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J. Biol. Chem., Vol. 279, Issue 46, 48376-48388, November 12, 2004

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AMP-activated Protein Kinase-regulated Phosphorylation and Acetylation of Importin 1

INVOLVEMENT IN THE NUCLEAR IMPORT OF RNA-BINDING PROTEIN HuR^*

Wengong Wang, Xiaoling Yang, Tomoko Kawai, Isabel López de Silanes, Krystyna Mazan-Mamczarz, Peili Chen¶, Yuh Min Chook||, Christina Quensel**, Matthias Köhler**, and Myriam Gorospe

From the Laboratory of Cellular and Molecular Biology, NIA Intramural Research Program, National Institutes of Health, Baltimore, Maryland 21224, the ^¶Sydney Kimmel Comprehensive Cancer Center, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21231, the ^||Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, and the ^**HELIOS Clinic Franz Volhard Clinic at the Max Delbrueck Center for Molecular Medicine, 13125 Berlin, Germany

Received for publication, August 6, 2004 , and in revised form, September 1, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Nuclear import of HuR, a shuttling RNA-binding protein, is associated with reduced stability of its target mRNAs. Increased function of the AMP-activated protein kinase (AMPK), an enzyme involved in responding to metabolic stress, was recently shown to reduce the cytoplasmic levels of HuR. Here, we provide evidence that importin 1, an adaptor protein involved in nuclear import, contributes to the nuclear import of HuR through two AMPK-modulated mechanisms. First, AMPK triggered the acetylation of importin 1 on Lys²², a process dependent on the acetylase activity of p300. Second, AMPK phosphorylated importin 1 on Ser¹⁰⁵. Accordingly, expression of importin 1 proteins bearing K22R or S105A mutations failed to mediate the nuclear import of HuR in intact cells. Our results point to importin 1as a critical downstream target of AMPK and key mediator of AMPK-triggered HuR nuclear import.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Post-transcriptional mechanisms of gene regulation, including mRNA export, turnover, and translation, are central to the implementation of specific gene expression patterns in mammalian cells. Among the post-transcriptional gene regulatory events, those affecting mRNA stability are emerging as highly effective means of altering mRNA abundance and consequently the levels of protein expressed (1–3). Labile mRNAs can be selectively stabilized or destabilized through the action of particular RNA-binding proteins that recognize specific RNA sequences. One of the major pathways regulating mRNA turnover relies on the presence of U-rich or AU-rich sequences (collectively known as AREs), typically present in the 3'-untranslated regions of transcripts encoding many cytokines, growth factors, and cell cycle regulatory proteins (4, 5). Several RNA-binding proteins that selectively bind to such instability sequences present on many labile mRNAs have been implicated in promoting their decay, including BRF1, AUF1 (hnRNP D), tristetraprolin, NF90, and KSRP (6–11). RNA-binding proteins that recognize AREs and enhance mRNA stability include the Hu proteins: HuR (HuA), which is ubiquitously expressed, as well as HuB, HuC, HuD, primarily expressed in neuronal tissues (12–15).

HuR, like all Hu proteins, contains three classic RNA recognition motifs and binds with high affinity and specificity to AREs in a variety of mRNAs, such as those encoding HSP70, vascular endothelial growth factor, tumor necrosis factor-, PAI-2, COX-2, p53, p27, p21, cyclin A, cyclin B1, GLUT-1, and c-Fos, and increases their stability, modulates their translation, or performs both functions (15–25). HuR is predominantly (>90%) localized in the nucleus of unstimulated cells. However, its influence on mRNA stabilization and translation has been linked to its cytoplasmic presence, so there has been much interest in identifying the mechanisms regulating its presence in each cellular compartment (16, 26–30). In the nucleus, HuR has been shown to bind proteins SET, SET, pp32, and APRIL (18). SET, SET, and pp32 have been identified as inhibitors of protein phosphatase 2A (31, 32), a serine/threonine phosphatase that becomes activated in response to various stimuli and dephosphorylates several major protein kinases (for review, see Ref. 33). Using cell-permeable peptides, HuR nuclear export was shown to involve the association of HuR with two of its nuclear ligands, pp32 and APRIL, which contain leucine-rich nuclear export signals that are recognized by the export receptor chromosome maintenance region 1 (CRM1).¹ Treatment with the CRM1 inhibitor leptomycin B caused the nuclear accumulation of pp32 and APRIL, as well as the increased association of HuR with pp32 and APRIL in the nucleus (34). Alternative pathways of nuclear export have been proposed to involve a shuttling sequence within HuR, the HuR nucleocytoplasmic shuttling sequence (HNS), which bears similarities with the M9 shuttling signal of hnRNP A1, (27, 34), but the export factor involved in HNS-dependent transport is currently unknown (35). Although a role for HuR in the export of target mRNAs awaits to be definitively demonstrated, evidence accumulated thus far strongly supports such a function. Therefore, the existence of multiple export pathways for HuR would ensure the rapid and effective export of HuR target mRNAs. Recently, transportin 1 (Trn1; also known as Karyopherin/Kap2) as well as the highly similar transportin 2 (Trn2) were shown to participate in the nuclear import of HuR (36). In a series of elegant in vitro studies, the HNS was found to mediate the import of HuR by transportins (35, 36).

In vivo, the levels of cytoplasmic HuR have been shown to be potently regulated by the activity of the AMP-activated protein kinase (AMPK), an enzyme that participates in the cellular response to metabolic stress (37). Believed to function as a "low fuel warning system" of the cell, AMPK activity is strongly elevated by conditions that elevate the cellular AMP:ATP ratio, such as exposure to metabolic poisons like arsenite and azide, depletion of growth factors or glucose, and treatment with the pharmacological agent AICAR (which mimics the effect of AMP) (38). In turn, active AMPK phosphorylates a number of metabolic enzymes causing both a global inhibition of biosynthetic pathways, thus conserving energy, and a global activation of catabolic pathways, thus generating more ATP (reviewed in Ref. 39). AMPK activity is ubiquitous, although different isoforms of its catalytic () and regulatory ( and ) subunits exhibit tissue-specific distribution, as well as preferential localization in different subcellular compartments (e.g. 2 subunits are partly nuclear, whereas 1 subunits are only found in the cytoplasm, etc.). Recently, we found that decreased AMPK activity led to an elevation in cytoplasmic HuR levels; conversely, AMPK activation through interventions such as treatment with AICAR and ectopic expression of a constitutively active isoform of AMPK caused a reduction in cytoplasmic HuR (40). The lowering of cytoplasmic HuR was accompanied by a reduction in complexes of HuR bound to target mRNAs encoding proliferative proteins (cyclins A and B1), a decrease in the stability of such mRNAs, a lessening in the expression of the corresponding protein products, and an ensuing inhibition of cell growth (40).

Given that HuR shuttles between the nucleus and cytoplasm (26, 27, 29, 30) and given that AMPK-triggered reductions in cytoplasmic HuR levels profoundly influence the stability of target mRNAs (40), we set out to investigate the mechanisms underlying the AMPK-mediated nuclear import of HuR. In this investigation, we have identified importin 1 as a key cytoplasmic HuR ligand mediating this process. Importin 1 functions as an adaptor that associates with importin , which transports bound cargoes through the nuclear pore complex (NPC). We provide additional evidence in support of a role for AMPK in dually modifying importin 1: AMPK triggered the acetylation of importin 1 through AMPK-stimulated activation of acetylase p300, and AMPK directly phosphorylated importin 1, both in vivo and in vitro. Importantly, overexpression of wild-type full-length importin 1, but not point mutants of importin 1 lacking the phosphorylation and acetylation sites, readily promoted the nuclear import of HuR in intact cells. Together, our data indicate that AMPK induces the acetylation and phosphorylation of importin 1 and strongly suggest that dual modification of importin 1 is required for the nuclear import of HuR.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell Culture, Treatment, Transfection, and Infection—Human colorectal carcinoma RKO cells were cultured in minimum essential medium (41), and human embryo kidney fibroblasts 293 cells (ATCC) were cultured in Dulbecco's modified essential medium, each supplemented with 10% fetal bovine serum (Hyclone, Logan, UT) and antibiotics. Sodium butyrate, trichostatin A, and AICAR were from Sigma, and calpain inhibitor I, N-Ac-Leu-Leu-norleucinal (ALLN) was from Calbiochem (La Jolla, CA). Adenoviruses expressing either the control gene GFP (AdGFP) or a constitutively active isoform of the AMPK 1 subunit (Ad(CA)AMPK) (42) were amplified and titered in 293 cells using standard methodologies. The infections were carried out in serum-free Dulbecco's modified Eagle's medium for 4 h. Infection efficiency of RKO cells was determined by infection with AdGFP at various plaque-forming units/cell and assessment of the percentage of GFP-expressing cells 48 h later. For >90% infection, 100 plaque-forming units/cell was required, in accordance with the low infection rates of RKO cells (41); 100 and 20 plaque-forming units/cell were used in all infections of RKO and 293 cells, respectively. Cytoplasmic, nuclear, and whole cell fractions were prepared as described (24).

Constructs and Recombinant Proteins—All of the purified recombinant proteins were produced in Escherichia coli. His-tagged (C-terminal) importin 1 was purified by nickel-nitrilotriacetic acid affinity chromatography followed by ion exchange and gel filtration chromatography. GST-Imp and GST-Kap2 were purified by glutathione-Sepharose affinity chromatography, cleaved with either Precission protease (Imp) or Tev protease (Kap2), and Imp and Kap2 were further purified by ion exchange and gel filtration chromatography (43).

Bacterial expression constructs were prepared by ligating PCR-amplified inserts into plasmid pBAD-TOPO (Invitrogen). For expression in mammalian cells, PCR-amplified full-length and truncated importin 1 cDNA products were inserted into plasmid pcDNA3.1/V5-His-TOPO (Invitrogen). Plasmid pCMV-p300 was used for expression of p300 in cultured cells (Upstate USA Inc., Lake Placid, NY).

For expression in mammalian cells, importin 1 proteins bearing either a lysine-to-arginine mutation on residue 22 (Imp1(K22R)) or a serine-to-alanine mutation on residue 105 (Imp1(S105A)), or both mutations (Imp1(S105A,K22R)) were generated using the QuikChange site-directed mutagenesis kit (Stratagene) and were expressed in plasmid pcDNA3.1/V5-His-TOPO (Invitrogen). All of the constructs were verified by sequencing.

Antibodies and Western Blot Analysis—For Western blotting, whole cell (20 µg), cytoplasmic (40 µg), and nuclear (10 µg) lysates, prepared as previously described (24), were size-fractionated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Monoclonal antibodies recognizing -tubulin (a control cytoplasmic protein), HDAC1 (a control nuclear protein), HDAC2, HDAC3, HDAC4, c-Myc, and cyclin A, as well as polyclonal antibodies recognizing CRM1 and p300, were from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibodies recognizing importin , transportin 1/Kap2, importin 1, NF-B, NTF2, Ran, Ran-BP1, Nup-98, Nup-88, and Nup-62 were from BD Pharmaceuticals. Polyclonal antibodies recognizing histone H4 and acetylated histone H4 were from Upstate Biotechnology Inc. (Lake Placid, NY). A monoclonal antibody recognizing HuR (Molecular Probes, Inc., Eugene, OR) was used. Anti-HA antibody was from Qiagen. Polyclonal antibodies recognizing importins 1, 3, 4, 5, and 7 were previously described (44). Following secondary antibody incubations, signals were detected by enhanced chemiluminescence. Rabbit polyclonal antiserum recognizing acetylated importin 1 was a generous gift from Dr. T. Kouzarides (45).

Immunoprecipitation—Whole cell lysates for immunoprecipitation (IP) were prepared by adding 200 µl of IP buffer (10 mM Hepes, pH 7.4, 50 mM -glycerophosphate, 1% Triton X-100, 10% glycerol, 2 mM EDTA, 2 mM EGTA, 10 mM NaF, 1 mM DTT, 1 mM Na₃VO₄, 20 nM microcystin-LR, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1 µM trichostatin A, 20 mM sodium butyrate, 5 µg/ml soybean trypsin inhibitor, and 0.1 mM benzamidine) supplemented with 0.5% SDS. Each sample was passed through a 27.5-gauge needle 20 times and then centrifuged (10 min, 4 °C, 21,000 x g). IP reactions were carried out by using 20 µl of the resulting supernatant, diluting it with 1 ml of IP buffer, and adding 2 µg of the antibodies indicated above. The washes were performed as follows: three times with IP buffer, four times with a high stringency buffer (100 mM Tris-HCl, pH 7.4, 500 mM LiCl, 0.1% Triton X-100, 1 mM DTT, 1 mM Na₃VO₄, 20 nM microcystin-LR, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1 µM trichostatin A, 20 mM sodium butyrate, 5 µg/ml soybean trypsin inhibitor and 0.1 mM benzamidine), and then three times with IP buffer.

Pulse Labeling of Endogenous Protein—RKO cells (200,000 cells/6-cm dish) were incubated in methionine- and cysteine-free medium containing 5% dialyzed fetal bovine serum for 30 min and then for an additional 20 min with 900 µCi of L-[³⁵S]methionine and L-[³⁵S]cysteine (NEG-072 Expre³⁵S³⁵S Protein labeling mix; PerkinElmer Life Sciences) per well and scraped into 500 µl of ice-cold phosphate-buffered saline. After centrifugation (1,800 rpm, 4 °C, 1 min), the pellets were shock-frozen in liquid nitrogen and resuspended in 100 µl of TSD lysis buffer (50 mM Tris, pH 7.5, 1% SDS, and 5 mM DTT). One hundred-µg protein aliquots were brought to a final volume of 1.2 ml in TNN buffer (50 mM Tris, pH 7.5, 250 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 2 ng/µl aprotinin, and 2 ng/µl leupeptin) and precleared with protein A/G (1:1)-Sepharose mix (Sigma) for 1 h at 4 °C. Following a brief centrifugation, the supernatants were incubated for 1 h at 4 °C with 0.4 µg of either a monoclonal antibody that recognizes HuR (Santa Cruz Biotechnology) or IgG1 (BD Biosciences Pharmingen) and precipitated using 50 µl of protein A/G (1:1) mix (Sigma) for 1 h at 4 °C. Following washes in TNN buffer, the IP material was resolved by electrophoresis in SDS-containing 12% polyacrylamide gels, transferred onto polyvinylidene difluoride filters, and visualized using a PhosphorImager (Molecular Dynamics).

AMPK and HDAC Activity Assays—AMPK was assayed as described (38, 40). Briefly, AMPK was immunoprecipitated from 5 µg of cell lysate by incubation with 1 µg of anti-1 and 1 µg of anti-2 polyclonal antibodies, and AMPK activity in the IP complexes was determined by phosphorylation of peptide HMRSAMSGLHLVKRR (SAMS (38)). Synthetic peptides encompassing Ser⁷⁷ (⁷¹QGTVNWSVDDIVKGI⁸⁵), Ser¹⁰⁵ (⁹⁸QAARKLLSREKQ PPID¹¹³), and Ser¹⁷⁹ (¹⁷²ASPHAHISEQAVWALG¹⁸⁷) were obtained from AnaSpec, Inc. (San Jose, CA) and used in phosphorylation assays employing AMPK as kinase, following the procedures described above for the SAMS peptide. Phosphorylated substrates were measured by scintillation counting. HDAC activity was measured with a histone deacetylase assay kit from Upstate Biotechnology Inc., following the manufacturer's instructions; histone H4 was used as a substrate.

Acetylation of Importin 1 by p300 —Following infection of RKO cells with either AdGFP or Ad(CA)AMPK, p300 was immunoprecipitated from 100 µg of nuclear lysate (prepared as previously described (24)) using 3 µg of an anti-p300 monoclonal antibody (Upstate Biotechnology, Inc.). p300 bound to G protein-coupled Sepharose beads was incubated with a reaction mixture containing 50 mM Tris-HCl, pH 8.0, 10% glycerol, 0.1 mM EDTA, 1 mM DTT, 0.5 µg of purified His-Imp1 protein, 20 µM acetyl-CoA, and 1 µCi of [³H]acetyl-CoA for 30 min at 30 °C in a shaking incubator. The reaction mixtures were then size-separated by 12% SDS-PAGE, and radiolabeled acetylated proteins were visualized using a phosphorimaging device (Molecular Dynamics).

Protein Phosphorylation Assays—Active AMPK was immunoprecipitated from 200 µg of lysate prepared from 293 cells that had been infected with Ad(CA)AMPK, using an anti-c-Myc antibody and G protein-coupled Sepharose beads. For in vitro phosphorylation assays, the proteins that were either immunoprecipitated from RKO cells or purified from bacteria were incubated with AMPK (bound to G protein-coupled Sepharose beads) in the presence of 10 µCi of [-³²P]dATP and kinase buffer containing 50 mM Hepes, 1 mM DTT, 0.02% Brij-35, 6.25 mM MgCl₂, and 0.25 mM AMP for 10 min at 30 °C. Phosphorylated products were size-fractionated by 15% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Phosphorylated products were visualized using a PhosphorImager (Molecular Dynamics).

For in vivo phosphorylation assays, cultured cells were incubated with 0.5 mCi/ml H₃³²PO₄ (³²P_i), for 3 h under standard conditions (37 °C, 5% CO₂), and phosphorylation of either p300 or importin 1 was detected by IP using antibodies from Upstate Biotechnology Inc. or BD Biosciences, respectively.

Solution Binding Assays—Solution binding assays were performed essentially as described (46). Two µg of either MBP-HuR or His-importin 1 immobilized on 10 µl of protein G-Sepharose beads using either anti-MBP or anti-His antibodies, respectively, were incubated with 2 µg of either Imp, Kap2, MBP (New England Biolabs, Beverly, MA), MBP-HuR, Imp1, or a combination of these proteins for 45 min at 25 °C. The proteins bound to products immobilized on beads, as well as proteins remaining unbound, were visualized after 12% SDS-PAGE and staining with Coomassie Blue dye.

Immunofluorescence—293 cells were seeded on coverslips and were transfected with the plasmids indicated. Forty-eight h after transfection, the cells were fixed for 15 min in phosphate-buffered saline containing 4% paraformaldehyde and permeabilized for 15 min in phosphate-buffered saline containing 0.4% Triton X-100. After incubation for 16 h in blocking buffer (phosphate-buffered saline containing 2% bovine serum albumin and 0.1% Tween 20), the coverslips were incubated for 1 h at 1:500 dilution of mouse anti-HuR (Santa Cruz Biotechnology.) prepared in blocking buffer. Following washes with PBS containing 0.1% Tween 20, the samples were incubated for 1 h with a mixture of horse anti-mouse Texas Red (1:200; Jackson Laboratories) and Hoechst 33342 (1:5,000; Molecular Probes). After washes with PBS containing 0.1% Tween 20, the coverslips were mounted in Vectashield (Vector Laboratories, Burlingame, CA) and visualized with an Axiovert 200 M microscope (Zeiss; 63x lens) using separate channels for the analysis of red fluorescence and blue fluorescence. The images were processed with the AxioVision 3.0 program (Zeiss). Representative photographs from three independent experiments are shown.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Reduced Cytoplasmic HuR Levels after AMPK Activation— Treatment of human colorectal carcinoma RKO cells with the AMP analog AICAR, a strong and highly specific activator of AMPK (38) (Fig. 1A), markedly reduced the levels of cytoplasmic HuR (Fig. 1B). Evidence that the AMPK-imposed reduction in cytoplasmic HuR abundance was not due to increased cytoplasmic degradation of HuR was obtained by using inhibitors of the proteasome and proteases. As shown in Fig. 1C, pretreatment with ALLN (which inhibits the activity of the proteasome, calpain, and cathepsins) failed to prevent the reduction in cytoplasmic HuR levels elicited through treatment with AICAR (Fig. 1C); use of proteasome inhibitor lactacystin or protease inhibitors ALLM or PD150606 similarly failed to alter the reduction in cytoplasmic HuR levels triggered by AICAR (not shown). In addition, the AMPK-imposed reduction in cytoplasmic HuR abundance was not due to decreased translation, as observed by assessing nascent HuR levels. De novo synthesis of HuR was monitored by pulse labeling (for 20 min) of RKO cells in the presence of L-[³⁵S]methionine and L-[³⁵S]cysteine, followed by detection of the newly synthesized HuR through IP using anti-HuR antibodies (Fig. 1D). These two sets of data lent support to the notion that HuR abundance in the cytoplasm was primarily governed via nucleocytoplasmic transport and not changes in HuR translation or cytoplasmic stability. In keeping with earlier studies demonstrating that increased AMPK activity reduces cytoplasmic HuR levels (40) and studies that HuR shuttles between the nucleus and the cytoplasm (26, 27, 29, 30), these findings supported the existence of an AMPK-triggered effect on nuclear transport of HuR. Given that AMPK-imposed reductions in cytoplasmic HuR levels profoundly impacted on the half-lives of HuR target mRNAs (40), we sought to examine the regulatory mechanisms whereby AMPK regulated the nuclear import of HuR.

View larger version (31K): [in this window] [in a new window]   FIG. 1. AMPK activity and subcellular localization of HuR in RKO cells treated with AICAR. A, 6 h after exposure of RKO cells to the indicated doses of AICAR, whole cell lysates were prepared, and AMPK activity was assayed after IP with a polyclonal antibody recognizing the AMPK 1 and 2 subunits, using the synthetic peptide SAMS as substrate (described under "Experimental Procedures" and in Ref. 38). The data represent the means and S.E. of three separate experiments. B, Western blot analysis to assess HuR levels in whole cell (Total,10 µg), cytoplasmic (40 µg), and nuclear (10 µg) lysates prepared from RKO cells that were treated for 6 h with the indicated concentrations of AICAR. Assessment of the levels of the cytoplasm-specific -tubulin and nucleus-specific HDAC1 after sequential stripping and reprobing of the filters served to verify the quality and equal loading of the cytoplasmic and nuclear preparations, respectively. All Western blotting signals were measured by densitometry scanning; HuR abundance in each compartment following AICAR treatment was calculated after normalization against the values of control proteins (either -tubulin or HDAC1) and is presented as a percentage of remaining signals relative to those measured in untreated samples of each lysate group. C, Western blot analysis to monitor HuR levels in either cytoplasmic or whole cell (Total) lysates prepared from RKO cells that were either pretreated for 1 h with 10 µM of the protease/proteasome inhibitor calpain inhibitor I, N-Ac-Leu-Leu-norleucinal (ALLN) or left untreated and then incubated for 6 h in the presence of the indicated doses of AICAR. Assessment of the levels of -actin and -tubulin served to monitor equal loading and transfer of cytoplasmic and total protein samples, respectively. D, RKO cells were treated for 6 h in the presence of the indicated concentrations of AICAR, and then incubated for an additional 20 min in the presence of L-[35S]methionine and L-[35S]cysteine, whereupon nascent HuR was visualized by IP as described under "Experimental Procedures"; the IgG1 lane represents the control IP material. The samples were resolved by electrophoresis in SDS-containing 12% polyacrylamide gels. Radiolabeled HuR signal is shown.

View larger version (33K): [in this window] [in a new window]   FIG. 2. Effect of histone deacetylase inhibitors on the cytoplasmic localization of HuR and AMPK activity. A, RKO cells were treated with histone deacetylase inhibitors SButyr (15 mM) or TSA (1 µM), and cytoplasmic (40 µg) and whole cell (Total, 10 µg) lysates were used to monitor HuR expression levels. Relative HuR abundance was calculated as explained in the legend of Fig. 1; -actin signals served to normalize for differences in loading and transfer of protein samples. B, 6 h after treatment of RKO cells with 2 mM AICAR, 15 mM SButyr, 1 µM TSA, or combinations of these drugs, the lysates were prepared, and HDAC activity ("Experimental Procedures") and AMPK activity (described in the legend of Fig. 1) were assayed. The data represent the mean of two experiments, each yielding similar results. C, RKO cells were treated for 6 h with 2 mM AICAR, 15 mM Sbutyr, or 1 µM TSA, alone or in combination, and HuR abundance was assessed in cytoplasmic (40 µg) and whole cell (10 µg) lysates. Relative HuR expression levels were calculated as explained in the legend of Fig. 1; -actin signals served to normalize for differences in the loading and transfer of protein samples. Ctrl, control.

HuR Associates Specifically with Importin 1—The above findings prompted a further analysis of components of the nuclear import machinery potentially involved in transporting HuR into the nucleus following AMPK activation. To identify the specific factor(s) that might mediate the import of HuR into the nucleus, we carried out a series of IP reactions coupled with Western blot analyses to investigate potential associations between HuR and other proteins. This survey included a number of transport proteins such as CRM1, NTF2, and members of the karyopherin , importin , and importin families. It also included several negative control proteins, including proteins involved in the structure and function of the nuclear pore complex (such as Ran, Ran-binding protein, and nucleoporins) and nuclear proteins such as HDACs, given the aforementioned evidence that cytoplasmic HuR levels were influenced by altered protein acetylation.

The potential interactions between these proteins and HuR were first assayed by IP using an anti-HuR antibody, followed by Western blotting using antibodies that specifically recognized the proteins of interest (Fig. 3A, left column, Panel). These interactions were further tested by carrying out individual IP reactions with antibodies recognizing each of the proteins listed and then performing Western blot analysis of the IP material to test for HuR presence (Fig. 3A). These studies revealed that importin , karyopherin 2, and importin 1 were associated with HuR, although importin appeared to bind less strongly with HuR. Fig. 3B depicts illustrative IP-coupled Western blot analyses performed to obtain the data summarized in Fig. 3A.

View larger version (22K): [in this window] [in a new window]   FIG. 3. Interaction of HuR with nuclear proteins and with proteins involved in nucleocytoplasmic transport. A, whole cell lysates prepared from RKO cells were subjected to two types of analyses. First (IP: HuR; IB: Panel column), HuR was immunoprecipitated along with associated proteins, whereupon the presence of nuclear proteins and proteins associated with the nucleocytoplasmic transport system (left column) was detected by performing Western blot (WB) analysis with the corresponding specific antibodies. Second (IP: Panel; IB: HuR column), IP reactions were performed using a collection of antibodies recognizing each of the proteins listed in the left column, followed by Western blot analysis to detect HuR in the immunoprecipitated material. The details are provided under "Experimental Procedures." Western blot signals: -, undetectable; +/-, weak; +, moderate; ++, prominent; ++++, very strong; ND, not done. B, Western blot analysis to illustrate IP-coupled Western blot analyses used to generate the list in A. Two hundred µg of whole cell lysate was used per each immunoprecipitation (IP) reaction, and the immunoprecipitated material was then used to perform Western blotting analysis to detect the presence of the proteins indicated. IP followed by WB analyses were carried out two to five times; representative Western blotting signals are shown. IB, immunoblot.

View larger version (35K): [in this window] [in a new window]   FIG. 4. Importin 1 and HuR interact in vitro. A, 2 µg each of purified recombinant Imp, MBP-HuR, or MBP were incubated with immobilized His-Imp1 and used in solution binding assays (described under "Experimental Procedures"). B,2 µg each of purified recombinant Imp, Kap2, His-Imp1, and GST-c-Jun were incubated with immobilized MBP-HuR. All bound and unbound fractions, prepared as described under "Experimental Procedures," were size-fractionated by 12% SDS-PAGE and detected by Coomassie Blue staining. H.C., heavy chain; MWM, molecular weight marker; *, truncated MBP-HuR product.

View larger version (40K): [in this window] [in a new window]   FIG. 5. Acetylation of importin 1 by p300 in an AMPK-dependent fashion. A, 24 h after transfection of 293 cells with 20 µg of either the insert-less plasmid pCMV (V), or plasmid pCMV-p300 (p300), expressing wild-type p300, the abundance of p300, total importin 1, acetylated importin 1, total histone H4, and acetylated histone H4, was assessed by Western blotting using 20 µg of whole cell lysate. Antibodies and procedures are described under "Experimental Procedures." B, AMPK activity in cells that had been infected with either an adenovirus expressing a constitutively active isoform of the subunit of AMPK (Ad(CA)AMPK) or control AdGFP adenovirus was calculated as explained in the legend of Fig. 1A. C, top panel, endogenous p300 was immunoprecipitated from RKO cells that had been infected with either Ad(CA)AMPK or AdGFP and then used for in vitro acetylation of 0.5 µg of purified His-importin 1 (His-Imp1) or the control protein MBPHuR. The protein molecular weight markers are indicated. Bottom panel, the presence of p300, His-Imp1, and MBP-HuR in the reaction materials was monitored by Western blotting. D, RKO cells infected with either Ad(CA)AMPK or control AdGFP were incubated with 32Pi for 3 h, whereupon phosphorylated p300 was detected by immunoprecipitation of p300, separation of IP material by 12% SDS-PAGE, and visualization using a PhosphorImager. 24 h after infection of 293 cells using either Ad(CA)AMPK or control AdGFP, the abundance of p300, total importin 1, acetylated importin 1, total histone H4, and acetylated histone H4 was assessed by Western blotting using 20 µg of whole cell lysate. The antibodies and procedures are described under "Experimental Procedures."

Importantly, overexpression of p300 also caused a decrease in cytoplasmic HuR levels, as determined by both Western blotting (Fig. 6A) and immunofluorescence (Fig. 6B). These findings strongly support the hypothesis that p300-mediated events influence the subcellular compartmentalization of HuR.

View larger version (37K): [in this window] [in a new window]   FIG. 6. Decreased cytoplasmic localization of HuR in cells overexpressing p300. A, Western blot analysis to assess HuR levels in whole cell (Total, 10 µg), cytoplasmic (Cytopl., 40 µg), and nuclear (10 µg) lysates prepared from 293 cells 48 h after transfection with 20 µg of either plasmid pCMV (V) or plasmid pCMV-p300 (p300). Assessment of the levels of the cytoplasm-specific -tubulin and nucleus-specific HDAC1 after sequential stripping and reprobing of the filters served to verify the quality and equal loading of the cytoplasmic and nuclear preparations, respectively. Western blotting signals were measured by densitometry scanning; HuR abundance, calculated after normalization against the values of control proteins (either -tubulin or HDAC1), is presented as the percentage of remaining signals relative to those measured in untreated samples of each lysate group. Shown are representative Western blotting signals; experiments were performed in triplicate. B, detection of HuR by immunofluorescence in 293 cells 48 h after transfection with either plasmid pCMV (V) or plasmid pCMV-p300 (p300). Left panels, HuR immunofluorescence; right panels, Hoechst staining to visualize nuclei. Representative photographs from three independent experiments are shown.

View larger version (39K): [in this window] [in a new window]   FIG. 7. AMPK phosphorylates importin 1 in vivo and in vitro. A, constitutively active AMPK, prepared from 293 cells that had been infected with Ad(CA)AMPK, was prepared by IP using an anti-Myc antibody. The immunoprecipitated material was then used in in vitro phosphorylation assays using purified His-Imp1 as well as control proteins Imp and MBP-HuR as substrates. B, endogenous importin 1 and constitutively active AMPK were immunoprecipitated independently, and the immunoprecipitated materials incubated to assess the phosphorylation of importin 1 in vitro. In vitro phosphorylation assays (A and B) were carried out as described under "Experimental Procedures"; the phosphorylated products were visualized through electrophoresis of the kinase reaction mixtures in SDS-containing 12% polyacrylamide gels that were subsequently transferred onto filters and subjected to both PhosphorImager scanning (top panel) and Western blotting (WB, bottom panel) to detect the proteins indicated. C, in vivo phosphorylation of importin 1. 293 cells infected with either Ad-AMPK(CA) or AdGFP were incubated with 1 mCi/ml 32Pi, whereupon whole cell lysates were immunoprecipitated (IP) and importin 1 abundance and phosphorylation status were analyzed by Western blotting (WB, top panel) and PhosphorImager scanning (bottom panel), respectively.

View larger version (31K): [in this window] [in a new window]   FIG. 8. AMPK-mediated in vitro phosphorylation of either full-length importin 1, truncated importin 1, or importin 1 peptides. A, schematic representation of bacterially produced His-tagged importin 1 constructs derived from plasmid pBAD-TOPO employed in this analysis. Full-length Imp1, as well as N-terminally truncated importin 1 mutant proteins (Imp1(62) and Imp1(205)) were produced as His-tagged proteins. B, left panel, His-tagged products were detected by IP+WB using anti-His antibodies. Right panel, 48 h after infection of 293 cells with Ad(CA)AMPK, lysates were prepared and the ability of AMPK to phosphorylate the proteins indicated was assayed in the presence of 32Pi; (CA)AMPK was immunoprecipitated using anti-c-Myc, and recombinant proteins, immunoprecipitated using anti-His, were used in in vitro phosphorylation assays ("Experimental Procedures"). C, 48 h after infection of 293 cells with Ad-(CA)AMPK, cell lysates were prepared, (CA)AMPK was immunoprecipitated using anti-c-Myc antibody, and AMPK activity (described in the legend of Fig. 1) was assayed using 0.25 mM of peptides SAMS (38), Ser77 (71QGTVNWSVDDIVKGI85), Ser105 (98QAARKLLSREKQPPID113), and Ser179 (172ASPHAHISEQAVWALG187), following the procedures described above for the SAMS peptide. The data shown are representative of five independent experiments.

View larger version (36K): [in this window] [in a new window]   FIG. 10. Influence of overexpression of wild-type importin 1 and point-mutated importin 1 on cytoplasmic HuR levels. A, His-tagged importin 1 constructs were prepared from vector pcDNA3.1/V5-His-TOPO to express either wild-type importin 1 (Imp1) or importin 1 proteins bearing point mutations as shown. B, 24 h after transfection of 293 cells with the constructs indicated, in vitro phosphorylation was assessed by using immunoprecipitated (CA)AMPK (obtained by performing anti-c-Myc IP from Ad(CA)AMPK-infected 293 cells; "Experimental Procedures") and His-tagged proteins were detected by Western blotting. C, twenty-four h after transfection of 293 cells with the constructs indicated, Western blot analyses were carried out to assess HuR levels in cytoplasmic (Cytopl., 40 µg) and whole cell (Total, 10 µg) lysates. To verify the quality and equal loading of the protein preparations, the blots were stripped and reprobed to detect -tubulin; an anti-Myc antibody was used to monitor the evenness of tagged protein expression in the transfected populations. Western blotting signals in the cytoplasmic preparations were measured by densitometry scanning; HuR abundance was calculated after normalization against the intensity values of -tubulin and indicated as a percentage of the remaining signals relative to those measured in vector-transfected samples. The experiment was repeated three times; representative Western blots are shown.

View larger version (35K): [in this window] [in a new window]   FIG. 9. Influence of overexpression of full-length importin 1 or truncated importin 1 on cytoplasmic HuR levels. A, 24 h after transfection of 293 cells with His-tagged importin 1 constructs derived from vector pcDNA3.1/V5-His-TOPO, full-length importin 1 (Imp1) or N-terminally truncated importin 1 mutant proteins (Imp1(62) and Imp1(205)) were detected using an anti-His antibody. B, 24 h after transfection of RKO cells with the constructs indicated, Western blot analyses were carried out to assess HuR levels in cytoplasmic (Cytopl., 40 µg), nuclear (10 µg), and whole cell (Total, 10 µg) lysates. Verification of the quality and equal loading of the protein preparations was carried out by assessing the levels of -tubulin (for cytoplasmic lysates) and HDAC1 (for nuclear and whole cell lysates) after stripping and reprobing the filters. Western blotting signals in the cytoplasmic preparations were measured by densitometry scanning; HuR abundance was calculated after normalization against the intensity values of -tubulin and indicated as a percentage of remaining signals relative to those measured in vector-transfected samples. The experiment was repeated three times, and representative Western blots are shown.

Analysis of the subcellular distribution of HuR by immunofluorescence (Fig. 11A) was in precise agreement with the Western blotting data (Fig. 10C). Overexpression of Imp1, but not vector, Imp1(K22R), Imp1(S105A), or Imp1(K22R, S105A), effectively led to an enhanced HuR import, as revealed by the marked reduction in cytoplasmic HuR in these populations. Similar findings were obtained in transient transfections to express the deletion mutants described in Fig. 9 (data not shown). Importantly, only wild-type importin 1 appeared to interact with HuR, as revealed by co-IP analysis using an anti-HuR antibody followed by Western blot analysis of the ectopically expressed (HA-tagged) importin 1 proteins present in the IP material using an anti-HA antibody. As shown in Fig. 11B, wild-type importin 1 appeared to interact with HuR, but no such interactions were detected with any of the point mutants tested. These findings lend support to the notion that wild-type importin 1, but not importin 1 mutants that cannot be phosphorylated or acetylated, are capable of binding HuR.

View larger version (25K): [in this window] [in a new window]   FIG. 11. Immunofluorescence detection of cytoplasmic HuR levels in 293 cells transfected with either full-length or truncated importin 1 proteins. A, HuR was detected by immunofluorescence 48 h after transfection of 293 cells with pcDNA3.1/V5-His-TOPO plasmids either lacking an insert (V) or carrying wild-type importin 1 (Imp1) or importin 1 proteins bearing point mutations (Imp1(K22R), Imp1(S105A),or Imp1(K22R,S105A)). Left panel, HuR immunofluorescence; right panel, Hoechst staining to visualize nuclei. Representative photographs from three independent experiments are shown. B, lysates from 293 cells that had been transfected 48 h earlier with the HA-tagged constructs described in A were subjected to IP using anti-HuR antibodies. The presence of the ectopically expressed wild-type or point mutant importin 1 proteins (all of them exhibiting the same molecular weight) in the IP materials was subsequently detected by Western blot analysis using an anti-HA antibody. H.C., heavy immunoglobulin chain.

View larger version (23K): [in this window] [in a new window]   FIG. 12. Proposed model of AMPK-triggered HuR nuclear import through its AMPK-mediated dual modification of importin 1. We propose that AMPK promotes the nuclear import of HuR via mechanisms involving the transport protein importin 1. AMPK achieves importin 1-mediated nuclear import through modification of importin 1 on two residues. One modification is the acetylation of importin 1, plausibly through AMPK-mediated phosphorylation of p300 (a modification that activates p300), which in turn acetylates importin 1 on residue Lys22. The second modification is the direct phosphorylation by AMPK on Ser105 of importin 1. We further propose that importin mediates the import of HuR-importin 1 complexes into the nucleus.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cellular macromolecules are transported between the nucleus and the cytoplasm through the nuclear pore complex (the NPC, a large structure composed of 30 proteins named nucleoporins), mediated by the action of several families of soluble transport molecules. One such family of transport molecules, represented by NTF2, is involved in the nuclear import of the small GTPase Ran (52, 53), whereas another comprises the TAP/NXF p15/NXT protein dimer, which participates in the nuclear export of mRNA (reviewed in Ref. 54). However, the largest family of transport factors is the family of proteins known as importins/exportins or karyopherins. In mammalian cells, many members of this family have been shown to be involved in nuclear import (such as importin , karyopherin 2, transportin 2, transportin-SR, importin 1, importin 5, importin 7, and importin 9), whereas others have been shown to mediate nuclear export (like CAS, CRM1, exportin-t, exportin 4, and exportin 5), and yet others been reported to mediate both import and export (like importin 13) (36, 49, 55).

Members of the importin family have been most extensively implicated in the "classical" import pathway of cytoplasmic import substrates (cargoes). In addition to receptor molecules such as those comprising the importin family, adaptor proteins such as those in the importin family (including importins 1, 3, 4, 5, 6, and 7 in humans (44)) mediate the recognition of nuclear localization signals such as the classical NLS but require importin to be transported t