ARHI (DIRAS3), an Imprinted Tumour Suppressor Gene, Binds to Importins and Blocks Nuclear Import of Cargo Proteins

Biosci. Rep. (2010) / 30 / 159–168 (Printed in Great Britain) / doi 10.1042/BSR20090008

ARHI (DIRAS3), an imprinted tumour suppressor gene, binds to importins and blocks nuclear import of cargo proteins

Shaoyi HUANG*, In Soon CHANG†, Wenbo LIN§, Wenduo YE§, Robert Z. LUO*, Zhen LU*, Yiling LU‡, Ke ZHANG†, Warren S.-L. LIAO*, Tao TAO§, Robert C. BAST, Jr*, Xiaomin CHEN† and Yinhua YU*1

*Department of Experimental Therapeutics, The University of Texas, M.D. Anderson Cancer Center, Houston, TX 77030, U.S.A., †Department of Biochemistry and Molecular Biology, The University of Texas, M.D. Anderson Cancer Center, Houston, TX 77030, U.S.A., ‡Department of Systems Biology, The University of Texas, M.D. Anderson Cancer Center, Houston, Texas 77030, U.S.A., §School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, People’s Republic of China, and Obstetrics and Gynecology Hospital of Fudan University, Shanghai 200011, People’s Republic of China

' $ Synopsis ARHI (aplasia Ras homologue member I; also known as DIRAS3) is an imprinted tumour suppressor gene, the expression of which is lost in the majority of breast and ovarian cancers. Unlike its homologues Ras and Rap, ARHI functions as a tumour suppressor. Our previous study showed that ARHI can interact with the transcriptional activator STAT3 (signal transducer and activator of transcription 3) and inhibit its nuclear translocation in human breast- and ovarian-cancer cells. To identify proteins that interact with ARHI in nuclear translocation, in the present study, we performed proteomic analysis and identified several importins that can associate with ARHI. To further explore this novel finding, we purified 10 GST (glutathione transferase)–importin fusion proteins (importins 7, 8, 13, β1, α1, α3, α5, α6, α7 and mutant α1). Using a GST-pulldown assay, we found that ARHI can bind strongly to most importins; however, its binding is markedly reduced with an importin α1 mutant that contains an altered NLS (nuclear-localization signal) domain. In addition, an ARHI N-terminal deletion mutant exhibits greatly reduced binding to all importins compared with wild-type ARHI. In nuclear-import assays, the addition of ARHI blocked nuclear localization of phosphorylated STAT3. ARHI also inhibits the interaction of Ran–importin complexes with GFP (green fluorescent protein) fusion proteins that contain an NLS domain and a β-like import receptor-binding domain, thereby blocking their nuclear localization. By conducting GST-pulldown assays, we found that ARHI could compete for Ran- importin binding. Thus ARHI-induced disruption of importin-binding to cargo proteins, including STAT3, could serve as an important regulatory mechanism that contributes to the tumour-suppressor function of ARHI. Key words: aplasia Ras homologue member I (ARHI), importin, nuclear import, nuclear translocation, Ran, signal transducer and activator of transcription 3 (STAT3) & %

INTRODUCTION [extracellular-signal-regulated kinase; also known as MAPK (mitogen-activated protein kinase)]} [4] occurs through impaired nuclear import, enhanced export, suppression of degradation and The transport of macromolecules between the nucleus and cyto- sequestration in protein aggregates. Conversely, secreted factors plasm is critical for the normal function of eukaryotic cells. such as CYR61 (cysteine-rich protein 61), CTGF (connective Two groups of karyopherins (importins and exportins) mediate tissue growth factor) and NOV (the protein encoded by nephro- RanGTPase-dependent transport through the nuclear pore [1]. blastoma overexpressed gene; also known as CCN3), and EGF During malignant transformation, aberrant nucleocytoplasmic (epidermal growth factor), FGFs (ﬁbroblast growth factors) and transport of transcription factors [such as STAT3 (signal trans- their receptors are often detected in the nuclei of cancer cells. ducer and activator of transcription 3) and E2F1 (E2F transcrip- Nuclear localization of these molecules has been correlated tion factor 1)] [2,3] and their regulatory kinases {such as SGK with tumour progression and poor prognosis for patient survival (serum- and glucocorticoid-induced protein kinase) and ERK [5,6].

...... Abbreviations used: ARHI, aplasia Ras homologue member I; BIB, beta-like import receptor binding; E2F1, E2F transcription factor 1; GFP, green ﬂuorescent protein; GST, glutathione transferase; IBB, importin β-binding; IL, interleukin; MAPK, mitogen-activated protein kinase; NLS, nuclear-localization signal; NTD, N-terminal deletion mutant; pSTAT, phosphorylated signal transducer and activator of transcription; RanGTP; small GTP-binding protein Ran. 1To whom correspondence should be addressed (email [email protected]).

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The classical nuclear-import pathway consists of importin place cargo proteins, including STAT3, and inhibit their nuclear α and importin β. Whereas importin α interacts with NLS localization. (nuclear-localization signal) in the cargo, importin β binds to the autoinhibitory domain on importin α and mediates the transport of the trimeric complex from the cytoplasm to the nucleus through MATERIALS AND METHODS the nuclear pores. Once inside the nucleus, RanGTP (small GTP- binding protein Ran) dissociates the complex by interacting with importin β. Importins α and β are shuttled separately back to the Cell lines and reagents cytoplasm [7]. The importin α family includes importins α1, α3, The SKBr3 human breast-cancer cell line was maintained as de- α4, α5, α6andα7 [8]. There are 20 members in the importin β scribed in [17]. HeLa cells were grown at 37◦C to near-confluence superfamily, including importins β1, β7, β8, β9, and β13 [9– in Dulbecco’s modified Eagle’s medium containing 10% (v/v) 13]. Importin α proteins are composed of a flexible N-terminal fetal bovine serum. Constructs of Ran and importin α1 mutant IBB (importin β-binding) domain. The flexible IBB domain in- that contained an altered NLS domain, as well as anti-importin teracts either in trans with importin β or in cis with the cNLS β1 antibody were a gift from Dr Karsten Weis (Department of (classical NLS)-binding groove [8]. Importin β proteins have in Molecular and Cell Biology, University of California, Berke- common an N-terminal Ran-binding domain. Importins direct ley, Berkeley, CA, U.S.A.). Constructs of GST (glutathione the import of various cargoes and may have different functions. transferase), GST–importin 7, GST–importin 8, GST–GFP For example, the importin β–importin 7 heterodimer is a func- (green fluoresecent protein), GST–GFP–BIB (beta-like import tional nuclear-import receptor for histone H1 [10]; importin β, receptor binding) and GST–GFP–NLS were a gift from Dr Keith transportin, importin 7, and importin 9 promoted efficient im- Yamamoto (Department of Cellular and Molecular Pharmaco- port of c-Jun into the nucleus; by contrast, importin α inhibited logy, University of California San Francisco, San Francisco, CA, nuclear import of c-Jun in vitro [11]. Importin 13, a recently iden- U.S.A.) [20]. Anti-importin 7 and anti-importin 9 antibodies tified importin β family member, regulates nuclear import of the were a gift from Dr Dirk Gorlich¨ (Department of Molecular Bio- glucocorticoid receptor in airway epithelial cells [12,13]. logy, University of Heidelberg, Heidelberg, Germany). Anti-Ran Ran is a small Ras-like GTP-binding protein that switches and anti-GST antibodies were purchased from Upstate Biotech- between a GTP-bound and a GDP-bound form by GTP hy- nology. Anti-ARHI antibody was produced by our group as drolysis and nucleotide exchange [14]. The GTPase Ran plays previously described in [17]. Adenovirus constructs expressing a crucial role in nucleo-cytoplasmic transport of tumour sup- LacZ, ARHI and NTD were prepared as described in [21]. The pressors, proto-oncogenes, signalling molecules and transcrip- reagents for a nuclear-import assay (digitonin, ATP,GTP,creatine tion factors. The RanGTPase cycle provides directionality to phosphate and creatine kinase) were purchased from Sigma. nucleocytoplasmic transport, regulating interactions between cargoes and nuclear-transport receptors of the importin β family. The Proteomic analysis common principle underlying these diverse functions through- Wild-type ARHI and its NTD were re-expressed in the breast- out the cell cycle is thought to be anisotropy of the distribution cancer cell line SKBr3 using a dual adenovirus system [21]. of RanGTP (the RanGTP gradient), driven by the chromatin- LacZ adenovirus-infected cells were used as a control. ARHI- associated guanine nucleotide-exchange factor RCC1 (regulator interacting proteins were immunoprecipitated using an anti- of chromatin condensation 1) [15]. ARHI monoclonal antibody. In brief, cell lysates were incubated ARHI (aplasia Ras homologue member I) is a maternally with anti-ARHI antibody at 4◦C on a rocker for 90 min, then imprinted tumour-suppressor gene that encodes a 26 kD pro- purified further with prewashed Protein G beads (Pierce) at 4◦C tein with 55–62% homology to Ras and Rap [16]. In contrast on a rocker for 90 min. After washing three times with immuno- with Ras, ARHI contains a 34 amino acid N-terminal extension precipitation wash solution [0.5% (v/v) Triton X-100, 0.5% (v/v) and inhibits the growth, motility and invasion of cancer cells Nonidet P40, 150 mM NaCl, 10 mM Tris/HCl, pH 7.4, 1 mM

[16,17]. Our recent work found that ARHI regulates autophagy EDTA, 1 mM EGTA, 1 mM Na3VO4, 1 mM PMSF and 10% and tumour dormancy in human ovarian-cancer cells by down- glycerol], immunoprecipitates were separated by SDS/PAGE regulating PI3K (phosphoinositide 3-kinase) and Ras/MAPK sig- (15% gel) and analysed by MS at the University of Texas M.D. nalling, thereby down-regulating mTOR (mammalian target of Anderson Cancer Center Proteomics Facility. rapamycin) [18]. ARHI can also interact with the transcription activator STAT3 and inhibit its nuclear translocation and tran- Western-blot analysis scription activity in human breast-cancer and ovarian-cancer cells Conﬂuent SKBr3 cells were lysed with lysis buffer [20 mM [19]. The ARHI NTD (N-terminal deletion mutant) has markedly Tris/HCl, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, reduced growth inhibitory activity, suggesting that this unique 0.5% (v/v) Nonidet P40, 0.5% (v/v) Triton X-100, 1 mM PMSF,

extension may contribute to the inhibitory effects of ARHI on 1mMNa3VO4 and aprotinin 10 μg/ml], the lysates were incub- STAT3-mediated transcriptional activities [19]. To identify addi- ated on ice for 20 min with occasional mixing and clariﬁed by tional ARHI-interacting proteins, we have performed proteomic centrifugation in a microcentrifuge for 5 min at 16000 g at 4◦C. analysis and found that ARHI is complexed with several importin Equal amounts of total cellular proteins were electrophoresed proteins. We have explored the possibility that ARHI might dis- by SDS/15% PAGE and transferred to a PVDF membrane

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Figure 1 ARHI and importin form complexes (A) Proteomic analysis. Wild-type ARHI and its NTD were expressed in SKBr3 breast-cancer cells using a dual adenovirus system. LacZ adenovirus-infected cells served as a control. Lane 1, ARHI complexes; lane 2, NTD complexes; lane 3, LacZ control. Arrows point to protein bands corresponding to the indicated novel binding proteins. kD, kDa. (B–D) Immunoprecipitation of ARHI–importin complexes. Protein lysates from (B) SKBr3 or (C) HeLa cells infected with ARHI, NTD or LacZ adenoviruses were incubated with anti-ARHI antibody. The immunoprecipitates were analysed by Western blotting with anti-importin (Imp) 7, 9 and β1 antibodies. Mouse IgG was used as a control. To confirm the efficiency of the assays, the immunoprecipitates were also analysed by Western blotting with anti-ARHI antibody. (D) Proteins immunoprecipitated with anti-importin 7 antibodies were analysed by Western blotting with anti-ARHI antibody. To confirm the efficiency of the assays, the immunoprecipitates were also analysed by Western blotting with anti-importin 7 antibody.

(Millipore). The membranes were immunoblotted with antibod- The ARHI and NTD cDNA sequences were PCR amplified ies, and signals were measured using an ECL® (enhanced chemi- from pcDNA3-ARHI plasmid DNA and cloned into pQE30 vec- luminescence) system (GE Healthcare) as described previously tor (Qiagen). The importin 7 coding sequence was retrieved from [17]. the GST–importin 7 plasmid by restriction digestion and inserted into pQE30 vector. For the nuclear import substrates, GST–GFP– BIB and GST–GFP–NLS proteins were purified by glutathione– Fusion-protein expression and purification Sepharose beads (GE Healthcare), as described in the manufac- GST, GST–importin α1, α3, α5, α6andα7, GST–importin turer’s instructions. pQE-Ran, pQE-importin 7, pQE-ARHI, and α1 mutant, and GST–importin β1, β7, β8andβ13 were all pQE-NTD were expressed in SG13009 competent cells and pur- expressed in Escherichia coli BL21 (DE3) cells (Stratagene). ified as described by Nachury and Weis [23]. Importin β1was Overnight bacterial cultures in LB (Luria–Bertani)/ampicillin cleaved from the GST–β1 fusion protein by PreScission Protease

(100 μg/ml) were diluted 1/100 and grown to an A595 of 0.6– (GE Healthcare) and importin α7 was cleaved from GST-α7fu- 0.8 at 37◦C. Cultures were cooled, induced with 0.25 mM iso- sion using TEV Protease (Invitrogen), and the mixture was loaded ◦ propyl β-D-thiogalactoside overnight at 16 C and harvested by on to a HiTrap Q cartridge (GE Healthcare) to separate GST from centrifugation at 5000 g for 30 min. Cell pellets were resuspen- the importin proteins. All fusion proteins were concentrated us- ded in lysis buffer [50 mM Tris, pH 8.0, 150 mM NaCl, 1 mM ing Centricon-30 concentrators (Millipore), divided into aliquots ◦ EDTA, 1 mM DTT (dithiothreitol), 1 μg/ml leupeptin, 1 μg/ml (50 μl), flash-frozen in liquid nitrogen and stored at −80 C. pepstatin, 1 μg/ml aprotinin and 1 mM PMSF], sonicated and cla- rified at 40000 g for 30 min at 4◦C. After centrifugation, the su- Labelling of pSTAT3β pernatant fraction was further purified on an AKTA¨ TM FPLC sys- pSTAT3β proteins were FITC-labelled using an FITC Protein tem (GE Healthcare) using a glutathione–Sepharose column and Labeling Kit (Pierce), as described in the manufacturer’s instruc- a SuperdexTM 200 gel-filtration column (both from GE Health- tions. care). Tyrosine phosphorylated STAT3β (pSTAT3β) (residues 127–722) was purified as described in [22]; briefly, the core frag- GST-pulldown assay ment of STAT3β was co-expressed with the Elk kinase in E. coli. GST–importin fusion proteins (2–4 μg) were mixed with 10– The identity of the phosphoprotein was confirmed by MS. 30 μg of cell lysates from SKBr3 cells in binding buffer [20 mM

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Hepes, pH 7.9, 150 mM NaCl, 0.5 mM EDTA, 10% (v/v) glycerol, 0.1% (v/v) Triton X-100 and 1 mM DTT]. The mixture was incubated with shaking at 4◦C for 1 h. A 15 μl volume of glutathione–sepharose beads, pre-equilibrated in TEE buffer (50 mM Tris/HCl, pH 7.9, 1 mM EDTA and 1 mM EGTA) were added to the mixture and incubated with shaking at 4◦Cforan- other 1 h. The beads were collected and washed 4–5 times with washing buffer [20 mM Hepes, pH 7.5, 100 mM KCl, 2.5 mM

MgCl2, 1% (v/v) glycerol and 1% (v/v) Triton X-100] and once with ice-cold PBS. The beads were resuspended in 20 μlof2× SDS loading buffer and then boiled for 5 min. The soluble proteins were separated on SDS/15% PAGE gels for Coomassie Blue staining or Western-blot analysis.

Nuclear-import assay HeLa cells were grown on glass coverslips for 40–42 h. Cells were then washed twice with ice-cold transport buffer (20 mM Hepes, pH 7.3, 110 mM KOAc, 5 mM NaOAc, 2 mM MgOAc, 2 mM DTT and 1 mM EGTA), incubated with 40 μg/ml digitonin in transport buffer for 6 min on ice, washed twice in cold transport buffer and kept on ice for 20 min. Coverslips with permeabilized cells were incubated for 30 min at 30◦C with a 30 μl nuclear- import reaction mixture [2 μM GFP-tagged import substrate or 4 μM FITC-labelled p-STAT3β and an ATP-regenerating system (1 mM ATP, 5 mM creatine phosphate, 10 units/ml creatine kinase and 0.5 mM GTP)]. Some experiments were conducted Figure 2 ARHI–importin binding analysed by GST-pulldown assays with 3 μM RanGDP, 3 μM importin α7, 2 μM importin β1, and (A) ARHI binds to importins. SKBr3 cells were infected with ARHI adenovirus and lysed. A total of ten GST–importin (Imp) fusion proteins and 3 μMARHIor3μM NTD. After the import reactions were com- anti-ARHI antibody were used to detect ARHI–importin binding (upper pleted, the cells were rinsed once in PBS, ﬁxed in 4% (v/v) para- panel). GST protein served as a negative control. Loading was measured with anti-GST antibody (lower panel). (B) Wild-type ARHI binds more formaldehyde in PBS, and analysed by ﬂuorescence microscopy strongly to most importins than does the NTD. Equal amounts of ARHI using an Olympus microscope. or NTD protein lysates were used in GST-pulldown assays. A total of nine GST–importin fusion proteins (upper panel: GST, Imp β1, Imp 7, Imp 8; lower panel: Imp α1, Imp α3, Imp α5, Imp α6, Imp α7) and anti-ARHI antibody were used to detect the binding. GST proteins in the complexes RESULTS were detected by SDS/PAGE. DAPI, 4,6-diamidino-2-phenylindole. kD, kDa.

ARHI interacts with importins binding was greatly reduced compared with wild-type ARHI– Our previous studies have shown that when ARHI and STAT3 importin interaction. were both expressed in SKOv3 cells, ARHI formed a complex with STAT3 in the cytoplasm and prevented IL-6 (interleukin-6)- ARHI can bind to multiple importins induced STAT3 accumulation in the nucleus [19]. To identify the To investigate the ARHI–importin interaction, 10 GST–importin protein(s) interacting with ARHI in the nuclear-import process, fusion proteins (importins α1, α1 mutant, α3, α5, α6, α7, β1, we performed proteomic analysis. Wild-type ARHI and its NTD 7, 8 and 13) were purified. GST protein was used as a negative were expressed in SKBr3 breast-cancer cells with a dual adenov- control. ARHI bound strongly to most of the importins in GST- irus system [21]. Cells infected with the LacZ adenovirus served pulldown assays. Binding of ARHI to importin α1 was stronger as a control. ARHI or NTD complexes were immunoprecipitated than binding of ARHI to an importin α1 mutant that contained using a specific ARHI antibody, and co-immunoprecipitated pro- an altered NLS domain (Figure 2A), suggesting that ARHI may teins were analysed by MS. Several novel interacting proteins bind to importins through the NLS domain. were identified in complexes with wild-type ARHI and showed weaker binding to the NTD mutant. Three of these proteins (Fig- ure 1A) belong to the family of importins: importin 7, importin 9 The N-terminus of ARHI may mediate the binding and importin α re-exporter. These results were further confirmed with importins by co-immunoprecipitation assays. As shown in Figures 1(B) and Our previous studies demonstrated that deletion of the unique 1(C), in SKBr3 and HeLa cells, ARHI antibody could immuno- 34 amino acid N-terminal extension of ARHI nearly abolished precipitate importins 7, 9 and β1, and anti-importin 7 antibody its inhibitory effect on cancer-cell growth [17] and its ability to could also immunoprecipitate ARHI (Figure 1D). NTD–importin block the DNA-binding activity of STAT3 [19]. Thus the ARHI

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Figure 3 ARHI can block pSTAT3 β nuclear translocation (A) STAT3-importin binding analysed by GST-pulldown assays. SKBr3 cells were treated with or without IL-6 (10 ng/ml) for 30 min before cells were harvested for cell lysate preparation. A total of ten GST–importin (Imp) fusion proteins and STAT3/pSTAT3 antibodies were used to detect binding between STAT3 and importins. GST protein was used as a control. Loading was measured with anti-GST antibody. (B) Nuclear-import assay for pSTAT3β–GFP. Nuclear-import assays were performed as described in the Materials and methods section. Puriﬁed pSTAT3β and RanGDP, importin α7 and importin β1 were incubated with permeabilized HeLa cells. Import assays were also performed by adding ARHI protein. DAPI, 4,6-diamidino-2-phenylindole.

N-terminal extension could be important for its biological func- ARHI can block the STAT3 protein tion. In the proteomic analysis and immunoprecipitation assays nuclear-localization signal (Figure 1), NTD protein exhibited greatly reduced binding to Using a similar GST-pulldown assay, STAT3 protein was shown importins. To test further the possible role of the N-terminus of to bind to importins β1, α1, α3, α6andα7, but not to the ARHI in binding importins, equal amounts of ARHI complexes other importins and importin α1 mutant (Figure 3A). After IL-6 and NTD complexes were tested in GST-pulldown assays. As stimulation, the level of pSTAT3 greatly increased. Importins β1, presented in Figure 2(B), wild-type ARHI bound more strongly α1, α3, α6, and α7 bound to both pSTAT3 (after IL-6 stimulation) to most importins than did the NTD mutant, consistent with the and non-phosphorylated STAT3 (without IL-6 stimulation), but possibility that the 34 amino acid N-terminal extension of ARHI these bindings to pSTAT3 were much stronger. We also produced mediates ARHI–importin binding. the pSTAT3β core fragment as described in [22]. Using native gel

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Figure 4 Nuclear-import assay for NLS–GFP The nuclear-import assay was performed as described in the Materials and methods section. NLS–GFP protein was mixed with RanGDP, importin (Imp) α7 and/or importin β1 and incubated with permeabilized HeLa cells. Import assays were also performed by adding an equal amount of ARHI or NTD proteins. DAPI, 4,6-diamidino-2-phenylindole.

electrophoresis, we conﬁrmed that this core fragment can bind puriﬁed ARHI protein blocked Ran-dependent pSTAT3β nuclear to a subset of the importins, namely α3, α6andα7 (K. Zhang translocation. and X. Chen, unpublished work). To investigate if ARHI could block nuclear translocation of STAT3 protein, pSTAT3β was ARHI can block the protein nuclear-localization labelled with FITC and evaluated in the nuclear-import assay. As signal shown in Figure 3(B), pSTAT3β protein alone was concentrated Several studies have shown that interaction of importins with the at nuclear pores; when importins and Ran were added to the Ran protein is required to facilitate the transport of cargo proteins system, pSTAT3β was found in the nucleus. The addition of into the nucleus [14]. Since ARHI and Ran belong to the same

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Figure 5 Nuclear-import assay for BIB–GFP The nuclear-import assay was performed as described in the Materials and methods section. BIB–GFP protein was mixed with RanGDP and/or importin (Imp) 7 and incubated with permeabilized HeLa cells. Import assays were also performed by adding an equal amount of ARHI or NTD proteins. DAPI, 4,6-diamidino-2-phenylindole.

family of small G-proteins, ARHI therefore might antagonize translocation (Figures 4 and 5). In comparison, NTD protein did the interaction of importins with Ran. To test this hypothesis, we not block these interactions or nuclear transport (Figures 4 and 5). have puriﬁed ARHI and NTD proteins and assessed their effects on nuclear transport of cargo proteins in HeLa cells. In nuclear- import assays, an NLS–GFP fusion protein could be imported ARHI can compete for importin–Ran binding into the nucleus in association with importin α7, importin β1and To determine whether ARHI binds to the same region of RanGDP (Figure 4), whereas the BIB–GFP fusion protein only importins as Ran and could compete for importin–Ran binding, needed help from importin 7 and RanGDP for nuclear localization we performed GST-pulldown competition assays using GST– (Figure 5). ARHI protein blocked the interaction of NLS with importin fusion proteins. pSTAT3, GFP–NLS and GFP–BIB Ran–importin β and α complexes, and also the interaction of proteins were bound to these GST proteins, and ARHI protein BIB with Ran–importin 7 complexes, preventing their nuclear was added for competition. NTD protein served as a control. As

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Using GST–Ran fusion proteins, importins 13 and β1 were strongly bound to Ran, and ARHI protein could re- duce these binding abilities by at least 50% (Figure 6C). NTD protein did not have competitive activity in these assays.

DISCUSSION

STAT3, a latent transcription factor, transduces signals from the cell surface to the nucleus and activates gene transcription, trig- gering proliferation, resistance to apoptosis, motility, invasion and angiogenesis [24,25]. Previous studies clearly demonstrate that STAT3 is required for both tumour initiation and promotion [24]. STAT3 is frequently phosphorylated and activated in the majority of breast and ovarian cancers [26], where cytokines and growth factors, such as IL-6, bind to specific receptors and activate JAK2 which, in turn, phosphorylates STAT3 and prompts translocation of pSTAT3 to the nucleus. The classic NLS–importin pathway has been reported to mediate the nuclear translocation of STAT3. Liu et al. [27] reported that STAT3 nuclear import is mediated by importin α3, whereas other studies have shown that the regulation of STAT3 nuclear import is through importins α5andα7 [28]. Few studies investigated factors that regulate the translocation of pSTAT3 [29,30]. From our previous studies, re-expression of the putative tumour suppressor gene ARHI in cancer cells markedly inhibited binding of pSTAT3 to STAT response elements in target gene promoters and down-regulated STAT3-dependent promoter activity without significantly affecting STAT3 phosphorylation [19]. When ARHI and STAT3 were co-expressed in SKOv3 ovarian-cancer cells, ARHI formed a complex in the cytoplasm with STAT3 and prevented IL-6-induced STAT3 translocation to the nucleus [19]. The present study has elucidated the mechanism by which ARHI prevents nuclear translocation of pSTAT3. Figure 6 GST-pulldown competition for Ran–importin binding (A) ARHI competes for pSTAT3-importin α7 binding. GST–importin α7 Nuclear-import assays have shown that pSTAT3 could be trans- (Imp α7) fusion proteins were bound to glutathione–Sepharose 4B, located to the nucleus in the presence of Ran and importins (Fig- then incubated with p-STAT3 protein, treated with an equal amount ure 3B), whereas non-phosphorylated STAT3 could not (results of ARHI or NTD proteins and then blotted with monoclonal antibodies to p-STAT3 and ARHI. The input of pSTAT3 was detected by Western not shown). ARHI protein bound to importins (Figures 1 and 2) blotting, and the input of ARHI and NTD was determined by Coomassie and blocked nuclear translocation of pSTAT3 (Figure 3B). ARHI staining. (B) ARHI competes for NLS–importin α1 and BIB–importin 7 also blocked nuclear translocation of NLS–GFP and BIB–GFP binding. GST–importin α1(Impα1) and GST–importin 7 (Imp 7) fusion proteins were bound to glutathione–Sepharose 4B, then incubated with in the presence of Ran and appropriate importins (Figures 4 and GFP–NLS or GFP–BIB proteins, treated with an equal amount of ARHI 5). Thus ARHI might affect nuclear localization of STAT3, but or NTD proteins and then blotted with anti-GFP monoclonal antibody. might also affect transport of other proteins that are required for The input of ARHI and NTD was determined by Coomassie staining, and the input of GFP–BIB and GFP–NLS was detected by Western blotting. oncogenesis. (C) ARHI competes for importin–Ran binding. GST–Ran fusion proteins The precise mechanism by which ARHI disrupts the interac- were bound to glutathione–Sepharose 4B, then incubated with importin tion of cargo proteins, Ran and importins remains to be elucid- β1(Impβ1)-His6 or importin 13 (Imp 13)-His6, treated with ARHI or NTD proteins, then blotted with monoclonal antibodies to His6 or a polyclonal ated. ARHI is a member of the Ras superfamily, but contains a antibody to importin 13. The input of ARHI and NTD was determined by unique 34 residue extension at the N-terminus. Like Ras, ARHI Coomassie staining. can bind to GTP with high affinity, but has low intrinsic GTP- ase activity [17]. ARHI associates with the cell membrane after Figures 6(A) and 6(B) show, ARHI can significantly block prenylation at the C-terminal cysteine residue. Mutation of the the binding of NLS–importin α1, as well as partially block the conserved CAAX box at the C-terminus leads to a loss of binding of BIB–importin 7 and pSTAT3–importin α7. its membrane-association ability and a modest decrease in its

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ability to inhibit cell growth [16]. Most strikingly, deletion of the and GM68556 (to X.C.)]; from the National Foundation for Can- unique N-terminal extension of ARHI nearly abolishes its inhib- cer Research [grant number LF 2004-00009224HM (to R.C.B.)]; itory effect on cell growth [17,19]. In the present study, the NTD from the National Natural Science Foundation of China [grant num- lost much of the ability of the wild type protein to bind to im- bers 3047085 and 90608007 (to T.T.)] and from the Ministry of portins and did not block the nuclear translocation of NLS–GFP Science and Technology of China [grant number 2006AA02A310 or BIB–GFP. This suggests that interaction of the N-terminal (to T.T.)]. extension of ARHI with importin may be required to prevent effective interaction of Ran–importin complexes with NLS and BIB. Interestingly, C-terminal deletion mutants of ARHI loc- alize in the nucleus (results not shown), raising the possibility that accumulation of pSTAT3 in the cytoplasm in the presence REFERENCES of wild-type ARHI protein could result from direct binding of ARHI to STAT3, which we have demonstrated [19], or to lack of an effective nuclear-transport mechanism. As wild-type ARHI 1 Terry, L. J., Shows, E. B. and Wente, S. R. (2007) Crossing the exhibits a nuclear localization signal on its N-terminal exten- nuclear envelope: hierarchical regulation of nucleocytoplasmic transport. Science 318, 1412–1416 sion, the ARHI might compete with cargo for binding to im- 2 Herrmann, A., Vogt, M., Monnigmann,¨ M., Clahsen, T., Sommer, U., portins in the presence of Ran. Once bound, ARHI could trap Haan, S., Poli, V., Heinrich, P.C.andMuller-Newen,¨ G. (2007) importins in the cytoplasm as the small G-protein is tethered to Nucleocytoplasmic shuttling of persistently activated STAT3. cell membranes through its prenylated C-terminus. J. Cell Sci. 120, 3249–3261 ARHI [also known as DIRAS3 (DIRAS family, GTP-binding 3 Ivanova, I. A., Vespa, A. and Dagnino, L. (2007) A novel mechanism RAS-like 3)] and Ran share a 22% sequence identity and of E2F1 regulation via nucleocytoplasmic shuttling: determinants of nuclear import and export. Cell Cycle 6, 2186–2195 are expected to have a similar overall structure. Although the 4 Buse, P., Maiyar, A. C., Failor, K. L., Tran, S., Leong, M. L. and crystal structure of ARHI is not yet available, those of the Firestone, G. L. (2007) The stimulus-dependent co-localization of closest neighbours in the Ras family, DIRAS1 and DIRAS2, serum- and glucocorticoid-regulated protein kinase (Sgk) and have been solved. 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Mol Cell. 16, 319–330 (Figure 6), suggesting that ARHI binds to the same region 10 Jakel,¨ S., Albig, W., Kutay, U., Bischoff, F. R., Schwamborn, K., of importins as does Ran, and ARHI-induced disruption of Doenecke, D. and Gorlich,¨ D. (1999) The importin beta/importin 7 heterodimer is a functional nuclear import receptor for histone H1. Ran–importin binding could serve as an important regulatory EMBO. J. 18, 2411–2423 mechanism that contributes to the tumour-suppressor function of 11 Waldmann, I., Walde,¨ S. and Kehlenbach, R. H. (2007) Nuclear ARHI. import of c-Jun is mediated by multiple transport receptors. J. Biol. Chem. 282, 27685–27692 12 Tao, T., Lan, J., Lukacs, G. L., Hache,´ R. J. and Kaplan, F. (2006) Importin 13 regulates nuclear import of the glucocorticoid receptor ACKNOWLEDGEMENTS in airway epithelial cells. Am. J. Respir. Cell Mol. Biol. 35, 668–680 We gratefully thank Dr Walter N. Hittelman, M.D. Anderson Cancer 13 Quan, Y., Ji, Z., Wang, X., Tartakoff, A. M. and Tao, T. (2008) Center, Houston, TX, U.S.A., for help with microscope observations. Evolutionary and transcriptional analysis of karyopherin β superfamily proteins. Mol. Cell. Proteomics 7, 1254–1269 14 Moore, M. S. and Blobel, G. (1993) The GTP-binding protein Ran/TC4 is required for protein import into the nucleus. Nature 365, 661–663 FUNDING 15 Kalab,´ P., Pralle, A., Isacoff, E. Y., Heald, R. and Weis, K. (2006) This work was supported by grants from the National Cancer In- Analysis of a RanGTP-regulated gradient in mitotic somatic cells. stitute [grant numbers CA 80957 (to Y.Y.), CA 64602 (to R.C.B.) Nature 440, 697–701

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16 Yu, Y., Xu, F., Peng, H., Fang, X., Zhao, S., Li, Y., Cuevas, B., Kuo, 24 Haura, E. B., Turkson, J. and Jove, R. (2005) Mechanisms of W. L., Gray, J. W., Siciliano, M. et al. (1999) NOEY2 (ARHI), an disease: insights into the emerging role of signal transducers and imprinted putative tumor suppressor gene in ovarian and breast activators of transcription in cancer. Nat. Clin. Pract. Oncol. 2, carcinomas. Proc. Natl. Acad. Sci. U.S.A. 96, 214–219 315–324 17 Luo, R. Z., Fang, X., Marquez, R., Liu, S. Y., Mills, G. B., Liao, 25 Xie, T. X., Wei, D., Liu, M., Gao, A. C., Ali-Osman, F., Sawaya, R. W. S., Yu, Y. and Bast, R. C. (2003) ARHI is a Ras-related small and Huang, S. (2004) Stat3 activation regulates the expression of G-protein with a novel N-terminal extension that inhibits growth of matrix metalloproteinase-2 and tumor invasion and metastasis. ovarian and breast cancers. Oncogene 22, 2897–2909 Oncogene 23, 3550–3560 18 Lu, Z., Luo, R. Z., Lu, Y., Zhang, X., Yu, Q., Khare, S., Kondo, S., 26 Alvarez, J. V., Febbo, P. G., Ramaswamy, S., Loda, M., Richardson, Kondo, Y., Yu, Y., Mills, G. B., Liao, W. S. and Bast, Jr, R. C. (2008) A. and Frank, D. A. (2005) Identiﬁcation of a genetic signature of The tumor suppressor gene ARHI regulates autophagy and tumor activated signal transducer and activator of transcription 3 in dormancy in human ovarian cancer cells. J. Clin. Invest. 118, human tumors. Cancer Res. 65, 5054–5462 3917–3929 27 Liu, L., McBride, K. M. and Reich, N. C. (2005) STAT3 nuclear 19 Nishimoto, A., Yu, Y., Lu, Z., Mao, X., Ren, Z., Watowich, S. S., import is independent of tyrosine phosphorylation and Mills, G. B., Liao, W. S., Chen, X., Bast, Jr, R. C. and Luo, R. Z. mediated by importin-α3. Proc. Natl. Acad. Sci. U.S.A. 102, (2005) ARHI directly inhibits STAT3 translocation and activity in 8150–8155 human breast and ovarian cancer cells. Cancer Res. 65, 28 Ma, J. and Cao, X. (2006) Regulation of Stat3 nuclear import by 6701–6710 importin α5 and importin α7 via two different functional sequence 20 Freedman, N. D. and Yamamoto, K. R. (2004) Importin 7 and elements. Cell Signal. 18, 1117–1126 importin α/importin β are nuclear import receptors for the 29 Kawashima, T., Bao, Y. C., Minoshima, Y., Nomura, Y., Hatori, T., glucocorticoid receptor. Mol. Biol. Cell. 15, 2276–2286 Hori, T., Fukagawa, T., Fukada, T., Takahashi, N., Nosaka, T. et al. 21 Bao, J. J., Le, X. F., Wang, R. Y., Yuan, J., Wang, L., Atkinson, E. N., (2009) A Rac GTPase-activating protein, MgcRacGAP,isa LaPushin, R., Andreeff, M., Fang, B., Yu, Y. and Bast, Jr, R. C. nuclear localizing signal-containing nuclear chaperone in the (2002) Reexpression of the tumor suppressor gene ARHI induces activation of STAT transcription factors. Mol. Cell. Biol. 29, apoptosis in ovarian and breast cancer cells through a 1796–1813 caspase-independent calpain-dependent pathway. Cancer Res. 62, 30 Liu, Y. P., Tan, Y. N., Wang, Z. L., Zeng, L., Lu, Z. X., Li, L. L., Luo, 7264–7272 W., Tang, M. and Cao, Y. (2008) Phosphorylation and nuclear 22 Becker, S., Corthals, G. L., Aebersold, R., Groner, B. and Muller,¨ translocation of STAT3 regulated by the Epstein–Barr virus latent C. W. (1998) Expression of a tyrosine phosphorylated, DNA binding membrane protein 1 in nasopharyngeal carcinoma. Int. J. Mol. Stat3β dimer in bacteria. FEBS Lett. 441, 141–147 Med. 21, 153–162 23 Nachury, M. V. and Weis, K. (1999) The direction of transport 31 Vetter, I. R., Arndt, A., Kutay, U., Gorlich,¨ D. and Wittinghofer, A. through the nuclear pore can be inverted. Proc. Natl. Acad. Sci. (1999) Structural view of the Ran-importin β interaction at 2.3 A˚ U.S.A. 96, 9622–9627 resolution. Cell 97, 635–646

Received 13 January 2009/22 April 2009; accepted 13 May 2009 Published as Immediate Publication 13 May 2009, doi 10.1042/BSR20090008

...... 168 C The Authors Journal compilation C 2010 Biochemical Society