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J. Biol. Chem., Vol. 282, Issue 9, 6308-6315, March 2, 2007

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Lamina-associated Polypeptide 2- Forms Homo-trimers via Its C Terminus, and Oligomerization Is Unaffected by a Disease-causing Mutation^*

Luc Snyers, Sylvia Vlcek, Thomas Dechat¹, Darko Skegro^¶, Barbara Korbei, Andreas Gajewski, Olga Mayans^¶2, Christian Schöfer, and Roland Foisner²³

From the Center for Anatomy and Cell Biology, Medical University of Vienna, Schwarzspanierstrasse 17, A-1090 Vienna, Austria, the Max F. Perutz Laboratories, Medical University of Vienna, Dr. Bohr-Gasse 9, A-1030 Vienna, Austria, and the ^¶Division of Structural Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland

Received for publication, June 16, 2006 , and in revised form, January 5, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The nucleoplasmic protein, Lamina-associated polypeptide (LAP) 2, is one of six alternatively spliced products of the LAP2gene, which share a common N-terminal region. In contrast to the other isoforms, which also share most of their C termini, LAP2 has a large unique C-terminal region that contains binding sites for chromatin, A-type lamins, and retinoblastoma protein. By immunoprecipitation analyses of LAP2 complexes from cells expressing differently tagged LAP2 proteins and fragments, we demonstrate that LAP2 forms higher order structures containing multiple LAP2 molecules in vivo and that complex formation is mediated by the C terminus. Solid phase binding assays using recombinant and in vitro translated LAP2 fragments showed direct interactions of LAP2 C termini. Cross-linking of LAP2 complexes and multiangle light scattering of purified LAP2 revealed the existence of stable homo-trimers in vivo and in vitro. Finally, we show that, in contrast to the LAP2-lamin A interaction, its self-association is not affected by a disease-linked single point mutation in the LAP2 C terminus.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The nuclear envelope comprises the inner and outer nuclear membranes, the nuclear pore complexes, and the nuclear lamina, which underlies the inner nuclear membrane (1, 2). The nuclear lamina is the major structural framework in the nucleus of multicellular eukaryotes and is composed of a filamentous meshwork of type V intermediate filament proteins, the lamins. B-type lamins, encoded by two human genes (LMNB1 and LMNB2), are essential for cell viability. In contrast, the four A-type lamins (A, C, C2, and A10), representing splicing isoforms of the LMNA gene, are dispensable for viability of individual cells but have crucial functions in tissue organization after birth (3, 4).

In addition to the lamins, the nuclear lamina contains a number of integral membrane proteins of the inner nuclear membrane, the best characterized of which are the Lamin B receptor, Lamina-associated polypeptide (LAP),⁴ and the three LEM domain-containing proteins LAP2, emerin and MAN1 (5, 6). All these proteins interact with lamin A/C and/or B and contribute to anchorage of the nuclear membrane to the lamina. The LEM domain, a conserved 40-amino-acid motif located near the N terminus of the LEM family proteins, interacts with the DNA-binding protein barrier-to-autointegration factor (BAF) and mediates the binding of these proteins to chromatin (7). In LAP2 proteins, a LEM-like segment at the very N terminus has been shown to interact with DNA directly (8).

The family of LAP2 proteins includes six alternatively spliced isoforms derived from the same gene (9). Most LAP2 isoforms are closely related structurally and functionally and are localized to the inner nuclear membrane, such as LAP2. In contrast, LAP2 shares only the N-terminal 187 amino acids with the other isoforms, including the LEM and LEM-like domains, but otherwise possesses a unique 506-amino-acid C-terminal region without a transmembrane domain (see Fig. 1A), encoded by one large exon found only in mammals (10).

LAP2 is exclusively located in the nucleoplasm in interphase and interacts with lamin A/C (11) and hypophosphorylated retinoblastoma protein (pRb) via distinct C-terminal domains (12, 13). The LAP2-lamin A/C-pRb complex is thought to regulate cell proliferation and differentiation in adult stem cells (3, 12). During mitosis, LAP2 dissociates from chromosomes in a phosphorylation-dependent manner and is redistributed throughout the mitotic cytoplasm, like most nuclear lamina components (14, 15). However, during anaphase, LAP2 associates with the telomeres of separated sister chromatids and subsequently forms stable structures associated with decondensing chromatin before the nuclear envelope is formed (14). Although LAP2 can interact with BAF and DNA via its common LEM and LEM-like motifs, its C terminus was shown to be essential and sufficient for chromatin association during mitosis (16, 17). Intriguingly, a mutation causing an amino acid substitution (Arg-690 to Cys) near the C terminus of LAP2 has been associated with dilated cardiomyopathy (DCM) (18), a condition also known to be caused by mutations in the LMNA gene (5). The mutation altered the observed LAP2 interaction with A-type lamins in vitro and may represent a rare cause of DCM.

In this study, we show that LAP2 is engaged in homo-oligomerization via its unique C-terminal domain and forms stable trimers in vivo and in vitro. These homo-trimers may be the building blocks of higher order structures containing LAP2 and other proteins.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Reagents—Mouse monoclonal anti-LAP2 (6E10) and rabbit polyclonal anti-Myc antibodies were obtained from Abcam. Rabbit anti-LAP2 antiserum and monoclonal anti-LAP2 antibody 15/2 and 12 were described (16, 17). Polyclonal anti-GFP antibodies were from Clontech; polyclonal anti-DsRed antibody was from BD Biosciences; anti-protein C affinity matrix (mouse monoclonal antibody HPC4, immobilized) and mouse monoclonal anti-Myc antibody (9E10) were from Roche Diagnostics; protein G-agarose conjugate was from Sigma.

Vector Construction and Expression of Recombinant Proteins—cDNA coding for monomeric red fluorescent protein 1 (mRFP1) was amplified from pRSETB-mRFP1 (a kind gift of Dr. R. Tsien, Howard Hughes Medical Institute, University of California, San Diego, CA) and inserted into the vector pCI (Promega). The resulting plasmid was used to create N-terminally tagged mRFP1-LAP2 and mRFP1-LAP2-(188–693) by ligating amplified fragments of LAP2 in-frame with mRFP1. mRFP1-LAP2 and mRFP1-LAP2-(188–693) were then excised and ligated blunt into the EcoRV site of eukaryotic expression vector pEF-puro.PL3 (19). Plasmids pEF.mRFP1-LAP2 and pEF.mRFP1-LAP2-(188–693) were transfected into HeLa cells using Lipofectamine (Invitrogen), and clones were selected in 2 µg/ml puromycin.

For the construction of PC-Myc-mRFP1-LAP2-(188–693) and PC-Myc-LAP2-(188–693), a sequence coding for protein C epitope EDQVDPRLIDGK fused to the Myc-tag EKLISEEDL was inserted into the EcoRV site of pEF-puro.PL3. The resulting plasmid was linearized with EcoRV and ligated in-frame with mRFP1-LAP2-(188–693) or LAP2-(188–693).

For GFP-LAP2 carrying the DCM mutation, vector gAG43 was constructed by shuttling wild-type LAP2 cDNAs from gAG41 (18) into GFP destination vector pcDNA-DEST53 by the LR reaction (Invitrogen). The bacterial expression vectors encoding His-tagged LAP2 fragments are described elsewhere (16).

For bacterial expression, proteins were expressed in Escherichia coli strain BL21(DE3) using the inducible T7 RNA polymerase-dependent pET vector system as described previously (11, 16). Protein expression was induced with 0.5 mM isopropyl--D-thiogalactopyranoside for 3 h. Bacteria were harvested by centrifugation at 4,000 rpm for 5 min (Heraeus Megafuge, 1.0R) and lysed in one-tenth of the original culture volume of Tris buffer (20 mM Tris-HCl, pH 8, 500 mM NaCl, 5 mM imidazole, 1 mM dithiothreitol, protease inhibitors) by freezing and thawing and the addition of 0.1 mg/ml lysozyme, 0.1% Triton X-100, 10 mM MgCl₂, 50 µg/ml DNase, and 20 µg/ml RNase. Following a 30-min incubation at 30 °C, the samples were centrifuged for 10 min at 14,000 rpm, and pellets were resuspended in one-tenth of the original culture volume of Tris buffer plus 7 M urea and incubated for 1 h at room temperature. Cell lysates were centrifuged at 45,000 rpm for 30 min, and supernatants were stored as aliquots at -20 °C. If fragments were soluble, urea was added directly to the cell extract prior to centrifugation at 14,000 rpm. Renaturation of recombinant proteins was achieved by dialyzing twice against KHM buffer (78 mM KCl, 50 mM HEPES, pH 7.4, 8.4 mM CaCl₂, 10 mM EGTA, 4 mM MgCl₂, 1 mM dithiothreitol) and cleared by centrifugation at 4,000 rpm for 5 min. For light scattering, full-length LAP2 was expressed in E. coli strain Rosetta 2 (DE3) (Novagen) grown in Luria-Bertani medium containing 100 mg/liter ampicillin at 30 °C. Expression was induced at an A₆₀₀ = 0.6 by the addition of 1 mM isopropyl--D-thiogalactopyranoside. Cultures were further grown at 20 °C for 20 h. Cells were harvested by centrifugation at 4,500 x g and 4 °C. The bacterial pellet was resuspended in 100 mM potassium phosphate, pH 8.0, 100 mM NaCl, 1 mM dithiothreitol (DTT), containing 50 µg/ml DNase and a protease inhibitor mixture (Roche Applied Science) and sonicated. The homogenate was clarified by centrifugation and filtering, and the resulting supernatant was applied to a Ni²⁺-chelating HiTrap column (GE Healthcare) equilibrated in lysis buffer. Upon serial column washes of buffer supplemented with 50 mM imidazole, the protein was eluted in 300 mM imidazole. The elutant was subjected to gel filtration in buffer supplemented with 10 mM DTT (Superdex 200, HiLoad 16/60 PG; GE Healthcare). The protein so obtained was pure according to SDS-PAGE.

Immunoblotting—Cell extracts in 20 mM Tris-HCl, pH 8.0, 130 mM NaCl, 1% Triton X-100, and protease inhibitor mixture were centrifuged at 13,000 x g for 10 min, and pellet and supernatant fractions were analyzed on 10% polyacrylamide gels and electrotransferred to nitrocellulose (Schleicher & Schuell). Blots were blocked for 60 min in Tris-buffered saline, pH 8.0, 0.1% bovine serum albumin, and 5% nonfat dry milk, incubated with primary antibody in Tris-buffered saline, 0.1% bovine serum albumin for 60 min and with anti-mouse (Sigma) or anti-rabbit (DAKO) alkaline phosphatase-conjugate, and visualized using BM purple AP substrate, (Roche Diagnostics). Semi-native gels contained SDS only in the running buffer (0.1% SDS); no SDS was present in the sample buffer or the polyacrylamide gels themselves.

Immunoprecipitations—Confluent cell monolayers in 10-cm dishes were lysed in TNCT (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Ca²⁺, 1% Triton X-100, and protease inhibitors) and centrifuged for 10 min at 13,000 x g. Supernatants were incubated with 25 µl of anti-protein C matrix for 1 h. Bound complexes were washed four times with TNCT and eluted by boiling in SDS-PAGE sample buffer. Samples were processed for immunoblotting. Alternatively, supernatants were divided in two parts; one half was incubated on ice with monoclonal anti-Myc (2 µg) or monoclonal anti-LAP2 (15 µg) antibody, and the other half was incubated with a control monoclonal antibody (mouse monoclonal anti-HRV2, a kind gift of Dr. Blaas, Max Perutz Laboratories, Vienna, Austria) for 30 min. Immunocomplexes were bound to 12.5 µl of protein G-agarose for 1 h, washed with TNCT, and eluted in sample buffer.

Immunofluorescence Microscopy—Cells on coverslips were fixed with 3% paraformaldehyde in phosphate-buffered saline and quenched in 50 mM NH₄Cl. They were permeabilized in 0.1% Triton X-100, blocked in 5% fetal calf serum, and incubated with monoclonal anti-Myc antibody at 1 µg/ml in phosphate-buffered saline containing 1% fetal calf serum. The secondary antibody was anti-mouse Alexa Fluor 488 (Molecular Probes). Preparations were mounted using Citifluor AP1 (PLANO) and examined with a Nikon Eclipse 800 fluorescent microscope.

Chemical Cross-linking—HeLa cells were resuspended in KHM buffer and homogenized on ice by pressing the suspension 10–15 times through a metal ball cracker (European Molecular Biology Laboratory (EMBL), Heidelberg, Germany). Cell lysates and dialyzed recombinant proteins were mixed with various concentrations of cross-linking agent dithiobis-(succinimidylpropionate) (DSP, Pierce) for 2 h on ice, and the reaction was stopped by quenching free active groups with 50 mM Tris-HCl, pH 6.8, for 1 h on ice. Samples were analyzed by SDS-PAGE in the presence or absence of 100 mM DTT.

Blot Overlay Assays—Recombinant polypeptides were resolved by SDS-PAGE and blotted to nitrocellulose membranes as described (11). Membranes were stained with Ponceau S, washed with phosphate-buffered saline containing 0.05% Tween 20, and incubated in overlay buffer (10 mM Hepes, pH 7.4, 100 mM NaCl, 5 mM MgCl₂, 2 mM EGTA, 0.1% Triton X-100, 1 mM DTT) for 1 h. After blocking with 2% bovine serum albumin in overlay buffer, membranes were probed 3 h with [³⁵S]methionine-labeled polypeptides diluted 1:50 in overlay buffer plus 1% bovine serum albumin. Membranes were washed extensively with overlay buffer, and bound proteins were detected by autoradiography.

In Vitro Transcription/Translation and GFP Immunoprecipitation—150 µl of protein A-Sepharose were coupled overnight with 6 µl of polyclonal anti-GFP antibody and 150 µl of protein G-Sepharose with 1 ml of monoclonal anti-LAP2 antibody 12. ³⁵S-labeled wild-type or mutated LAP2 was expressed by in vitro transcription/translation using the TNT quick-coupled transcription/translation reaction mix (Promega) either alone or together with GFP-tagged wild-type LAP2 (gAG49, gAG49 and gAG43, gAG50, gAG50 and gAG43). After incubation at 30 °C for 3 h, binding buffer (50 mM HEPES, pH 7.4, 50 mM NaCl, 5 mM MgCl₂, 1 mM EGTA, 0.1% Triton, 1 mM DTT, and 1 mM phenylmethylsulfonyl fluoride) was added to 225 µl. For preclearing, samples were incubated with 20 µl of protein A-Sepharose or protein G-Sepharose for 15 min and centrifuged at 1,000 rpm for 3 min. Supernatants were incubated with antibody-coupled beads (see above) for 2 h at 4 °C, and beads were centrifuged through a 30% sucrose cushion. Supernatants and beads were mixed with SDS sample buffer and analyzed by gel electrophoresis and autoradiography.

Size Exclusion Chromatography Combined with Multiangle Light Scattering (SEC-MALS)—The oligomeric state of LAP2 in solution was determined by SEC-MALS measurements performed on an ÁKTA explorer 10 system (GE Healthcare) connected to a tri-angle light scattering detector and a differential refractometer (miniDAWN Tristar and Optilab, respectively; Wyatt Technology). A Superdex 200 10/300 GL column (GE Healthcare) was equilibrated in 100 mM potassium phosphate, pH 8.0, 100 mM NaCl, 10 mM DTT at a flow rate of 0.5 ml/min. A sample volume of 100 µl was injected at a concentration of 1 mg/ml. Data were processed using ASTRA software (Wyatt Technology) assuming a specific refractive index increment (dn/dc) of 0.185 ml/g. To determine the detector delay volumes and the normalization coefficients for the MALS detector, a bovine serum albumin sample (Pierce) was used as reference. Neither despiking nor band broadening correction was applied.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The C Terminus of LAP2 Is Involved in the Formation of Oligomeric Complexes in Vivo—Our previous studies revealed the formation of stable, chromatin-associated LAP2 structures during early nuclear assembly stages (14, 17), indicating oligomerization of the protein. To analyze oligomeric LAP2 structures in vivo and to determine the domain of LAP2 responsible for oligomerization, either full-length LAP2 or the LAP2-specific C-terminal domain, LAP2-(188–693), fused to the C terminus of monomeric red fluorescent protein 1 (mRFP1) (Fig. 1A), were stably expressed in HeLa cells. Unlike GFP and DsRed, mRFP1 remains entirely monomeric and therefore precludes misinterpretations of LAP2 oligomerization due to dimerization or tetramerization of the tag (20). Immunoblot analyses of cell extracts prepared from stable cell clones revealed the presence of fusion proteins of expected sizes, in amounts comparable with that of the endogenous LAP2 (Fig. 1B, left panel). Both constructs reacted with polyclonal antibodies against LAP2 C terminus (anti-LAP2)) and antibodies against red fluorescent protein (anti-RFP), whereas a monoclonal antibody directed against the N-terminal region of LAP2 that is common to all LAP2 isoforms recognized only mRFP1-LAP2 plus the endogenous protein (anti-LAP2). Both mRFP1-LAP2 and mRFP1-LAP2-(188–693) were readily extracted in buffer containing 1% Triton X-100 and 130 mM NaCl (Fig. 1B, left panel). However, although a small fraction of endogenous LAP2 and of mRFP1-LAP2 remained in the pellet fraction, mRFP1-LAP2-(188–693) was completely soluble, indicating that full-length LAP2 is more tightly bound to nuclear complexes than the LAP2 C terminus.

Next, we examined the cellular distribution of the tagged proteins at various stages of the cell cycle by using fluorescence microscopy. As shown in Fig. 1C, both mRFP1-LAP2-(188–693) and mRFP1-LAP2 localized to the nucleoplasm in interphase, to the cytoplasm in metaphase, and to chromatin in anaphase/telophase as reported previously for the endogenous protein and GFP-LAP2 (14, 16, 17, 21). Unlike the full-length protein, a fraction of mRFP1-LAP2-(188–693) remained in the cytoplasm during anaphase (Fig. 1C, arrow), again indicating a less stable integration of the C terminus to chromatin-associated structures as compared with full-length protein. Nevertheless, these data show that the C terminus is capable of interacting with chromatin or nuclear lamina components during nuclear assembly in vivo.

View larger version (34K): [in this window] [in a new window]   FIGURE 1. Stable expression of LAP2 and the -specific C terminus in HeLa cells. A, schematic presentation of fusion proteins of full-length LAP2 and LAP2-(188–693) with mRFP1, protein C epitope, and Myc tag, and for comparison, the domain organizations of LAP2 and LAP2 are shown. The light gray region represents the LAP2-specific C terminus; gray and black boxes show positions of LEM, LEM-like, and transmembrane domains, respectively. The arrow indicates the epitope recognized by the monoclonal anti-LAP2 common domain antibody. B, untransfected HeLa cells (HeLa) or HeLa cells stably expressing the indicated constructs described in A were lysed in 1% Triton X-100 and total cell lysates (T) or pellet (P) and supernatant (S) fractions following centrifugation were analyzed by immunoblotting using polyclonal anti-LAP2 (anti-LAP2), monoclonal anti-LAP2 common domain (anti-LAP2), and polyclonal anti-DsRed (anti-RFP) or polyclonal anti-Myc antibodies, as indicated. The numbers show molecular masses in kDa. C, cells expressing mRFP1-LAP2 or mRFP1-LAP2-(188–693) or PC-Myc-LAP2-(188–693) as indicated, were analyzed at various cell cycle stages by fluorescence microscopy. Left images show mRFP1 or anti-Myc staining, and right images show 4',6-diamidino-2-phenylindole-stained DNA. The arrow indicates anaphase cells with remaining cytoplasmic staining. Bar, 10 µm.

View larger version (25K): [in this window] [in a new window]   FIGURE 2. Co-immunoprecipitation of endogenous LAP2 with expressed LAP2 C terminus from HeLa cell extracts. A, soluble cell extracts of non-transfected HeLa cells, two HeLa clones expressing PC-Myc-mRFP1-LAP2-(188–693), and one clone expressing PC-Myc-LAP2-(188–693) were incubated with anti-protein C matrix, and immunoprecipitates were analyzed by immunoblotting using anti-LAP2 common domain antibody. Note that in SDS-PAGE, LAP2 runs seemingly faster in the first two lanes as compared with lane 3 due to comigration of PC-Myc-mRFP1-LAP2-(188–693) with endogenous LAP2 under these conditions. Furthermore, the amount of PC-Myc-mRFP1-LAP2-(188–693) is higher in the sample shown in lane 2 as compared with that in lane 1. B, cell extracts were incubated with monoclonal anti-Myc (+) or a control monoclonal antibody (-), and immunoprecipitates were probed with anti-LAP2 antibody. C, extracts were incubated with monoclonal anti-LAP2 (+) or with control (-) antibody, and immunoprecipitates were probed with anti-Myc antibody.

View larger version (78K): [in this window] [in a new window]   FIGURE 3. Analysis of LAP2 complexes by chemical cross-linking and semi-native gel electrophoresis. A, HeLa cell lysates were analyzed on semi-native polyacrylamide gels (Endogenous complex, left panel) or incubated with the indicated amounts of DSP for 2 h, dissolved in sample buffer containing (+) or lacking (-) DTT, and analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting using monoclonal anti-LAP2 antibody 15/2. B, bacterially expressed full-length LAP2 and LAP2 fragments, as indicated, were incubated with 1 mM DSP, mixed with sample buffer with (+) or without (-) DTT, and analyzed by immunoblotting using monoclonal anti-LAP2 antibody 15/2 (left and central panels) or anti-LAP2 antibody 12 (right panel). C, in vitro translated, [35S]methionine-labeled LAP2 was analyzed by semi-native PAGE and autoradiography. The numbers denote molecular masses in kDa, the arrows indicate the position of monomeric proteins, and the arrowheads indicate the position of complexes.

To test whether LAP2 is able to form oligomeric complexes of similar sizes in vitro, we cross-linked highly enriched recombinant, bacterially expressed LAP2. Immunoblot analyses revealed a LAP2 complex of more than 200 kDa in non-reducing conditions (Fig. 3A, -DTT), whereas mostly the monomeric protein of 80 kDa was detected in reducing conditions (Fig. 3B). Intriguingly, the C terminus of LAP2-(188–693) also formed larger complexes in non-reducing conditions, whereas the N-terminal region (1–187) remained mostly monomeric under these conditions. Since both the N terminus and the C terminus contain lysine residues, which can be targeted by the cross-linker, we concluded that LAP2 forms higher order, oligomeric complexes through self-association of its C-terminal region.

To further show the homo-oligomerization of LAP2, we generated LAP2 by in vitro translation in a reticulocyte lysate and analyzed the complex by semi-native PAGE (Fig. 3C). Interestingly, a major band between 200 and 250 kDa was detected in addition to the 80-kDa monomer. Since the reticulocyte lysate does not contain any nuclear proteins, which may be specific interaction partners for LAP2, it is very likely that the >200-kDa complex represented homo-oligomeric LAP2 complexes.

The LAP2 Self-interaction Is Mediated by the C-terminal Region—Our data suggest direct interaction of LAP2 C-terminal regions mediating homo-oligomerization of the protein. To further demonstrate that self-interaction of LAP2 polypeptides occurs via the C terminus, we performed additional in vitro binding assays. Bacterially expressed full-length LAP2 (1–693), LAP2 C terminus (188–693), and LAP2 N terminus (1–187) were separated by SDS-PAGE, blotted onto a membrane, and overlaid with radioactively labeled in vitro translated full-length LAP2 or LAP2 C terminus. Autoradiography revealed binding of both labeled proteins to full-length LAP2 and to the C terminus, whereas the N terminus did not interact (Fig. 4A). Thus, only the C terminus of LAP2 can mediate self-interaction of the protein. To narrow down the self-interaction domain within the LAP2 C terminus, we expressed different fragments of LAP2 in bacteria and performed solid phase overlay assays with radioactively labeled, in vitro translated, full-length LAP2 or LAP2 C terminus. Results presented in Fig. 4B and summarized in Fig. 4C show that removal of 78 residues from the C terminus of LAP2 only moderately affected binding of the truncated protein to LAP2 and LAP2 C terminus, removal of the C-terminal 279 residues significantly reduced binding (for LAP2 C terminus to an undetectable level), and deletion of the last 439 residues abolished binding completely. On the other hand, the C-terminal 284 residues of LAP2-(410–693) showed a binding that was slightly weaker than that of the entire C terminus (188–693). These results show that the extreme C-terminal domain of LAP2-(410–693) has some binding affinity to the LAP2 C terminus, but strong interaction requires upstream regions between residues 254 and 410. Vimentin as a negative control did not interact with LAP2.

Purified Soluble LAP2 Is a Homo-trimeric Complex—Our results demonstrate a self-association of LAP2, and cross-linking experiments revealed complexes of larger than 200 kDa, which suggests trimeric structures. To show the level of assembly of LAP2 in solution accurately, we performed SEC-MALS and refractive index measurements on soluble, purified samples of LAP2. This technique yields an accurate determination of the molecular mass independent of molecular shape or hydrodynamic parameters. SEC-MALS data revealed that LAP2 migrates as a monodisperse molecule with an estimated molecular mass of 207.2 kDa (Fig. 5). Since the calculated theoretical mass of a LAP2 trimer is 226.0 kDa, this result shows that LAP2 exists as a stable homo-trimer in solution. It is also in accordance with the molecular mass observed in the cross-linking and the semi-native gel electrophoresis experiments (Fig. 3).

View larger version (41K): [in this window] [in a new window]   FIGURE 4. LAP2 self-associatesviaits-specific C terminus in vitro. A,invitro translated [35S]methionine-labeled LAP2 or the -specific domain LAP2-(188–693) were overlaid onto immobilized bacterially expressed full-length LAP2, LAP2-(188–693), and LAP2-(1–187). An autoradiogram of an SDS-polyacrylamide gel of in vitro translated proteins (Autoradiogram), a Coomassie Blue-stained gel of bacterially expressed proteins (Coomassie), and autoradiograms of overlays (Overlay) are shown. B, transblotted vimentin and recombinant LAP2 fragments, as indicated, were overlaid with in vitro translated [35S]methionine-labeled recombinant full-length LAP2 or the -specific domain LAP2-(188–693). Ponceau S stain of blotted LAP2 fragments and autoradiograms of overlays are shown. The numbers show molecular masses in kDa. C, schematic diagrams of immobilized LAP2 and LAP2 fragments used in B are shown. The numbers denote amino acid position. The right panel indicates binding of the in vitro translated proteins to immobilized proteins, based on signal intensities on autoradiograms of the overlays (++++, strongest interaction; +, weakest interaction; -, no interaction). The calculated molecular masses (Mr) of LAP2 and LAP2 fragments are shown in kDa.

View larger version (12K): [in this window] [in a new window]   FIGURE 5. Purified LAP2 exists as a homo-trimer in solution. Purified full-length LAP2 was analyzed by SEC-MALS. The refractive index (arbitrary units) of the corresponding size exclusion chromatogram (Superdex 200 10/300 GL, GE Healthcare) performed in 100 mM potassium phosphate, pH 8.0, 100 mM NaCl, 10 mM DTT is shown as solid line. The weight-averaged molecular mass measured inside the peak area at volume intervals is displayed by the line above the peak. The theoretical molecular mass for a trimer of LAP2 as calculated from the amino acid sequence is indicated by a dashed horizontal line.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The studies presented here revealed a so far unappreciated biochemical property of LAP2, the formation of homo-oligomeric structures, and revealed the presence of LAP2 homotrimers in cell lysates and in solution in vitro. Co-immunoprecipitation of endogenous LAP2 with exogenously expressed full-length LAP2 or different fusion proteins containing the -specific C-terminal domain demonstrated that higher order LAP2 structures occur in living cells, are stable, and require the unique C terminus of the protein. Cross-linking of LAP2 complexes in cell lysates and size exclusion chromatography of purified LAP2 combined with multiangle light scattering measurements showed that LAP2 forms stable homo-trimers.

LAP2 C Terminus Is Involved in Multiple Interactions—In vitro overlay experiments suggested that the homo-trimerization of LAP2 is mediated through the direct interaction of its C-terminal -specific domain. Although the last 284 amino acids were sufficient for self-interaction in vitro, additional upstream regions within the unique LAP2 domain contribute to the formation of stable oligomers. The C terminus has been shown to contain several additional binding domains. The last 78 amino acids of LAP2 are involved in the interaction with lamin C (11), whereas further upstream regions mediate the interaction with pRb (12). One may argue that the binding regions of LAP2 for pRb, for lamin A/C, and for self-association may overlap and influence or even compete with each other. However, our observation that stable LAP2 trimers exist in the cell argues that these trimers form the building block of higher order LAP2 complexes. Thus, it is much more likely for a LAP2 trimer versus a monomer to interact simultaneously with pRb and lamin A/C and act as a platform for the assembly of transcriptional regulator complexes as proposed in previous studies (12).

View larger version (26K): [in this window] [in a new window]   FIGURE 6. A disease (DCM)-causing mutation in LAP2 does not affect its self-interaction. Wild-type (WT) LAP2 or DCM mutant LAP2 was in vitro translated and labeled with [35S]methionine in a reticulocyte extract either alone or with GFP-LAP2, and complexes were immunoprecipitated using anti-GFP antibody. Input samples and supernatants (S) and pellet (P) fractions were analyzed by SDS-PAGE and autoradiography. The numbers indicate molecular masses in kDa.

Size of Stable Homo-oligomeric LAP2 Structures—Analysis of HeLa cell extracts after chemical cross-linking or in seminative non-denaturing gels showed the presence of stable LAP2-containing complexes of molecular masses between 200 and 250 kDa. Complexes of the same size were detected after cross-linking of bacterially expressed LAP2 and by seminative PAGE of LAP2 expressed in reticulocyte lysate, indicating that LAP2 is the only protein in the 200–250-kDa complex. According to their size, these complexes might accommodate three LAP2 molecules of 75–80 kDa, suggesting that the protein exists as a homo-trimeric complex. Size exclusion chromatography combined with light scattering analyses of highly purified bacterially expressed LAP2 revealed a high monodispersity of the sample with a molecular mass consistent with a trimeric organization.

Functional Implication of the LAP2 Self-interaction—By fluorescent microscopy, LAP2 C-terminal domain stably expressed in HeLa cells revealed a cellular distribution identical to that of endogenous LAP2, except for anaphase-telophase, where it showed a slightly less efficient association with chromosomes. This observation supports previous findings, showing that the C terminus is required and sufficient for chromosome association of LAP2 during nuclear assembly (17). The less efficient binding of LAP2 C terminus to chromatin also indicates that, although the C terminus is sufficient for targeting LAP2 to chromosomes, its stable association also requires the N-terminal common domain. However, based on the data presented here, we cannot rule out completely that the C-terminal fragment associated with chromosomes mainly through its interaction with full-length endogenous LAP2 during nuclear assembly in vivo.

In any case, the engagement of LAP2 in a self-interaction has important implications for its previously reported functions. The formation of stable LAP2 trimers as basic building blocks for higher order structures clearly affects the chromatin binding properties of LAP2. A trimer brings three LEM and LEM-like domains in close vicinity in a complex and generates multiple binding sites for BAF and DNA, thus significantly increasing the affinity of the complex for chromatin. Secondly, the N termini of the oligomeric complex could interact simultaneously with several DNA fibers and thus perform efficient cross-linking of chromatin regions. In line with this model, it was shown that full-length LAP2 and BAF are essential components of the preintegration complex of retroviruses (22). The proposed role of LAP2 in stabilizing the interaction of BAF with preintegration complexes required both the N-terminal common domain and the -specific C-terminal domain of LAP2. Our results, showing that the C terminus is the determinant for oligomerization of LAP2, provide a possible explanation for this observation. Finally, the proposed role of LAP2 as a transcriptional regulator of E2F target genes (12) by forming complexes with pRb and lamin A/C can be better accomplished by a higher order structure, such as a trimer, than by a monomer.

In conclusion, our data suggest a model where a homo-trimeric core complex of LAP2, formed via self-association of the C-terminal domain, serves as the building block for higher order homo- and hetero-oligomeric structures of LAP2 that are involved in chromatin organization and in transcriptional regulation.

	FOOTNOTES

 
^* This study was supported in part by Austrian Science Research Fund Grant FWF P17871 [GenBank] (to R. F.), Austrian National Bank Grant 9840 (to C. S.), and from the Swiss National Foundation Grant 3100A0-100852 (to O. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Dept. of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, IL.

² Supported by the EURO-Laminopathies research project of the European Commission (Contract LSHM-CT-2005-018690).

³ To whom correspondence should be addressed. Tel.: 43-1-4277-61680; Fax: 43-1-4277-9616; E-mail: roland.foisner{at}meduniwien.ac.at.

⁴ The abbreviations used are: LAP, Lamina-associated polypeptide; BAF, barrier-to-autointegration factor; DCM, dilated cardiomyopathy; LEM, Lamina-associated polypeptide2-Emerin-MAN1; pRb, retinoblastoma; SEC-MALS, size exclusion chromatography combined with multiangle light scattering; GFP, green fluorescent protein; DTT, dithiothreitol; DSP, dithiobis-(succinimidylpropionate); RFP, red fluorescent protein; mRFP, monomeric RFP.

	ACKNOWLEDGMENTS

 
We thank Michael Mrosek, Biozentrum Basel, for critical discussion of the work.

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