its anti-Crm1 as opposed to anti-GSK3 effects.
As import always precedes export, the inhibition of CnA nuclear import by peptide competition for the binding of the nuclear import protein importin1 presents a sophisticated approach to abolishing the deleterious effects of exaggerated NF-AT transcriptional activity. Nevertheless, assessment of the specific action of the NLS peptide on the calcineurin/NF-AT interaction must be performed before further experiments can be undertaken in vivo.
Acknowledgments
We thank J. M. Redondo (Universidad Autónoma de Madrid, Spain) for providing parts of the plasmid constructs, T. Renné (University of Wuerzburg, Germany) for advice on peptide synthesis, and C. Gebhardt for excellent technical support.
Sources of Funding
This work was supported by grants from the German Research Society (Ri 1085/3-1) and the Interdisziplinares Zentrum für Klinische Forschung Wuerzburg (E-25 to O.R. and S.E.).
Disclosures
None.
Footnotes
Original received March 10, 2006; revision received August 11, 2006; accepted August 16, 2006.
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