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Nesprin-2 Interacts with �-Catenin and Regulates WntSignaling at the Nuclear Envelope*□S
Received for publication, March 3, 2010, and in revised form, August 9, 2010 Published, JBC Papers in Press, August 26, 2010, DOI 10.1074/jbc.M110.119651
Sascha Neumann‡, Maria Schneider‡, Rebecca L. Daugherty§, Cara J. Gottardi§, Sabine A. Eming¶, Asa Beijer‡,Angelika A. Noegel‡1, and Iakowos Karakesisoglou�2
From the ‡Institute of Biochemistry I, Medical Faculty, Center for Molecular Medicine, University of Cologne, Cologne ExcellenceCluster on Cellular Stress Responses in Aging-associated Diseases, and the ¶Department of Dermatology, University of Cologne,50931 Cologne, Germany, the §Department of Medicine, Northwestern University Feinberg School of Medicine,Chicago, Illinois 60611, and the �Department of Biological Sciences, School of Biological and Biomedical Sciences,University of Durham, DH1 3LE Durham, United Kindgom
Nesprins and emerin are structural nuclear envelope pro-teins that tether nuclei to the cytoskeleton. In this work,we identified the cytoskeleton-associated �-N/E-catenins asnovel nesprin-2-binding partners. The association involvesthe C termini of nesprin-2 giant and �-N/E-catenins. �-E/T/N-catenins are known primarily for their roles in cadherin-mediated cell-cell adhesion. Here, we show that, in addition,�-catenin forms complexes with nesprin-2 that include�-catenin and emerin. We demonstrate that the depletion ofnesprin-2 reduces both the amount of active �-catenin insidethe nucleus and T-cell factor/lymphoid-enhancing factor-de-pendent transcription. Taken together, these findings suggestnovel nesprin-2 functions in cellular signaling. Moreover, wepropose that, in contrast to emerin, nesprin-2 is a positive reg-ulator of the Wnt signaling pathway.
Nesprin-1, -2, -3, and -4 (nuclear envelope spectrin repeat-containing proteins) are integral nuclear membrane proteins,greatly varying in size and domain composition (1–3). The larg-est nesprin-1 (1.01 MDa) and nesprin-2 (796 kDa) isoformsencompass an N-terminal paired calponin homology domainthat mediates F-actin binding. The C-terminal KASH (Klar-sicht/ANC-1/Syne homology) domain targets nesprins to thenuclear envelope (NE).3 These conserved domains are sepa-rated by a long stretch of spectrin repeats.
Nesprins play pivotal roles in maintenance of NE integrity(4), nuclear positioning (5), and anchorage to the cytoskeletonand the centrosome (2). Although compelling evidence under-lines nesprin roles in several human diseases that range fromcerebellar ataxia to dystrophy-like phenotypes; the underlyingmolecular mechanisms remain elusive (6).Here, we unravel novel nesprin-2 interactions with �-cate-
nin. The latter, together with �-catenin, is known for its role incell-cell adhesion. Cadherins are transmembrane proteins ofthe plasma membrane (PM), which play key roles in cell adhe-sion. The cytoplasmic tail of cadherin binds to�-catenin, whichin turn associates with �-catenin. This adhesion complex isdynamically connected to the cortical actin cytoskeleton (7).
�-Catenin has an additional role in the canonicalWnt signalingpathway by transferring signals from the PM into the nucleus.When theWntpathway is not active, cytoplasmic�-catenin levelsare kept low by protein degradation. Upon Wnt pathway activa-tion, �-catenin accumulates in the cytoplasm and enters thenucleus, where it acts as a transcription factor (8).In this work, we demonstrate �-catenin interactions that
involve �-catenin and NE-associated nesprin-2 and emerin.From our data, we propose a mechanism by which these NEassociations regulate nuclear �-catenin levels and Wnt signal-ing-dependent transcription.
EXPERIMENTAL PROCEDURES
Plasmids—The amino acid positions of nesprin-2 proteinsrefer to nesprin-2 giant (9). GST, GFP, and yeast two-hybridfusion proteins were cloned into pGEX-4T (GE Healthcare),pEGFP-C (Clontech), and pGBKT-7 and pGADT-7-Rec (Clon-tech). Nesprin-2-SR-(6146–6799), SR1-(6146–6241), SR2-(6247–6347), SR2–3-(6348–6552), SR1�2-(6146–6347), SR2�3-(6247–6656), and SR3�4-(6553–6799)were used in this study.The amino acid positions of �-N-Cat* proteins refer to �-N-catenin-4 (NCBI accession number AK295181.1), �-N-Cat*-(706–860), �-N-Cat*�Ex18-(706–765), �-N-Cat*�Ex18/15-(706–730), �-N-Cat*�Ex14-(731–860), Myc-�-E-catenin,and GFP-�-N-catenin-1 (according to IMAGE clone accessionnumber 6187158). For SP-GFP-SUN-1-C, the signal peptide(SP) sequence of torsinA and enhancedGFPwere isolated frompcDNAV5-His-GFP-hTORA (a gift fromDr.W. T. Dauer) (10)and combined with SUN-1-C (11) in pcDNA3.1 (Invitrogen).
* The work was supported by the Center for Molecular Medicine, University ofCologne, the Cologne Excellence Cluster on Cellular Stress Responses inAging-associated Diseases, and Bundesministerium fur Bildung und Fors-chung-Deutsches Zentrum fur Luft- und Raumfahrt Grant MD-NET R23 (toA. A. N. and I. K.), National Institutes of Health Grant GMS076561 (toC. J. G.), and an American Heart Association predoctoral award (to R. L. D.).
□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. S1–S5.
1 To whom correspondence may be addressed: Inst. for Biochemistry I, Med-ical Faculty, University of Cologne, Joseph-Stelzmann-Str. 52, 50931Cologne, Germany. Tel.: 49-22-1478-6980; Fax: 49-22-1478-6979; E-mail:[email protected].
2 To whom correspondence may be addressed: Dept. of Biological Sciences,School of Biological and Biomedical Sciences, University of Durham, SouthRd., DH1 3LE Durham, UK. Tel.: 44-19-1334-1299; Fax: 44-19-1334-1201;E-mail: [email protected].
3 The abbreviations used are: NE, nuclear envelope; PM, plasma membrane;SR, spectrin repeat; Ex, exon; SP, signal peptide; TRITC, tetramethylrhod-amine isothiocyanate.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 45, pp. 34932–34938, November 5, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
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SP-GFP-SUN-1-C comprises the C terminus of SUN-1,including the coiled-coil regions and the SUN domain (see Fig.4C). The N terminus is replaced with a GFP tag and a signalpeptide derived from torsin A (10) that targets the protein tothe perinuclear space (Fig. 4E, arrow) and the endoplasmicreticulum (Fig. 4E, arrowhead). Based on its structure, theSP-GFP-SUN-1-C protein is not anchored in the NE but ispresent in the perinuclear space and endoplasmic reticulum,resulting in a displacement of the nesprins from the NE (Fig.4E�, asterisk). SP-GFP is a fusion protein comprising the signalpeptide of torsin and GFP. Untransfected and transiently SP-GFP- or SP-GFP-SUN-1-C-expressing HaCaT cells were frac-tionated into the cytoplasm and nuclei.Yeast Two-hybrid Screening—Matchmaker Two-hybrid
System 3 was used following the yeast protocols handbook(PT3024-1, Clontech). SR was cloned into the yeast pGBKT-7plasmid (9). This bait was used to screen a pretransformedhuman brain cDNA library in pGADT-7-Rec expression vec-tors used as a prey. Positive clones were isolated, sequenced,and retransformed with the bait to confirm the interaction.Cell Culture—The following cell lineswere employed:COS-7
(12) and HaCaT (13). Primary human keratinocytes were pro-vided by Nils Buchstein and cultivated according to Rheinwaldand Green (14).Cell Transfection—COS-7 cells were transfected usingGene-
Pulser� II (Bio-Rad) at 170 V and 950microfarads. HaCaT cells
were transfected twice at intervals of 3 days using the Amaxacell lineNucleofector� kit V (Lonza) according to themanufac-turer’s instructions.GSTPulldown—COS-7 cells expressingMyc- orGFP-tagged
proteins were lysed in lysis buffer (50 mM Tris-HCl (pH 7.5),150 mM NaCl, 1% Nonidet-P40, and 0.5% sodium deoxy-cholate). After preclearing lysates for 1 h with beads, sampleswere incubated with GST fusion proteins, GST-coupled beads,or beads alone at room temperature for 2 h. Finally, the beadswere washed with lysis buffer and PBS. For Fig. 4A (1st ap-proach), the pulldown included two incubation steps. The firstincubationwas performed as described above to ensure bindingofGFP-�-N-catenin-1 (Input 1) toGST-SR1�2. For the secondincubation (Input 2), COS-7 cells were lysed in 0.5 ml of PBSwith 0.075% Nonidet-P40 (the detergent concentration wasreduced by doubling the volume with PBS). Lysates were incu-bated with GST fractions from the first incubation step over-night at 4 °C and washed with PBS containing 0.075% NonidetP-40 and PBS. For Fig. 4A (2nd approach), we used only thesecond incubation step as described above. Samples were ana-lyzed by Western blotting using the antibodies indicated.Co-immunoprecipitation—HaCaT cells were lysed in lysis
buffer or in PBS containing 0.075% Nonidet P-40. The volumewas doubled to decrease the detergent concentration. Afterpreclearing the lysates for 1 h with protein A-Sepharose CL-4Bbeads (GE Healthcare), lysis buffer lysates were incubated for
FIGURE 1. �-N-Cat*, a novel nesprin-2-binding partner. A, nesprin-2 giant and nesprin-2-SR schematic (modified from Ref. 4) focusing on the most conservedinstead of all predicted spectrin repeats (28). Nesprin-2 domains and antibody epitopes (inverted Y) are indicated. B and C, GFP-SR fusion proteins accumulatein the cytoplasm (arrowhead), at the PM (arrow), and along the NE (asterisk) in COS-7 cells. The DNA was stained with DAPI (Merge panel). D, SR and �-N-Cat*interact in a yeast two-hybrid assay. Panel A, growth on selection plates; panel B, �-galactosidase assay. E, schematic of CTNNA2 (�-N-catenin) and its isoforms.
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2 h with 6 �g of pAbK1 to allow antigen-antibody coupling,followed by a second incubation for 2 h with protein A-Sepha-rose beads. Subsequently, the beads were washed twice withPBS and incubated overnight with PBS lysates. Finally, thebeads were washed five times with PBS, and the fractions wereanalyzed by Western blotting.Subcellular Fractionation—Cells were resuspended in hypo-
tonic buffer (10mMHEPES (pH 7.9), 1.5mMMgCl2, and 10mM
KCl) and incubated for 10 min. Cells were lysed by passagethrough a needle (27Gx3/4�, 10 times) and centrifuged (10min,1000 rpm) 5 min after the 10-min incubation step. Pellets(nuclei) were washed four to six times with PBS. Supernatants(cytoplasm) were centrifuged (20min, 12000 rpm) and used forprotein analysis.Immunofluorescence and Immunoblotting Reagents—The
primary antibodies used were directed against �-catenin(epitope amino acids 890–902, Sigma), nesprin-2 (pAbK1,
K20–478) (1, 9), Myc (9E10) (15),GFP (K3–184-2), emerin (4G5,Abcam), �-catenin (6F9, Sigma),active �-catenin (8E7, Millipore),actin (Ac-74, Sigma), Lap-2 (27, BDTransduction Laboratories), LamB1(Abcam), GAPDH (71.1, Sigma),�-tubulin (WA3,U. Euteneuer), andBrdU (BD Biosciences). The sec-ondary antibodies used were AlexaFluor 488/568 conjugates (Sigma).Nuclei were stained with DAPI,and actin was stained with TRITC-phalloidin (Sigma). Immunofluo-rescence analysis and microscopywere performed as described (1).RNA Interference—Human nesprin-
2 knockdown was obtained by plas-mid-based RNAi. Oligonucleotideswere cloned into pSHAG-1 (BseRIand BamHI) (16): Nes-2ct1-s, GAG-AAGAAACTCAAACAGTGAAG-CTTGACTGTTTGAGTTTCTT-CTCTTTTTT; Nes-2ct1-as, GAT-CAAAAAAGAGAAGAAACTCA-AACAGTCAAGCTTCACTGTT-TGAGTTTCTTCTCCG;Nes-2ct2,CCAGCCTCCTGCAACATCCG-AAGCTTGGGATGTTGCAGGA-GGCTGGTTTTTT; Nes-2ct2-as,GATCCCAGCCTCCTGCAACA-TCCCAAGCTTCGGATGTTGC-AGGAGGCTGGCG; Control-s,ATCTACTCGACGTGAGCGTG-AAGCTTGACGCTCACGTCGA-GTAGATTTTTT; and Control-as,GATCAAAAAATCTACTCGAC-GTGAGCGTCAAGCTTCACGC-TCACGTCGAGTAGATCG.TOP/FOP Promoter Assay—
HEK293T were transiently trans-fected with C-terminal nesprin-2 shRNA1 or shRNA2 or withthe corresponding control using Lipofectamine 2000 (Invitro-gen). The cells were transfected twice at intervals of 3 days. Thefirst transfection was with shRNA alone. The second transfec-tion was with shRNA-, TOPflash-, or FOPflash-luciferasereporters and a polymerase III-Renilla luciferase controlreporter. 5 days after the first transfection, cells were treatedovernight with 30 mM LiCl to induce the Wnt pathway. TheTOP/FOP promoter assay was carried out using the Dual-Lu-ciferase reporter assay system (Promega) according to theman-ufacturer’s instructions. The experiments were done threetimes each in duplicate. A representative graph is shown.BrdU Incorporation Assay—HaCaT cells were transiently
transfected with C-terminal nesprin-2 shRNA1 or the corre-sponding control, plated on glass plates, and incubated with 10�g/ml BrdU (Sigma) under growing conditions for 2 h. Thecells were fixed with 4% paraformaldehyde and permeabilized
FIGURE 2. Nesprin-2 interacts in vitro with �-N-Cat*. A and B, scheme of GST-nesprin-2-SR and GFP-�-N-Cat*fusion proteins, respectively. Spectrin repeats are shown are red ovals, and polypeptides encoded by exons 14,15, and 18 are shown as blue ovals. The amino acid positions refer to nesprin-2 giant and �-N-catenin-4.C, GST-nesprin-2 pulldown of GFP-�-N-Cat* and C-terminally truncated �-N-Cat* proteins. Supernatants (S)and precipitates (�-N-Cat*, �-N-Cat*�Ex18, and �-N-Cat*�Ex18/15) were analyzed by Western blotting (WB)with GFP-specific mAb K3–184-2. D, GST-nesprin-2 pulldown of N-terminally truncated GFP-�-N-Cat* protein(�-N-Cat*�Ex14)-expressing lysates.
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with 0.4% Triton X-100. To allow the anti-BrdU antibodyaccess to the antigen, cells were washed with PBS and treatedwith 2 M HCl for 30 min at room temperature, followed byfurther washing with PBS. Immunofluorescence analysis wasperformed as described above.
RESULTS
�-N-catenin, a Novel Nesprin-2-binding Partner—To gaininsights into nesprin-2 biology, we performed a yeast two-hy-brid screening using a nesprin-2 giant C-terminal fragment(SR), which resembles nesprin-2�TM1 (17), as bait (Fig. 1A).Nesprin-2-SR represents a domain common to most nesprin-2isoforms (4) and accumulates at the NE of COS-7 cells, al-though it lacks a KASH domain (Fig. 1, B and C, asterisks). Weconfirmed that the NE localization was not mediated by theGFP tag by transfecting the empty vector into COS-7 cells,where GFP alone could not be detected at the NE (supplemen-tal Fig. S1, A and B, asterisks).The yeast two-hybrid screening revealed a C-terminal frag-
ment of the neuron-specific �-N-catenin termed �-N-Cat*(Fig. 1,D and E). The corresponding gene,CTNNA2, comprises18 exons and gives rise to three alternatively spliced isoforms(18). In contrast to �-N-catenin-1, �-N-catenin-2 harbors aC-terminal 48-amino acid insertion (exon 17) (Fig. 1E) (19).�-N-catenin-3 possesses an alternative transcription initiation
site (Fig. 1E, triangle) and does not contain the first six exonsand exon 17 (18). �-N-Cat* appears to belong to an additionalisoform (NCBI accession number AK295181.1) lacking exons16 and 17, which we designated as �-N-catenin-4 (Fig. 1E). Weverified the interaction of nesprin-2 and�-N-Cat* byGST pull-down and narrowed down the respective binding sites (Fig. 2).The studies revealed that several �-N-Cat* proteins, namely�-N-Cat*, �-N-Cat*�Ex18, and �-N-Cat*�Ex18/15, showedthe strongest binding for SR1�2. Further analysis demon-strated that SR2 is sufficient for mediating the interaction,although the binding appeared to be weaker compared withthe larger proteins (Fig. 2C). Moreover, two distinct nesprin-2-binding sites were identified in �-N-Cat*, including theexon 15- and 18-encoded (Fig. 2D) and exon 14-encoded(Fig. 2C) regions. The interaction is not unique to�-N-Cat*, asinteractions with �-N-catenin-1 and �-E-catenin could also bedemonstrated (supplemental Fig. S2A). Because �-E/T/N-catenins are conserved and tissue-specific (20), we concludethat nesprin-2 interactions can take place in a tissue-indepen-dent manner.In an immunoprecipitation using the anti-nesprin-2 polyclonal
antibody pAbK1, which detects a large number of isoforms,including the 800-kDa nesprin-2 giant, we precipitated endoge-nous �-catenin from HaCaT cells (supplemental Fig. S2B). From
FIGURE 3. �-Catenin and nesprin-2 localize to the NE and PM in primary human keratinocytes. A, upon Ca2� level reduction, adherens junction formationwas prevented, and �-catenin colocalized with nesprin-2 at the NE (A and A�, arrow). B, under high Ca2� conditions, �-catenin localized to PM junctionalcomplexes (arrow). Colocalization with nesprin-2 (B�) at the NE was no longer detectable (B�). C and D, optical sections through cells kept under low Ca2�
conditions. C� and D�, phalloidin staining indicated the lack of cell-cell contacts (D�, arrowhead). Nesprin-2, visualized with pAbK1 (Nesprin-2-K1), localized to theNE (C, asterisk). High Ca2� triggered cell contact formation (E�, arrow). Nesprin-2 remained at the NE (E, arrowhead) and additionally localized to the PM (E,asterisk). F, HaCaT keratinocytes were transiently transfected with Myc-�-E-catenin (arrow). No differences in the localization of nesprin-2 between untrans-fected cells (F�, arrowhead) and �-catenin-overexpressing cells (F�, asterisk) were detectable. G, GFP-SR accumulated in the cytoplasm (arrowhead), the PM(arrow), and the NE (asterisk) of HaCaT cells. The localization of endogenous �-E-catenin (G�) along the PM was unaffected. A�–G�, merge, scale bars � 10 �m.
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this, we additionally conclude that, in vivo, the interactionbetween both proteins is not limited to isoforms composed ofthe nesprin-2-SR sequence.
�-Catenin and Nesprin-2 Localize at the NE and PM—Bear-ing in mind that �-catenin is a core adherens junctions compo-nent, we analyzed its subcellular distribution in primary humankeratinocytes. Cells were grownunder low (50�MCaCl2) andhigh (2mM CaCl2) Ca2� conditions toinhibit or favor adherens junctionformation, respectively. Under highCa2� conditions, �-catenin local-ized to the PM (Fig. 3B, arrow); sig-nals surrounding the nucleus couldonly be observed sporadically(supplemental Fig. S3A, arrow).Nesprin-2 localized along the NE(Fig. 3B�). In contrast, under lowCa2� conditions, the depletion ofcell-cell contacts triggered a dra-matic redistribution of �-cateninfrom the PM to a perinuclear posi-tion (supplemental Fig. S3B,arrows). A continuous NE labelingwas observed sporadically (Fig. 3, Aand A�, arrows).Next, different GFP- and Myc-
tagged �-catenin proteins were tran-siently expressed in COS-7 cells andHaCaT keratinocytes, which differmarkedly in theirability to formadhe-rens junctions (supplemental Fig. S4).In HaCaT, GFP-�-N-catenin-1 (sup-plemental Fig. S4A, arrow) and Myc-�-E-catenin (Fig. 3F, arrow) werealmost found exclusively at the PM.COS-7 cells exhibited a different�-catenin distribution, as both �-E-catenin-1 and �-N-catenin-1 local-ized along the NE in the majority ofcells (supplemental Fig. S4, B, C, E,and F, arrows, and I). Intriguingly,N-terminally truncated �-N-cate-nin fusion proteins were distributedthroughout the cytoplasm (supple-mental Fig. S4D, arrowhead) andalong the NE in HaCaT cells (sup-plemental Fig. S4, D, arrow, and I).In COS-7 cells, C-terminal �-cate-nin polypeptides were much morefrequently found along the NE, par-alleling the full-length proteins(supplemental Fig. S4, G and H,arrows, and I). In HaCaT keratino-cytes, �-catenin seemed to be keptprimarily at the PM, probably byinteractions mediated by its Nterminus.
To determinewhether nesprin-2 could be detected at the PMof primary human keratinocytes as well, its localization wasassayed under different Ca2� conditions. Under low Ca2�
conditions (Fig. 3,C andD), nesprin-2was found at theNE (Fig.3C, asterisk). Different Z-sections are shown to demonstratethe absence of cell contacts, whichwere visualized by phalloidin
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(Fig. 3D�, arrowhead). Upon Ca2� addition, nesprin-2 wasdetected at the NE (Fig. 3E, arrowhead) and along the PM (Fig.3E, asterisk) by pAbK1 when NE staining was overexposed.Because nesprin-2 was detectable at the PM in pAbK1-stainedcells (Fig. 3E) but not in mAb K20–478-stained cells (Fig. 3, A�and B�), we assume that nesprin-2 isoforms lacking the actin-binding site localized primarily to the PM. Notably, ectopicallyexpressed nesprin-2-SR or�-catenin did not have an impact onthe localization of the respective endogenous binding partner(Fig. 3, F and G). In summary, we conclude that nesprin-2 and�-catenin can be found at the PM and NE.Nesprin-2 Regulates �-Catenin-dependent Transcription—
By performing GST pulldown experiments, we next demon-strated the simultaneous binding of SR1�2 to �-catenin,�-catenin, and emerin (Fig. 4A, 1st approach). Because we orig-inally found �-catenin to be a novel interaction partner ofnesprin-2, we additionally explored whether the interactionbetween nesprin-2 and �-catenin and emerin is mediated by�-catenin (Fig. 4A, 2nd approach). We found that, in precipi-tates missing �-catenin, �-catenin and emerin could still bedetected together with SR1�2, indicating that �-catenin doesnot act as the sole link between these proteins. Emerin is aninteraction partner of both nesprin-2 (9, 21) and�-catenin and,importantly, is a component of the �-catenin nuclear exportmachinery (22). Interestingly, �-catenin seems to have animpact on the Wnt pathway as well (23).Intrigued by these correlations, we explored the role of
nesprin-2. Because �-catenin has to pass the NE to get into thenucleus, we testedwhether a nesprin displacement from theNEmight affect the amount of �-catenin in the nucleus. For this,we assayed for unphosphorylated �-catenin, which representsactive �-catenin that enters the nucleus (24).The experiments were performed in HaCaT cells, in which
active �-catenin was detectable in the nucleus without furtherstimulation by Wnt ligands (Fig. 4, D and G). Nesprin-2 wasdisplaced from theNE (Fig. 4E�,asterisk) by the dominant inter-fering effects of SP-GFP-SUN-1-C. The SUN domain ofSP-GFP-SUN-1-C (Fig. 4C) competes with endogenous SUNproteins for binding to the nesprin KASH domains. SP-GFP(Fig. 4C) was used as a control. Cells expressing SP-GFP-SUN-1-C showed a reduction in the amount of nuclear active�-cate-nin (Fig. 4D). Because the SUN-1 C terminus interacts with theKASH domain of nesprin-1, -2, and -3 (25), we chose a morespecific approach. Cells were transiently transfected with ashRNA directed against the nesprin-2 C terminus or a control.By efficiently depleting nesprin-2 (Fig. 4F), we validated the
�-catenin reduction in the nuclear fraction of nesprin-2-silenced cells (Fig. 4G).To further verify the nesprin-2 role in controlling �-cate-
nin-dependent transcription, we performed TOP/FOP pro-moter assays. HEK293T cells possessing the above-men-tioned proteins (supplemental Fig. S5) were transfected withplasmids encoding shRNAs targeted to the C terminus ofnesprin-2 or a control, followed by incubation with LiCl toinduce the Wnt pathway. The �-catenin-dependent tran-scriptional activity is defined by the ratio between TOPflashand FOPflash. In nesprin-2-depleted cells the TOP/FOP ratiowas strongly reduced, indicating a reduced transcriptionalactivity of the reporter genes (Fig. 4I).The Wnt pathway plays fundamental roles in many biologi-
cal processes such as cell proliferation and migration and stemcellmaintenance (26).Wenext explored the role of nesprin-2 inone of these processes under physiological conditions. WetreatedHaCaTcells, which showed reduced amounts of�-cate-nin in the nuclear fraction uponnesprin-2 displacement/silenc-ing (Fig. 4,D andG), with a C-terminal nesprin-2 shRNA or thecorresponding control and examined the proliferation poten-tial in a BrdU incorporation assay (Fig. 4H). The silencing ofnesprin-2 resulted in a decrease in BrdU-positive cells (33.64%)compared with control cells (52.64%).
DISCUSSION
Here, we have shown novel NE roles in �-catenin-dependenttranscriptional regulation. �- and �-catenins play crucial rolesin cell adhesion andWnt signaling (7).When theWnt pathwayis inactive,�-catenin levels are kept low. In contrast, activeWntsignaling inhibits its cytoplasmic degradation and unfolds�-catenin transcriptional activities by increasing its nuclearlevels (Fig. 5C). C-terminal nesprin-2 isoforms and emerin arecommon to both NE membranes (9, 27). Emerin interactsdirectly with �-catenin and facilitates nuclear �-catenin export(22). In this study, we identified �-catenin as a novel nesprin-2-binding partner. Moreover, localization studies showed bothproteins at the PM and NE (Fig. 5B). Because nesprin-2 associ-ates with emerin (21), it is not surprising to identify also�-cate-nin in the nesprin-2 interactome. This evidence illustrates theNE interplay with key Wnt pathway elements.Depletion of nesprins from theNE results in the reduction of
active nuclear �-catenin levels (Fig. 5D). We propose that,when the Wnt pathway is activated, �- and �-catenins bind tonesprin-2 and emerin located at the outer nuclear membrane,forming a quaternary protein complex fromwhich �-catenin is
FIGURE 4. Nesprin-2 forms a quaternary protein complex with Wnt pathway components at the NE and influences nuclear �-catenin accumulation.A, GST pulldown analysis of nesprin-2 interactions with GFP-�-N-catenin-1. SR1�2 co-precipitated with GFP-�-N-catenin-1 and endogenous �-catenin andemerin (1st approach). The absence of �-catenin did not disturb the interaction of SR1�2, �-catenin, and emerin (2nd approach). The Coomassie Blue gel (A) andthe supernatant fractions (B) show input of equal protein amounts. C, schematic of SUN-1, SP-GFP, and the dominant-negative nesprin-interfering SP-GFP-SUN-1-C proteins. D, subcellular fractionation of WT and SP-GFP- and SP-GFP-SUN-1-C-expressing cells. Nesprin NE displacement resulted in decreased nuclear�-catenin levels. E, SP-GFP-SUN-1-C localized to the NE (arrow) and the endoplasmic reticulum (arrowhead) and displaced nesprin-2 (E�, asterisk) from the NE.Nesprin-2-K1, nesprin-2 visualized with pAbK1. E� merge, F and G, HaCaT cells were treated with control or nesprin-2 (Nes-2)-specific shRNA. F, nesprin-2silencing efficacy was shown by Western blotting (WB) using pAbK1. G, Western blot analysis of fractionated cell lysates. Nuclear �-catenin content in nesprin-2shRNA-treated cells was reduced. Total cell lysates (F) or cytoplasmic and nuclear fractions (G) were prepared using equal cell numbers. H, knockdown ofnesprin-2 antagonized cell proliferation. BrdU incorporation was measured in HaCaT cells transiently treated with nesprin-2-specific shRNA or the correspond-ing control. The statistical significance was analyzed using Student’s t test (p � 0.0016). I, TOP/FOP reporter assay of WT, control shRNA-treated, and nesprin-2shRNA-treated HEK293T cells. The TOP/FOP ratio (y axis), in which the TOPflash-measured �-catenin-dependent transcriptional activity was normalized againstunspecific FOPflash signals, was reduced when nesprin-2 was absent. TL, total lysates.
Nesprin-2 Interacts with �-Catenin
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efficiently released to reach into the nucleus (Fig. 5C). The lossof nesprin-2 may impair �/�-catenin heterodimer binding tothe NE and result in an inefficient translocation of �-catenininto the nucleus. Because outer nuclear membrane-residentnesprin-2 is known to tether emerin (4), nesprin-2 deficiencymay, in addition, also increase the inner nuclear emerin pooland thus favor �-catenin nuclear export. Clearly, additionalresearch is required to address these caveats.In conclusion, ourwork proposes a further regulatory layer at
the NE for �-catenin on its way into the nucleus (Fig. 5). Moreimportantly, our data provide evidence for a cross-talk betweenNE- and PM-associated proteins and show how these compart-ments might be linked. Such signaling may be the key to eluci-date the pathogenesis of NE-mediated diseases.
Acknowledgments—We thank Martina Munck for expert technicalassistance, C. Niessen for providing reagents, and C. Niemann forhelpful discussion.
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FIGURE 5. Model of nesprin-2 and �-catenin roles in Wnt signaling. A, �-and �-catenins localize to adherens junctions linking the adhesion complexto the actin cytoskeleton. Nesprin-2 and emerin localize to the NE. Innernuclear membrane-associated emerin supports the export of �-catenin fromthe nucleoplasm to the cytoplasm. Cytoplasmic �-catenin levels are kept lowby degradation. B, nesprin-2 can be detected at the nuclear and plasma mem-branes. �-Catenin is kept primarily at the PM. Disassembling these connec-tions by dissolving cell contacts allows �-catenin to reach the NE. C, upon Wntpathway activation, cytoplasmic �-catenin levels increase. A quaternary com-plex of nesprin-2, emerin, and �- and �-catenins can be formed at the NE.When released from this complex, �-catenin can enter the nucleus. D, loss ofnesprin-2 results in a reduced amount of �-catenin in the nucleus, probablydue to an inefficient binding of the �/�-catenin heterodimer at the NE. LRP,lipoprotein receptor-related protein.
Nesprin-2 Interacts with �-Catenin
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Eming, Asa Beijer, Angelika A. Noegel and Iakowos KarakesisoglouSascha Neumann, Maria Schneider, Rebecca L. Daugherty, Cara J. Gottardi, Sabine A.
Envelope-Catenin and Regulates Wnt Signaling at the NuclearαNesprin-2 Interacts with
doi: 10.1074/jbc.M110.119651 originally published online August 26, 20102010, 285:34932-34938.J. Biol. Chem.
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