Importance of class II transactivator leucine-rich repeats for dominant-negative function and nucleo-cytoplasmic transport

Margarita M. Camacho-Carvajal1,3, Sebastian Klingler1, Felix Schnappauf1, Sandra B. Hake2 and Viktor Steimle1,4

1 Hans-Spemann-Laboratories, Max-Planck-Institut für Immunbiologie, Freiburg D-79108, Germany 2 Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA 3 Present address: Laboratory for Physiological Chemistry and Centre for Biomedical Genetics, Utrecht 3584CG, The Netherlands 4 Present address: Département de Biologie, Université de Sherbrooke, QC J1K 2R1, Canada

Correspondence to: V. Steimle, Département de Biologie, Université de Sherbrooke, 2500 Boulevard Université, Sherbrooke, QC J1K 2R1, Canada. E-mail: viktor.steimle{at}usherbrooke.ca
Transmitting editor: L. Glimcher


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Class II transactivator (CIITA), the master regulator of MHC class II (MHC-II) gene transcription, shows a complex behavior in terms of self-association, nucleo-cytoplasmic transport and MHC-II gene transactivation. Here, we analyzed the mechanisms of dominant-negative function and nucleo-cytoplasmic transport of CIITA with emphasis on the role of the C-terminal leucine-rich-repeat (LRR) region in these processes. First, we determined nucleo-cytoplasmic transport of endogenous CIITA and thus validated results obtained with epitope-tagged CIITA constructs. LRR mutations in potential protein–protein contact positions lead to either completely blocked or reduced nuclear import, but can also give rise to increased nuclear export. Surprisingly, N-terminally truncated CIITA mutants show dominant-negative inhibition of wild-type CIITA, whether they are located in the nucleus or in the cytoplasm. Integrity of the LRR is necessary for the dominant-negative function of both types of mutants. LRR mutations are dominant over the effect of an exogenously added N-terminal nuclear localization signal (NLS) leading to cytoplasmic localization. Taken together, our results show that the LRR regulate the function of one or several NLS within CIITA, and control both nuclear import and export. Self-association is not affected in these mutants; we therefore suggest that interaction of the LRR with an unknown protein partner may be necessary for import and transactivation function of CIITA.

Keywords: immune response genes, MHC class II gene regulation, transcriptional regulation


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
MHC molecules present peptide antigens to T lymphocytes and thus play a key role in eliciting an immune response. MHC class II molecules (MHC-II) present mainly exogenous antigens to CD4+ Th cells. They are expressed constitutively only on a restricted subset of cells of the immune system, such as B lymphocytes, macrophages and dendritic cells. MHC-II expression is inducible in other cell types upon stimulation with cytokines, such as IFN-{gamma}. Expression of MHC-II genes is controlled mainly at the level of transcription via a set of conserved promoter elements known as the W (S), X, X2 and Y boxes [reviewed in (1,2)].

The MHC-II transactivator CIITA was identified as the first gene responsible for hereditary MHC-II deficiency [or bare lymphocyte syndrome (BLS)] (3). CIITA is the master regulator of MHC-II expression, since its expression is necessary and sufficient to induce expression of all MHC-II promoter-containing genes in multiple cell lines and tissues (46). CIITA does not bind DNA directly, it rather acts as a co-activator through protein–protein interactions with MHC-II promoter-binding proteins (1,2).

Several functional domains have been characterized within CIITA: an N-terminal transcriptional activation domain consisting of an acidic domain (amino acids 1–160) (79) and a region rich in prolines, serines and threonines (P/S/T domain, amino acid 160–320), a central GTP-binding domain (GBD, amino acids 400–730) (10,11), and a C-terminal region with at least four leucine-rich-repeat motifs (LRR) (12,13).

CIITA shows a very complex behavior in terms of self-association, nucleo-cytoplasmic transport and MHC-II gene transactivation. Structure–function analyses revealed that all three processes involve large parts of the protein and are most probably closely interdependent. The acidic activation domain can be replaced by a viral activation domain (8) and was shown to interact with components of the basal transcription machinery [reviewed in (2,14)]. Deletions of the acidic or the acidic together with the P/S/T domain lead to dominant-negative phenotypes (10,1517). Sequences within the N-terminal domain, the GBD and the LRR have been shown to be involved in CIITA self-association (1821).

Sequence motifs and domains distributed over the whole length of the CIITA protein have been implicated in the regulation of its subcellular localization and nucleo-cytoplasmic transport. Nucleo-cytoplasmic transport occurs via the nuclear pore complex (NPC). Transport of substrates >30– 40 kDa is mediated by a specialized transport machinery in a specific and energy-dependent manner (22). Nuclear import requires the presence of a specific nuclear localization signal (NLS), often a cluster of basic amino acids, within the protein to be imported. Import of cargo proteins across the NPC is mediated by importins (karyopherins) (23). Importin-{alpha} isoforms act as adapter molecules binding both to NLS-containing cargo proteins and to importin-ß. Importin-ß is the transport receptor that directs importin-{alpha}/cargo complexes from the cytoplasm to the nuclear side of the NPC (23). Nuclear export is also a specific, receptor-mediated process. Leucine-rich sequence motifs have been shown to function as nuclear export signals (NES) (24,25). The nuclear export factor CRM-1 mediates the export of many different substrates and CRM-1-mediated export can be specifically blocked by the drug leptomycin B (LMB) (26).

N-terminally tagged, wild-type CIITA localizes both in the cytoplasm and in the nucleus (27). Numerous mutations within CIITA lead to altered subcellular localization, most often to aberrant preferential cytoplasmic localization (12,20,2733). Three NLS have been described within CIITA (2729), but their precise role in subcellular transport is not clear. N-terminally tagged CIITA is also actively shuttled out of the nucleus, since treatment with LMB leads to nuclear accumulation of the protein, and CIITA fragments spanning amino acids 1–114 and 408–550 have been shown in vitro to interact with CRM-1 (20).

LRR, which are found in diverse classes of proteins, have been described as protein–protein interaction domains (34,35). CIITA contains at least four LRR in its C-terminus, which have been implicated in self-association (1820), transactivation (12) and subcellular localization (12,13). Here, we analyzed the mechanisms of dominant-negative function and nucleo-cytoplasmic transport of CIITA with emphasis on the role of the LRR in these processes.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cell culture and transfections
The CIITA cell lines HEK293-EBNA (Invitrogen, Carlsbad, CA) and HeLa were grown in DMEM medium supplemented with 2 mM L-glutamine, 10% FCS, 10 U/ml penicillin and 10 µg/ml streptomycin. HEK293-EBNA cells were transfected by calcium phosphate precipitation and HeLa cells with Lipofectamine (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions. Cells were analyzed 2 days after transfection.

Expression vectors and cDNA expression constructs
cDNA constructs were expressed using Epstein–Barr virus episomal expression vectors EBO-PL, EBS-PL or EBS-NPL (3,12,15). All amino acid positions within CIITA refer to the 1130-amino-acid form (form III) (3). CIITA LRR mutants with each two to four alanine replacements within the LRR (MT1 to MT13) have been described (12). Mutants MT2 and MT11 show instability on the protein level as EGFP-CIITA fusion proteins (12) and were therefore excluded from the analysis. CIITA constructs with unmodified N-termini were expressed as form III constructs. EGFP-CIITA (12) and CIITA mutants from BLS patients BCH [allele 2(BCH-2)] and Sa have been described (30,36). Myc6-CIITA contains six consecutive Myc epitope tags fused N-terminally to methionine 1 of CIITA form III (37). NLS-L335 initiating at amino acid 335 and containing an N-terminal SV40 NLS (MPKKKRKVHP335EPV, NLS underlined) has been described (15). L335 contains the following context of initiation: MGP335EPV. EGFP-NLS-L335 and EGFP-L335 were generated by inserting an N-terminal EGFP open reading frame. LRR mutants in EGFP-L335 or EGFP-NLS-L335 were generated by replacing the C-terminus with the corresponding fragment obtained from the EBS-CIITA LRR mutants described in (12). Strep-TagII-L335 was generated by PCR mutagenesis. It contains the streptavidin-binding peptide Strep-TagII (38) fused N-terminally to position 335 (MASWSHPQFEKP335EPV, Strep-TagII underlined). All constructs were verified by sequencing.

Generation of CIITA-specific antisera
A His6-tagged fusion protein comprising amino acid 25–408 of CIITA was expressed in Escherichia coli, purified by metal-affinity chromatography under native conditions using Ni-NTA (Qiagen, Hilden, Germany) following the manufacturer’s recommendations and used to immunize rabbits. The resulting polyclonal antiserum (serum K5) was absorbed on HeLa acetone powder.

FACS analysis
Cell staining and flow cytometric analysis were performed as described previously (3) on a FACSCalibur (Becton Dickinson). Half a million cells were stained with anti-human HLA-DR coupled to Quantum Red (Clone HK14; Sigma, Taufkirchen, Germany) at a dilution of 1:1000.

Immunofluorescence microscopy
Immunolocalizations were performed on HeLa cells transfected with untagged CIITA constructs or induced with IFN-{gamma} (500 U/ml, 2 days). For experiments with LMB (Sigma) cells were treated with a concentration of 10 ng/ml for 2 h prior to fixation. Cells grown on glass cover slides were fixed with 3% paraformaldehyde in PBS, permeabilized with methanol containing 2 µg/ml bisbenzimidine (Hoechst 33258) for nuclear staining, blocked in PBS/1% BSA and stained with CIITA antiserum K5 (1:2000 dilution, 30 min at room temperature. After washing (PBS/1% BSA), slides were incubated with Alexa 488 goat anti-rabbit (Molecular Probes, Leiden, The Netherlands; 1:2000, 1 h at room temperature). For the immunolocalizations of Myc6-CIITA, anti-myc mAb 9E10 (1:280) and a sheep anti-mouse F(ab)–Cy3 conjugate (1:250) (Sigma, Taufkirchen, Germany) were used. Cells were observed under a Zeiss Axioplan microscope. Digital images were acquired using a Hamamatsu C4880 CCD camera and OpenLab software (Improvision, Coventry, UK).

Immunoprecipitations and immunoblotting
Immunoprecipitations were performed using anti-GFP (Roche, Basel, Switzerland), anti-Myc 9E10 and Dynabeads (Dynal, Oslo, Norway) or with Streptactin Sepharose beads (IBA, Göttingen, Germany) as described (12). Immunoprecipitated proteins were eluted by boiling the beads in protein loading buffer and immediately loaded on an SDS–PAGE. Immunoblotting was performed as previously described (12) using anti-CIITA antisera #21 (15).

Real-time PCR
Total-RNA from 1 x 106 transiently transfected cells was isolated (Absolutely RNA kit; Stratagene, La Jolla, CA) and first-strand cDNA generated (Expand reverse transcriptase, Roche, Basel, Switzerland) according to manufacturers recommendations. Real-time PCR analysis was performed using LightCycler FastStart DNA Master SYBR Green I kit on a LightCycler instrument (Roche). HLA class II DRA mRNA expression was analyzed with primers HLA-DRA-LC-F690 5'-ccctgggcctgactgtgg-3' and HLA-DRA-LC-R931 5'-ctatagggctggaaaatgctgaag-3'. RNA from the MHC-II+ cell line Raji was used as quantification standard.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Immunolocalization of endogenous CIITA and CIITA mutants
So far, subcellular localization of CIITA and CIITA mutants has only been analyzed using N-terminally tagged versions of CIITA, mostly in combination with significant overexpression (12,20,2733). Since the presence of an N-terminal tag on CIITA leads to a considerable increase in protein half-life (37), it was important to establish the subcellular localization and intracellular transport of unmodified CIITA expressed at physiological protein levels. CIITA expression is known to be induced in non-specialized antigen-presenting cells by treatment with IFN-{gamma} (46). We treated HeLa cells with IFN-{gamma} and performed immunolocalization of CIITA on fixed cells after 2 days of induction using the CIITA-specific polyclonal antiserum K5 (Fig. 1A). Subcellular localization of transfected, tagged EGFP-CIITA and untagged CIITA was compared with that of endogenous CIITA (Fig. 1A). Endogenous CIITA and transfected untagged CIITA are evenly distributed between the nucleus and the cytoplasm (Fig. 1A). EGFP-CIITA is also present both in the nucleus and the cytoplasm, but shows a somewhat stronger nuclear localization. Thus, the presence of a tag on CIITA does not affect its subcellular localization dramatically.



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Fig. 1. Nucleo-cytoplasmic transport of endogenous and tagged CIITA. (A) The subcellular localization of transiently transfected CIITA (EGFP-CIITA or untagged CIITA) and IFN-{gamma} induced endogenous CIITA (+IFN-{gamma}) in HeLa cells was determined by indirect immunofluorescence using a rabbit polyclonal anti-CIITA antiserum (K5) and a secondary anti-rabbit antibody coupled to Alexa 488 or via GFP fluorescence of EGFP-CIITA transfected cells (left hand panels). Where indicated (+LMB), cells were treated with LMB for 2 h prior to fixation (–IFN-{gamma}; unstimulated HeLa cells). The right-hand panels show nuclear staining (Hoechst 33258) of the same images. (B) Comparison of subcellular localization of EGFP-tagged and untagged CIITA mutants.

 
Previously, the subcellular localization of various CIITA LRR mutants had been analyzed in HEK293 cells, using N-terminal EGFP fusions (12). Here we analyzed the subcellular localization of CIITA LRR mutants, and additional mutants derived from CIITA-deficient BLS patients Sa (L469P) (30) and BCH-2 ({Delta}1079–1106) (36) as untagged proteins by immunofluorescence in HeLa cells, and compared it with the subcellular localization of the corresponding EGFP fusions (Fig. 1B and data not shown). CIITA LRR mutants can be grouped into two groups: those which are transcriptionally completely inactive (MT1–MT3, MT5, MT7 and MT10–MT13) and those with residual transactivation potential (MT4, MT6, MT8 and MT9) (12). Patient mutant Sa shows also residual transactivation potential, while BCH-2 is completely inactive (30,36). With respect to their subcellular localization, the untagged mutants MT13, Sa and BCH-2 are predominantly cytoplasmic (Fig. 1B), and only MT4 shows both nuclear and cytoplasmic staining (Fig. 1B). All mutants show accumulation around the nuclear envelope. The EGFP-fusions show a similar phenotype, but the cytoplasmic staining is more pronounced (cf., e.g. MT4 with EGFP-MT4).

Influence of CIITA LRR on intracellular shuttling and transactivation
CIITA has been reported to shuttle out of the nucleus in a CRM1-dependent manner (20). We analyzed the effect of the CRM1 inhibitor LMB on endogenous, transfected untagged and EGFP-tagged wild-type CIITA and CIITA mutants (Figs 1A and 2A). Endogenous CIITA induced by IFN-{gamma} shows little nuclear accumulation in the presence of LMB (Fig. 1A). Nuclear accumulation is more pronounced with transfected, untagged CIITA and even stronger with EGFP-CIITA.

Treatment of LRR mutants and patient-derived CIITA mutants (untagged and EGFP-CIITA constructs) with LMB revealed three different phenotypes with respect to nuclear import–export. (i) Mutants with blocked import: the protein does not accumulate in the nucleus in the presence of LMB (BCH-2, MT1, MT3, MT5, MT7, MT10, MT12 and MT13; Figs 1B, 2A and B, and data not shown). These mutants are completely inactive in terms of MHC-II transactivation (Fig. 2C and data not shown) (12,36). (ii) Mutants with impaired import: the mutants show a reduced, but significant nuclear accumulation in the presence of LMB (Sa, MT6, MT8 and MT9; Figs 1B, 2A and B, and data not shown). These mutants transactivate MHC-II at various intermediate levels (Fig. 2C) (30). (iii) Increased export: the protein accumulates at levels comparable to the wild-type in the presence of LMB; therefore increased cytoplasmic staining in untreated cells is most probably due to increased protein export (MT4; Fig. 2A and B). MT4 shows a MHC-II transactivation potential which is very similar to wild-type CIITA (Fig. 2C). Transfections with reduced amounts of DNA (as low as 10 ng) showed exactly the same transactivation potential of the different constructs at low levels of EGFP-CIITA expression, but had a reduced number of cells with high levels of expression of the transfected constructs (data not shown).



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Fig. 2. Nucleo-cytoplasmic transport and transactivation potential of CIITA LRR mutants. (A) EGFP-CIITA LRR mutants were transiently transfected into HeLa cells and their subcellular localization was determined by fluorescence microscopy. Where indicated (+LMB), cells were treated with LMB for 2 h prior to fixation. (B) Schematic representation of the CIITA LRR mutants. The positions of the alanine mutations and the correlation to the nuclear transport phenotype (no import, impaired import or increased export) are indicated. (C) Transactivation potential of CIITA LRR mutants. HEK293 cells were transiently transfected with (5 µg) EGFP-CIITA LRR mutants and analyzed by flow cytometry 2 days after transfection for EGFP expression (x-axis) and cell-surface expression of HLA-DR (y-axis) as a measure of the transactivation potential of the mutants.

 
Influence of LRR mutations on subcellular localization and dominant-negative potential of N-terminally deleted CIITA mutants
We had previously generated a potent dominant-negative mutant of CIITA by deleting the N-terminal 334 amino acid and adding an exogenous N-terminal SV40-derived NLS (construct NLS-L335) (15). An EGFP-tagged version of NLS-L335 (EGFP-NLS-L335) is localized exclusively nuclear with strong staining of intranuclear structures (Fig.3A). The localization of NLS-L335 could not be determined by immunofluorescence since the CIITA-specific antisera used here recognizes mostly determinants in the N-terminal domain (data not shown). In contrast to EGFP-NLS-L335, a construct without the exogenous NLS (EGFP-L335) localizes in the cytoplasm and does not accumulate in the nucleus in the presence of LMB (Fig. 3A). In addition, an EGFP fusion protein to the first 339 or 408 amino acids of CIITA shows subcellular localization very similar to that of the full-length wild-type protein [data not shown, see (20)]. This indicates that the N-terminal 334 amino acids of CIITA play a major role in the nuclear localization of full-length CIITA. Our results also show that nuclear localization of the C-terminal part (335–1130) can be enforced through addition of an exogenous NLS.



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Fig. 3. Subcellular localization and dominant-negative potential of N-terminal CIITA deletion mutants L335 and NLS-L335. (A) EGFP-CIITA, EGFP-L335 and EGFP-NLS-L335 were transiently transfected into HeLa cells. Subcellular localization was determined as described in Fig. 1. (B) Dominant-negative potential of EGFP-L335 and EGFP-NLS-L335 was determined in co-transfection with wild-type CIITA by real-time PCR and specific primers for HLA-DRA mRNA. The values are expressed as percentages of the value of wild-type CIITA co-transfected with empty vector (100%). Mean values and SE bars of four independent transfections are shown. (C) Wild-type Myc6-CIITA was co-transfected with EGFP-L335 or EGFP-NLS-L335. The subcellular localization of Myc6-CIITA was determined by indirect immunofluorescence using an anti-Myc mAb and a Cy3-coupled secondary antibody (left). The dominant-negatives were visualized via EGFP fluorescence (right).

 
We analyzed the capacity of both NLS-L335 and L335 to inhibit the function of wild-type CIITA in co-transfection experiments. MHC-II HLA-DRA mRNA was measured by quantitative real-time PCR as a read-out of the dominant-negative potential. Surprisingly, L335 shows a dominant-negative effect almost as strong as NLS-L335, despite the completely different subcellular localization (Fig. 3B). Comparable transfection efficiencies were ascertained by FACS analysis of EGFP expression, and western blotting analysis for CIITA and EGFP protein expression (data not shown). Dominant-negative inhibition of CIITA function can therefore be achieved in different cellular compartments and may be due to different mechanisms. One possibility to account for the dominant-negative mechanism of L335 would be sequestration of wild-type CIITA in the cytoplasm through self-association. To address this question, we co-transfected Myc-tagged full-length CIITA with EGFP-tagged L335 and NLS-L335. Myc6-CIITA expressed alone shows similar localization to untagged or EGFP-tagged CIITA (Fig. 3C). Co-expression with EGFP-L335 leads to similar or even slightly increased nuclear staining of Myc6-CIITA (Fig. 3C). Myc6-CIITA co-expressed with EGFP-NLS-L335 shows an increased nuclear localization of Myc6-CIITA (Fig. 3C). Therefore, L335 does not inhibit CIITA function by retaining full-length CIITA in the cytoplasm.

We had shown that the dominant-negative function of NLS-L335 depends on the integrity of the LRR (12). Therefore, we tested whether LRR mutations affect the subcellular localization of NLS-L335 and L335, and whether the dominant-negative function of the cytoplasmic L335 construct is also dependent on the integrity of the LRR. As expected, LRR mutations do not affect the cytoplasmic localization of EGFP-L335 either in the presence or absence of LMB (data not shown). On the other hand, introduction of LRR mutations on the EGFP-NLS-L335 backbone leads to a strongly increased cytoplasmic localization for all mutants, with the exception of EGFP-NLS-L335-MT4 (Fig. 4A and data not shown). Treatment with LMB leads to a modest increase in nuclear staining for mutants EGFP-NLS-L335-MT6, -MT8 and -MT9. For EGFP-NLS-L335-MT13, the large majority of the cells show completely cytoplasmic staining, which is not influenced by LMB treatment. However, rare cells in untreated or LMB-treated cells show staining in both nucleus and cytoplasm (Fig. 4A). Only mutant EGFP-NLS-L335-MT4 remains nuclear, despite the LRR mutation. In contrast to EGFP-NLS-L335, this mutant does not stain subnuclear structures (Fig. 4A). Therefore, the LRR mutations are not only clearly dominant over the effect of an endogenous NLS at the N-terminus of the full-length protein, but also in N-terminally truncated CIITA proteins containing an exogenous NLS.



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Fig. 4. Subcellular localization and dominant-negative potential of EGFP-NLS-L335-LRR mutants. (A) Subcellular localization of EGFP-NLS-L335-LRR mutants was determined by fluorescence microscopy in HeLa cells. The images for MT13 show cells with the typical cytoplasmic localization, but also rare cells with nuclear staining. (B) The dominant-negative potential of LRR mutants in the L335 or NLS-L335 backgrounds was determined by co-transfecting Myc6-CIITA with EGFP-(NLS)-L335 LRR mutants into HEK293 cells and measuring HLA-DRA mRNA by real-time PCR as described in Fig. 3.

 
Subsequently, the dominant-negative potential of N-terminally deleted LRR mutants was tested (Fig. 4B). Introduction of the MT4 LRR mutation on the N-terminally deleted backbone reduces the dominant-negative effect by ~50% compared to the construct containing wild-type LRR both in NLS-L335 and in L335 (Fig. 4B). The MT13 mutation considerably reduces the dominant-negative effect, and shows a residual reduction of HLA-DRA mRNA expression of ~30% on both the NLS-L335 and L335 background (Fig. 4B). Introduction of LRR mutations from MT6, MT8 or MT9 leads to intermediate phenotypes between those of MT4 and MT13 in both types of construct (data not shown). This demonstrates that the integrity of LRR is necessary for dominant-negative function also in the cytoplasmic L335 construct.

CIITA LRR mutations do not affect self-association
Since mutations in the LRR decrease the dominant-negative potential of L335 and NLS-L335, and self-association of CIITA has been shown to be mediated by both the GBD and the LRR (1820), we reasoned that mutations in the LRR might alter the self-association between the dominant-negative L335 or NLS-L335 and the wild-type. Two forms of wild-type full-length CIITA (untagged CIITA and EGFP-CIITA) interacted efficiently with each other in our co-immunoprecipitation assay, confirming results obtained by other groups (1821) (Fig 5A). Next, the self-association potential of L335 or NLS-L335 with full-length CIITA and with themselves was examined. HEK293 cells were co-transfected with combinations of wild-type Myc6-CIITA together with Strep-TagII-L335 or Strep-TagII-L335 together with EGFP-L335. Whole-cell lysates were prepared and immunoprecipitated using anti-Myc or anti-GFP antibodies coupled to magnetic beads or Streptactin Sepharose. Both L335 and NLS-L335 were able to associate both with full-length CIITA (Fig. 5B) and with themselves (Fig. 5C). Self-association was not influenced by the completely different subcellular localization of the L335 (cytoplasmic) or NLS-L335 (nuclear, see Fig. 4) constructs.



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Fig. 5. Mutations in the LRR do not affect self-association. (A) EGFP-CIITA and unmodified CIITA were transiently co-transfected into HEK293 cells. Whole-cell lysates were prepared and immuno precipitated with anti-GFP-coupled magnetic beads. The western blot was probed using an anti-CIITA rabbit polyclonal antiserum (In: input, 1% of the lysate; IP: GFP, eluate from the immunoprecipitation using anti-GFP antibodies). The left-hand side (inputs) shows a longer exposure of the same western blot. The two bands apparent for unmodified CIITA are derived from translation initiation at the first and second AUG. (B) Myc6-CIITA and Strep-L335 were transiently co-transfected into HEK293 cells. Whole-cell lysates were used for co-immunoprecipitation or affinity purification with anti-Myc-coupled magnetic beads or with Streptactin Sepharose. Immunoblots were performed using anti-CIITA rabbit polyclonal antisera (Inp: input, 1% of lysate; IP-Myc: eluate from the immunoprecipitation using anti-Myc antibodies; Strep eluate: eluate from the Streptactin Sepharose beads). (C) Co-immunoprecipitation using anti-GFP coupled magnetic beads (IP GFP) on whole-cell lysates from HEK293 cells co-transfected with EGFP-L335 and Strep-L335. Immunoblot against CIITA. (D) Co-immunoprecipitations of Myc6-CIITA and EGFP-(NLS)-L335 with mutations MT4 or MT13 as indicated. Immuno precipitations and immunoblot were performed as described in (B). Inputs correspond to 1% of the total cell lysate. Note that the CIITA-specific antiserum recognizes mostly determinants in the N-terminal domain. The amount of the N-terminally truncated forms is therefore underestimated in the immunoblots.

 
To test if mutations in the LRR that decreased the dominant-negative potential of L335 and NLS-L335 also decreased the self-association with the full-length wild-type protein, LRR mutants in the L335 or NLS-L335 background were co-transfected with full-length Myc6-CIITA and immunoprecipitated with a GFP-specific antibody. EGFP-L335 or EGFP-NLS-L335 constructs efficiently associate with full-length CIITA, independently of the presence or absence of the MT4 or MT13 LRR mutations (Fig. 5D).


    Discussion
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 Abstract
 Introduction
 Methods
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 Discussion
 References
 
We have recently shown that CIITA is a short-lived protein and that blocking of the N-terminus by generating fusion proteins considerably stabilizes the protein (37). It was therefore important to establish the nucleo-cytoplasmic transport behavior of unmodified or even endogenous CIITA. Using indirect immunofluorescence we show here for the first time the subcellular localization of endogenous and unmodified CIITA (Fig. 1A). CIITA induced by IFN-{gamma} in HeLa cells is evenly distributed between the cytoplasm and nucleus so that it is difficult to identify the nucleus clearly in these stainings. In transfected HeLa cells, unmodified CIITA shows a somewhat more pronounced nuclear staining, which is even stronger with EGFP-CIITA. Treatment with the export inhibitor LMB leads to considerable nuclear accumulation of EGFP-CIITA and transfected unmodified CIITA, while there is only a very mild effect on endogenous CIITA (Fig. 1A). Analysis of unmodified CIITA mutants by immunofluorescence revealed a staining similar to the tagged versions (Fig. 1B). However, the EGFP-tagged forms tend to show an even more pronounced cytoplasmic staining than the unmodified forms. Taken together, these results validate the existing body of evidence obtained with the N-terminally tagged form of CIITA, but show also some interesting differences.

CIITA contains in its C-terminal region at least four LRR spanning amino acids 988–1097 (12). However, the actual number of LRR in CIITA is probably higher. Two additional LRR in this region were noted by Harton et al. (13). Sequence comparison between CIITA and the consensus sequence for the ribonuclease inhibitor subfamily of LRR sequences listed in the Conserved Domain Database (cd00116) shows an even longer region of homology between these two sequences extending from CIITA position 788 to the C-terminus. Therefore, CIITA may contain a substantially higher number of LRR than previously appreciated by us (12), or Harton et al. (13), with potentially up to 12 LRR in this region. The LRR which we described earlier (12) show the highest homology to the consensus and would correspond to LRR #8–11 of the extended homology region. Definitive information on the number of LRR in CIITA will depend on the elucidation of the three-dimensional structure of CIITA. In order to facilitate comparison with our earlier work (12), we continue to refer to the LRR analyzed here as LRR #1–4 (see Fig. 2).

Structurally, each LRR unit consists of an N-terminal ß-strand region and a central ‘loop’ region, which contain the protein–protein contact positions, followed by a C-terminal {alpha}-helical region that forms the backbone of the horseshoe-like structure of LRR proteins (34,35). The CIITA LRR alanine mutations analyzed here are not directed to structurally conserved positions like the leucines, but mainly to potential contact positions of protein–protein interactions (12).

Earlier we described the subcellular localization of EGFP-CIITA LRR mutants in HEK293 cells (12). In general, the CIITA subcellular localization was similar in HEK293 and HeLa cells as shown here, except for mutants that show residual transactivation activity (MT4, MT6, MT8 and MT9). In HEK293, these mutants show preferential cytoplasmic localization and only in cells with low protein expression levels was some protein observed in the nucleus (12). In HeLa cells, probably due to the lower expression levels, the mutant proteins are also mainly cytoplasmic, but show a more significant residual signal in the nucleus (Fig. 2A).

Using the nuclear export inhibitor LMB we analyzed the nucleo-cytoplasmic trafficking of our CIITA LRR mutants, distinguishing three different phenotypes: (i) blocked nuclear import, (ii) reduced nuclear import and (iii) increased nuclear export (Fig. 2). While the first group of mutants is completely non-functional in terms of MHC-II transactivation, the other two groups show residual transactivation potential up to nearly wild-type level. Involvement in nucleo-cytoplasmic transport of the LRR was also analyzed by Harton et al. with other LRR mutants, focusing mostly on the contribution of structurally conserved leucines within the ß-strands and {alpha}-helical regions (13). With these different LRR mutants they showed either blocked or reduced nuclear import (13).

MT4 is the only mutant we have analyzed so far that shows almost wild-type transactivation potential and an increased nuclear export phenotype (Fig. 2). Leucine-rich export signals consist of 4–5 hydrophobic residues within a 10-amino-acid region. The LRR ß-strand consensus (LxxLxL) shows similarity to the NES consensus (Lx(2,3)-[LIVFM]x(2,3)Lx[LI]) (39). Therefore, the alanine mutations (underlined positions) in MT4 (1042LAASLLRLSLYNNC) may have led to the unmasking of a cryptic NES signal.

The other LRR mutations analyzed here reduce or block the nuclear import of CIITA (Fig. 2A). Three NLS have been described within CIITA: a bipartite NLS extending from positions 141 to 159 and requiring multiple lysine acetylation (NLS1) (28), a sequence 405KEHRRPRETR (NLS2) (29) preceding the NBD, and at the C-terminus (955RDLKK, NLS3) (27).

NLS3, the first NLS described for CIITA (27), may not be a ‘classical’ NLS. Region 959RDLKK overlaps with the ß-strand of the repeat unit immediately N-terminal to the first LRR described in (12) (repeat #7 of the extended homology region). The BLS patient mutation F962S (patient Fern) (40) also targets a conserved position within the same ß-strand region. The mechanism causing cytoplasmic localization of the 959RDLKK mutant may therefore be similar to those found in other CIITA LRR mutants, such as the ones described here.

Mutations in the LRR are clearly dominant over the endogenous NLS in CIITA, but also over an exogenously added SV40 NLS, be it on the L335 background as shown here or added N-terminally to the full-length protein (data not shown). This demonstrates that the LRR play a dominant role for the nucleo-cytoplasmic transport of CIITA. Since we were not able to enforce nuclear localization of full-length CIITA LRR mutants by adding an exogenous NLS, it was not possible to analyze the contribution of the LRR to transactivation isolated from their involvement in nucleo-cytoplasmic transport (data not shown).

Several scenarios can be envisaged how the CIITA LRR control nucleo-cytoplasmic transport. These could involve binding to other proteins, dimerization and/or conformational changes, as has been shown for other proteins. For example, the NLS of NF-{kappa}B are masked through binding of I{kappa}B (41,42). Stimulation triggers degradation of I{kappa}B and unmasking of the NLS (43). The nuclear import of the eukaryotic translation initiation factor 4E (eIF4E) occurs via a piggyback mechanism mediated by the 4E-T transporter protein (44). eIF4E is imported into the nucleus via the importin-{alpha} pathway only in the presence of 4E-T (44). The signal transducer and activator of transcription STAT1 resides in the cytoplasm as a monomer, but following IFN-{gamma} stimulation it is tyrosine phosphorylated and dimerizes. Dimerization induces a conformational change that reveals an NLS on each monomer. The dimer is transported into the nucleus via an importin-{alpha}5/ß pathway (45).

The CIITA LRR have been described to be involved in both homotypic (with themselves) and heterotypic (with the GDB) interactions within CIITA (1820). It is not clear to what extent intra- and/or inter-molecular interactions are important for CIITA function. We show here by co-immunoprecipitations that both L335 and NLS-L335 efficiently associate with full-length wild-type CIITA and this despite their completely different subcellular localization. Since mutations in the LRR reduce the dominant-negative potential (Fig. 4B), we tested whether LRR mutations, which affect both subcellular localization and dominant-negative function of the N-terminally truncated forms, might also be responsible for reduced self-association between the wild-type and (NLS)-L335. Our results indicate however that this is not the case, since in co-immunoprecipitation experiments (NLS)-L335, (NLS)-L335-MT4 and (NLS)-L335-MT13 show similar self-association with the wild-type (Fig. 5C). A similar observation was made by Ting et al., who found that a number of L -> P or L -> A mutations of structurally conserved leucines in the ß-strand or {alpha}-helical regions of the LRR—while abolishing transactivation and leading to aberrant cytoplasmic localization—did not inhibit self-association of CIITA (13,18).

These results might indicate that the CIITA LRR are actually involved in the interaction with another, as yet unknown, protein partner. In this context it is very interesting that both NLS-L335 and L335 show dominant-negative inhibition of wild-type CIITA. NLS-L335 is very efficiently recruited to the MHC-II promoter (46) and acts presumably by out-competing the binding of wild-type CIITA. The situation is less clear for L335. L335 does not enter the nucleus, even in the presence of LMB (Fig. 3A); therefore, we do not think it likely that L335 acts by out-competing wild-type CIITA for binding to the promoter. L335 does not inhibit nuclear localization of wild-type CIITA (Fig. 3C). We show that L335 efficiently associates with full-length CIITA in co-immunoprecipitation experiments (Fig. 5). Increased nuclear localization of full-length CIITA, which is observed in co-transfection experiments with NLS-L335 (Fig. 3C), indicates also that heterodimerization occurs in vivo. Thus it is possible that CIITA/L335 heterodimers are still able to enter the nucleus, but that this heterodimer is transcriptionally inactive. Another possibility would be that L335, which is expressed in large excess over wild-type CIITA (37), acts as a ‘sink’ in the cytoplasm for a protein partner of CIITA, which is not necessary for nuclear import, but for transactivation. We had earlier obtained evidence for an unknown protein of 33 kDa that binds to CIITA in an LRR-dependent manner (12). Unfortunately, the nature of this protein remains elusive.

LRR mutations with residual transactivation potential and residual nuclear import show also residual dominant-negative function both on the L335 and the NLS-L335 background, while mutants that are transcriptionally completely inactive and show a ‘no-import’ phenotype, such as MT13, reduce the dominant-negative effect drastically in both types of N-terminally truncated mutants. The observation that L335 is dominant-negative, but does not enter the nucleus, suggests that the effect on the nucleo-cytoplasmic transport is not directly related to the dominant-negative effect. This makes it more likely that the interaction with an unknown protein partner, which can also take place in the cytoplasm, is necessary for the dominant-negative effect. It is not clear at present why similar mutations in different repeats have such drastically different effects. For example, mutant MT7 targets the same relative positions within the ß-strand region as MT4 in a different repeat, but is transcriptionally completely inactive, shows a no-import phenotype and abolishes dominant-negative function on the NLS-L335 background [(12) and data not shown]. An explanation for such differences will only be possible once the structure, the exact functions and the protein-binding partners of the CIITA LRR are known.

Mutations in CIITA LRR might also affect nucleo-cytoplasmic transport through conformational changes. Different point mutations along the whole protein cause nuclear exclusion, pointing to a tight regulation of nuclear import–export based on the protein conformation. CIITA might posses ‘conformational’ NLS in addition to linear NLS, thus minor changes in the protein conformation might impair its binding to importins or to adapter molecules that carry an NLS themselves and therefore can target CIITA to the nuclear compartment. Our attempts to detect conformational changes in CIITA induced by LRR mutation through limited proteolysis have so far not been successful (data not shown). Further understanding of the exact regulation of nucleo-cytoplasmic transport and the influence of the overall conformation of the protein still awaits the elucidation of a crystal structure for CIITA.


    Acknowledgements
 
We thank Claudia Kammerbauer and Ralph Löw for excellent technical assistance, and Antoine Fouillet for help with some experiments. This work was supported by the Max-Planck-Gesellschaft zur Förderung der Wissenschaften.


    Abbreviations
 
BCH-2—BCH allele 2

BLS—bare lymphocyte syndrome

CIITA —class II transactivator

eIF4E—eukaryotic translation initiation factor 4E

GBD—GTP-binding domain

LMB—leptomycin B

LRR—leucine-rich repeat

NES—nuclear export signal

NLS—nuclear localization signal

NPC—nuclear pore complex


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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