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Genes to Cells (2008) 13, 931-940. doi:10.1111/j.1365-2443.2008.01216.x
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BCL11A is a SUMOylated protein and recruits SUMO-conjugation enzymes in its nuclear body

 Takeshi Kuwata and Takuro Nakamura*

Division of Carcinogenesis, The Cancer Institute, Japanese Foundation for Cancer Research, 3-10-6 Ariake, Koto-ku, Tokyo 135-8550, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
BCL11A/EVI9 is a zinc-finger protein predominantly expressed in brain and hematopoietic cells. Previous studies show that BCL11A is involved in acute myelomonocytic leukemia and chronic lymphoid leukemia in mouse and human, respectively. Moreover, BCL11A is localized in the characteristic nuclear body in which BCL6 is co-localized. However, the significance of BCL11A in leukemogenesis and nuclear function remains unknown. In this study we show that BCL11A interacts with UBC9, a small ubiquitin-like modifier (SUMO) E2 conjugating enzyme, and recruits SUMO1 into the nuclear body. A lysine residue at amino acid 634 of BCL11A is SUMOylated but not required for the SUMO1 recruitment. The N-terminal region of BCL11A is responsible for SUMO1 recruitment as well as its nuclear body formation. We also show that SENP2, a SUMO specific peptidase, is co-localized in the nuclear body. These results suggest that BCL11A could be involved in the SUMO conjugation system, and that BCL11A might play an important role in protein modification.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Post-translational modifications influence many aspects of the protein function and abundance. Conjugation of small ubiquitin-like modifier (SUMO), called SUMOylation, is considered to be involved in the broad range of the protein function; for example, subcellular distribution, DNA-binding and transcriptional activity (Seeler & Dejean 2001, 2003; Verger et al. 2003; Gill 2004; Hilgarth et al. 2004; Falender et al. 2005; Treuter & Gustafsson 2007). SUMO molecules are covalently conjugated to the lysine residue within a consensus amino acid sequence, {psi}KxE ({psi} is a hydrophobic amino acid and x is any kind of residues), named the SUMO consensus motif (SCM). So far four members of SUMO family genes have been cloned. SUMO1, SUMO2 and SUMO3 are expressed ubiquitously, although SUMO4 is an intron-less gene and expressed in limited organs (Bohren et al. 2004). The SUMOylation process requires SUMO E1 activating enzyme, AOS1/UBA2, and an E2 conjugating enzyme, UBC9. Several proteins have been reported to possess E3 ligase activities and enhance SUMOylation of specific target proteins, although their functional significance has not yet been fully understood.

BCL11A (also called EVI9/CTIP1) is a zinc finger protein that is predominantly expressed in hematopoietic cells as well as brain (Nakamura et al. 2000; Avram et al. 2002). It was initially identified as a protein related to hematopoietic malignancies (Nakamura et al. 2000; Satterwhite et al. 2001; Nelson et al. 2007). Several lines of evidence including a knockout mouse study suggest that BCL11A is indispensable for B-cell development (Liu et al. 2003; Hystad et al. 2007).

It has been reported that BCL11A recognizes and binds to a specific DNA sequence through its zinc-finger domains, and that it functions as a transcriptional repressor (Avram et al. 2002; Senawong et al. 2003). In addition, BCL11A is present in the unique speckle structures in the nucleus (nuclear body) (Nakamura et al. 2000). However, it has not been fully examined if BCL11A has other functions than transcriptional regulation, especially in the nuclear body.

In this study we show that BCL11A is SUMOylated and recruits SUMO1 as well as UBC9 into the nuclear body. SUMOylation of BCL11A itself is not necessary for nuclear body formation or SUMO1 recruitment, whereas the N-terminal region of BCL11A is required for both nuclear body formation and SUMO1 recruitment. Our results suggest the contribution of BCL11A in SUMOylation process other than transcriptional regulation.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
BCL11A is a SUMOylated protein

For clarifying the function of BCL11A, we first looked for the proteins interacting with BCL11A by using yeast two hybrid system. Screening 3 x 106 clones of a mouse B-lymphocyte cDNA library, 19 independent clones containing the UBC9 coding sequences, a SUMO E2 conjugating enzyme, were identified as a BCL11A interacting protein using the N-terminal region of BCL11A that contains a single zinc finger domain (Nakamura et al. 2000). Subsequently, two SUMO E3 ligases, PIAS3 (two clones) and PIASy (one clone), were identified using the middle and the C-terminal zinc finger domain of BCL11A as baits, respectively. UBC9 was in part co-localized with BCL11A in its specific nuclear body structure in NIH 3T3 cells (Fig. 1A). Immunoprecipitation analysis showed that PIAS3 was co-precipitated with BCL11A, suggesting that these proteins were interacting with each other (Fig. 1B). Although it was of our interest to identify the endogenous complex containing BCL11A and UBC9, and/or SUMO E3 ligases, we failed to show the interactions. This could be explained by the facts that BCL11A is expressed in limited cell types such as B-cells and its endogenous expression level is low, and that endogenous expression of PIAS3 and PIASy are not always detectable.


Figure 1
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  		Figure 1  (A) BCL11A is co-localized with UBC9 in NIH 3T3 cells. EGFP-UBC9 and myc-BCL11A were expressed in NIH 3T3 cells and stained with the anti-myc antibody. (B) BCL11A interacts with PIAS3. The cell lysates from HEK293T cells expressing myc-BCL11A and/or Flag-PIAS3 were immunoprecipitated with the anti-Flag antibody. Precipitated proteins were subjected to immunoblotting using the anti-Flag- (top) and anti-myc (below) antibodies.

 
As the SUMO E2 conjugating enzyme and E3 ligases are co-localized or interacted with BCL11A, we next examined whether BCL11A itself could be SUMOylated. When BCL11A was expressed together with SUMO1, several higher mobility-shifted bands appeared, suggesting that SUMO might be conjugated to BCL11A (Fig. 2A). The band approximately 150 kDa that corresponds to the shifted BCL11A band was identified with anti-SUMO1 (Fig. 2B). As SUMO1 induces mono-SUMOylation in contrast to SUMO2 and SUMO3 that cause poly-SUMOylation, multiple shifted bands suggest that the presence of several SUMOylation sites in BCL11A. Indeed, when we carried out an in vitro SUMOylation assay using BCL11A and each SUMO proteins, only a single shifted band was appeared with SUMO1 molecules. In contrast, SUMO2 or SUMO3 induced multiple shifted bands were observed (Fig. 2C). These results strongly suggest that BCL11A might have only a single SUMOylation site.


Figure 2
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  		Figure 2  (A) BCL11A is SUMOylated in vivo. Myc-BCL11A and/or 6xHis-SUMO-1gg are expressed in HEK293T cells. BCL11A was detected by the anti-myc antibody. (B) The lysate of HEK293T cells co-transfected with myc-BCL11A and 6xHisSUMO1gg was immunoprecipitated with an anti-myc antibody. The precipitated proteins were examined by Western blottnig by using anti-myc (left panel) and anti-SUMO1(right panel) antibodies. (C) BCL11A is SUMOylated in vitro. [35S-methionine]-labeled in vitro transcribed/translated myc-BCL11A is incubated in the solution containing indicated SUMO proteins with or without SUMO E1 and E2 ligases. SUMOylation of BCL11A is detected by an autoradiography after SDS-PAGE.

 
BCL11A recruits SUMO1 into its nuclear body

As BCL11A is localized in the specific nuclear body structure, we examined if BCL11A expression affects subcellular distribution of SUMO1. When SUMO1 was expressed alone, it was mainly located in the nucleus but not in any specific structures as was reported previously (Fig. 3A) (Matunis et al. 1996; Shen et al. 2006). Surprisingly, when SUMO1 was expressed with BCL11A, it was recruited and co-localized together with BCL11A in the nuclear body structure. BCL6, which is localized in the BCL11A nuclear body (Nakamura et al. 2000), failed to recruit SUMO1 into its own nuclear body, suggesting that BCL11A and BCL6 present in different compartment in the absence of their partners. Alternatively, BCL6 by itself is unable to interact with SUMO1.


Figure 3
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  		Figure 3  BCL11A recruits the SUMO1gg but not the SUMO1aa mutant. Myc-BCL11A and 6xHis-SUMO1gg (A) or 6xHis-SUMO1aa (B) are expressed in U2OS cells. Cells were stained with anti-myc and anti-6xHis antibodies.

 
SUMO1 is processed from its precursor protein by hydrolysis resulting in the production of the C-terminal double-glycine motif that is necessary for its conjugation to the target proteins (Verger et al. 2003). When the SUMO1aa mutant in which the double-glycine motif was substituted with double-alanine was expressed, it was no longer co-localized with BCL11A in the nuclear body, indicating that co-localization of BCL11A and SUMO1 requires SUMO conjugation to its target proteins (Fig. 3B).

The BCL11A K637 is SUMOylated but not required for SUMO1 recruitment into the nuclear body

Because BCL11A was interacted with SUMO1 as well as SUMO E2 conjugating enzymes and E3 ligases, we examined whether BCL11A itself would be the target of SUMOylation. A search of the SCM showed that there are three putative sites in the BCL11A sequence. We constructed the mutants which have a lysine residue substituted to arginine for each putative SCM (Fig. 4A). SUMOylation analysis in vivo showed that the shifted bands were significantly diminished in the K637R mutants, whereas the other mutations (K123R and K833R) did not affect SUMOylation of BCL11A at all (Fig. 4B). The result indicates that the lysine residue at 637 is the target of SUMOylation in BCL11A. This was further supported by the result that aa 376–740 of BCL11A containing K637 exhibited the shifted band corresponding to the SUMO binding fragment (Fig. 4C). Moreover, the shifted bands disappeared in K637R and K637/833R mutants but not in K123R and K833R mutants (Fig. 4D). Surprisingly, the K637R mutant was located in the similar nuclear body and still recruited SUMO1, suggesting that SUMOylation of BCL11A itself might not be required for nuclear body formation nor SUMO1 recruitment (Fig. 5).


Figure 4
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  		Figure 4  (A) Three putative SUMO consensus motifs are present in BCL11A. Black boxes indicate zinc finger motifs. (B) The lysine residue at amino acid 637 of BCL11A is SUMOylated in vivo. BCL11A mutants with lysine to arginine substitution at indicated lysine residues are co-transfected with 6xHis-SUMO1 in HEK293T cells. BCL11A is detected by an anti-myc antibody. (C) Myc-tagged BCL11A (aa 376–740) containing lysine 637 was expressed with or without 6xHis-SUMO1gg in HEK293T cells. Cell lysates were examined by Western blotting with anti-myc or anti-6xHis antibodies. (D) Each lysine to arginine mutants of myc-tagged BCL11A was examined for in vitro SUMOylation with either SUMO1 (left) or SUMO2 and SUMO3 (right). Un-SUMOylated (closed triangle) and SUMOylated forms (open triangle) of BCL11A were shown by Western blotting with an anti-myc antibody.

 

Figure 5
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  		Figure 5  The BCL11A K637R mutant still recruits SUMO1. Myc-BCL11A K637R is expressed with 6xHis SUMO1gg in U2OS cells. Each protein is detected by anti-myc and 6xHis antibodies.

 
The N-terminal region of BCL11A is responsible for nuclear body formation and SUMO1 co-localization

To clarify the region responsible for BCL11A nuclear body formation, several N- and C-terminal deletion mutants were expressed and the nuclear body formation was assessed. The N-terminal 210 amino acid sequences (210R) were sufficient for nuclear body formation, although some part of the protein accumulated in the extra-nuclear regions presumably because of degradation. In contrast, when the N-terminal 168 amino acids were deleted, diffuse nuclear localization of the protein was observed, suggesting that the region is necessary for BCL11A nuclear body formation. We then examined whether these deletion mutants could recruit SUMO1. Interestingly all the mutants that formed nuclear body could recruit SUMO1 into the structure, indicating that the N-terminal region of BCL11A is also important for SUMO1 recruitment (Fig. 6). In addition, UBC9 was recruited into the body formed by 286E (Fig. 7), confirming the authenticity of the yeast two hybrid experiment (see above). However, 286E was not co-localized with BCL6 (Fig. 7). The result is consistent with the data by which the zinc finger domains located in the C-terminal but not the N-terminal half of BCL11A are important for BCL6 interaction (Nakamura et al. 2000, and data not shown).


Figure 6
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  		Figure 6  The N- and C-terminal deletion mutants of BCL11A and their localization. Full length and deletion mutants of BCL11A are expressed as DsRedNuc fusion proteins in U2OS cells (left). Co-localization of SUMO1 is examined by using EGF-SUMO1gg (right).

 

Figure 7
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  		Figure 7  The BCL11A C-terminal deletion mutant, 235E, recruits UBC9 but is not co-localized with BCL6.

 
A SUMO specific protease is co-localized with BCL11A

Several SUMO/Sentrin specific peptidases (SENP) have been cloned recently and these studies showed that each SENP shows different subcellular localization (Hay 2007). SENP2 and SENP7 are involved in the BCL6 complex (Miles et al. 2005), and we found that SENP2 but not SENP7 was located in nuclear speckles. SENP2 was co-localized with BCL11A as well as BCL6 but not with SUMO1 (Fig. 8A, and data not shown). Interestingly, the 286E mutant of BCL11A was not co-localized in SENP2-forming nuclear speckles (Fig. 8B), suggesting that the region is required for BCL11A nuclear body formation and SUMO1 recruitment might be different from the region for co-localization with SENP2. When BCL11A, SENP2 and SUMO1 were co-expressed simultaneously, all three proteins were co-localized in the same speckles (Fig. 8C), suggesting that SENP2 by itself might be localized in its own speckles in the absence of BCL11A, and it might interact with BCL11A but not with SUMO1. Consistent with the idea, when the 286E mutant was co-expressed with SUMO1 and SENP2, the majority of SENP2 was localized independently from the speckles formed by 286E and SUMO1 (Fig. 8D).


Figure 8
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  		Figure 8  (A) SENP2 is co-localized with BCL11A but not with SUMO1. HA-SENP2 is expressed alone or with myc-BCL11A or 6xHis SUMO1 in U2OS cells, and visualized with mouse anti-myc or anti-His and rabbit anti-HA antibodies. (B) The BCL11A C-terminal deletion mutant, 286E, is not co-localized with SENP2. (C) and (D) EGFP-SUMO1, DsRed-BCL11A (C), 286E (D) and/or HA-SENP2 were expressed in U2OS cells. SENP2 was detected using an anti-HA antibody followed by the Alexa647 conjugated anti-mouse antibody.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
Although the functional relevance of SUMO conjugation has not been fully understood, it has been shown that a number of gene products involved in oncogenesis are SUMOylated and associated with nuclear bodies (Stelter & Ulrich 2003). SUMOylation is necessary for PML to form the nuclear body, where PML collaborates with its functional partners (Shen et al. 2006), and interaction with UBC9 and SUMOylation modulates the transcriptional activity and nuclear body assembly of TEL (Chakrabarti et al. 1999, 2000). Abnormalities in SUMO modifying proteins have also been reported. UBC9 up-regulation is observed in certain lung cancers (McDoniels-Silvers et al. 2002). It has also been reported that UBC9 is indispensable for mouse embryogenesis, especially for maintaining proper nuclear architecture and accurate chromosome segregation (Nacerddine et al. 2005). More directly, a SUMO/Sentrin-specific protease SENP1 was found as the target of chromosomal translocation t(12;25) (q13;q25) in a case of human infantile teratoma (Veltman et al. 2005). Thus the SUMO pathway is important for cellular homeostasis and it is very likely that dysfunction in the SUMO signaling is important in tumorigenesis.

Among the proteins associated with the BCL11A nuclear body, SUMO1 and UBC9 are also involved in the PML body. PML requires SUMOylation process for its nuclear body formation that is disrupted in acute promyelocytic leukemia (Sternsdorf et al. 1999; Zhong et al. 2000). As our previous study showed that the BCL11A nuclear body is a distinct structure from the PML body, it is possible that UBC9 is sequestrated between these two structures and its function was regulated by proper spatial organization.

Like ubiquitin SUMO molecules are conjugated to lysine residues within SCM of target proteins (Sternsdorf et al. 1999). There are three putative SCM in BCL11A and K637 is confirmed to be, in fact, SUMOylated. It is reported that SUMO1 conjugation is a mono-SUMOylating reaction (single SUMO molecule is conjugated to a lysine residue), whereas SUMO2 and SUMO3 generate poly-SUMOylation because they have SUMOylation sites on their own sequences and cause auto-SUMOylation (Saitoh & Hinchey 2000; Tatham et al. 2001). Our in vivo experiment showed that co-expression of SUMO1 with BCL11A resulted in producing several shifted bands whereas the in vitro assay showed a single shifted band (see Fig. 2A,B) and the K637R mutant showed no shifted band in vivo (Fig. 4B). The reason for the difference between in vivo and in vitro assays is yet to be determined. One possibility is that SUMO1 conjugation causes conformational change of BCL11A and subsequently triggers endogenous SUMO2/3 conjugation onto K637. It has been reported that there are functional differences between SUMO1 and SUMO2/3 (Kamitani et al. 1998; Saitoh & Hinchey 2000). Although it has been reported that BCL11A enhances COUP-TCFII-mediated transcriptional repression (Senawong et al. 2003), SUMOylation of BCL11A did not affect the repressional activity (data not shown). We could not detect endogenous SUMOylation of BCL11A, probably because of the minor fraction of endogenous SUMO molecules are conjugated to BCL11A.

By using several deletion constructs of BCL11A, we identified that the N-terminal region of BCL11A is responsible for its nuclear body formation as well as SUMO1 recruitment. Recently it has been reported that several proteins including PML could interact with SUMO molecules non-covalently through a SUMO interacting motif (SIM) (Minty et al. 2000; Song et al. 2004; Shen et al. 2006; Kerscher 2007). However, no SIM could be identified in BCL11A within the range of our search. Although the mechanism how BCL11A recruits SUMO1 into the nuclear body is yet to be clarified, one possibility is that the N-terminal region of BCL11A binds to another molecule that contains an SIM and results in indirect recruitment of SUMO1.

In conclusion, we have shown that several SUMO-related molecules interact and/or are co-localized with BCL11A. UBC9, PIAS3 and PIAsy are involved in the SUMO conjugation process. Senp2 has an activity to remove SUMO molecules from SUMOylated proteins, moreover, it hydrolyzes SUMO precursors to expose the di-glycine motif, facilitating SUMO conjugation onto target proteins. These results underscore the importance of the SUMOylation signaling in regulation and modification of the BCL11A function.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Cell culture and transfection

NIH 3T3 cells were maintained in minimum essential medium (Invitrogen, Carlsbad, CA) with 10% calf serum and Penicillin–Streptomycin mixture (Nacalai Tesque, Kyoto, Japan). HEK293T and U2OS cells were maintained in Dulbecco's modified Eagles medium (Sigma, St Louis, MO) supplemented with 10% fetal bovine serum, 1x non-essential amino acid solution, 1 mM sodium pyruvate (Sigma) and Penicillin–Streptomycin mixture. The cells were cultured at 37 °C under 5% CO2 humid atmosphere. Transfection of plasmid DNA to HEK293 and U2OS cells was carried out using LipofectAmine2000 reagents (Invitrogen) and FuGeneHD transfection reagents (Roche, Basel, Switzerland), respectively.

Antibodies

Mouse monoclonal antibodies for myc-, HA- and 6xHis-tag sequences were purchased from Roche, Invivogen (San Diego, CA) and Amersham Bioscience (Little Chalfont, UK), respectively. A rabbit polyclonal anti-SUMO1 antibody was from Alexis Biochemicals (San Diego, CA). Rabbit polyclonal antibodies for myc- and HA-tag sequence were purchased from Sigma. Alexa-488/546 conjugated anti-mouse/rabbit Ig antibodies were purchased from Invitrogen.

Oligonucleotide–PCR primers

The sequences of the oligonucleotides and primers used for tagging or in vitro mutagenesis are listed in Supplementary Information.

Plasmids

pcDNA-myc-Bcl11a and pcDNA-BCL6-myc were described previously (Nakamura et al. 2000). pBCL6-EGFP was produced by transferring the BCL6 cDNA to pcDNA-CTmyc and pEGFP-C1 (Clontech, Mountain View, CA), respectively, by using a PCR technique. pcDNA-HA-SENP2 was produced by ligating the PCR-amplified coding region of Senp2 to pcDNA-HA. pEGFP-SENP2 was produced by transferring HindIII/ApaI fragment of pcDNA-HA-SENP2 to the pEGFP-C1 vector. pSG5–6xHis-SUMO-1gg was gifted from Dr Issay Kitabayashi (National Cancer Center Research Institute, Japan). pEGFP- and pGEX-SUMO1gg were generated by ligating the PCR-amplified SUMO1gg sequence to pEGFP-C1 and pGEX4T-1 (Amersham Bioscience), respectively. For generating pGEX-SUMO2gg and SUMO3gg, coding sequences of SUMO2gg and SUMO3gg were produced by PCR and ligated to the pGEX4T-1 vector. pET28-AOS1 and pET11-ubc2 were gifted from Dr Flank Melchior (Max-Planck Institutes for Biochemistry, Germany). pET28-UBC9 was generated by ligating the PCR-amplified UBC9 sequence to the pET28a vector (Merck, Darmstadt, Germany). Accuracies of the PCR-amplified DNA sequences were verified by DNA sequencing.

In vitro mutagenesis

The SUMO1aa mutant was produced by a KOD + mutagenesis kit (Toyobo, Tokyo, Japan) with specific primers. Lysine to arginine mutants of BCL11A (pcDNA-Bcl11a-K123R, K637R and K833R) were generated by using a GeneEditor mutagenesis kit (Promega, Madison, WI) according to the manufacture's instruction. For making DsRedNuc-Bcl11a N- or C-terminal deletion mutants (pDsRedNuc-FL, 740R, 283E, 210R, 431–740 and 169–740), PCR-amplified Bcl11a sequences with corresponding primer sets were ligated with the pDsRedNuc vector (Clontech). Sequences of the mutant products were verified by DNA sequencing.

Cell lysate, immunoprecipitation and immunoblotting

Twenty-four hours after transfection, the cells were washed with PBS and collected by scraping. For Western blotting, the cells were directly lysed in the Laemmlli's sample buffer and boiled for 5 min. For the immunoprecipitation assay, the cells were lysed in TNE buffer (10 mM Tris, pH 7.5, 140 mM NaCl, 0.1 mM EDTA and 1% NP-40). Five millimolar of N-ethylmaleimide was added in the buffer for showing SUMOylated form of BCL11A. The cell lysate was pre-cleared with ProteinA-sepharose beads, incubated with anti-myc antibody for 2 h at 4 °C, and precipitated by incubating with ProteinA-sepharose beads for 1 h at 4 °C. The precipitated proteins were eluted from the beads by directly boiling in the Laemmeli sample buffer. Proteins were separated by SDS-PAGE and blotted onto nitrocellulose membranes. The membranes were first blocked with 3% skim milk and incubated with indicated primary antibodies followed by HRP-conjugated corresponding secondary antibodies. The detection was carried out using the ECL plus system (Amersham).

Recombinant protein purification and in vitro SUMOylation assay

Recombinant SUMO1/2/3gg, AOS1, UBC2 and UBC9 proteins were expressed in Escherichia coli strain BL21. SUMO1gg proteins were purified with glutathione-sepharose beads (Amersham) and recovered by thrombin cleavage. AOS1/UBC2 complexes were formed by mixing each bacterial lysates and dialysis against PBS containing 10% glycerol overnight at 4 °C. AOS1/UBC complexes and UBC9 were purified by using Ni-NTA agarose beads and eluted in the imidazole buffer. All the purified recombinant proteins were dialysed against PBS containing 10% glycerol. BCL11A and its mutants were produced by using the TNT in vitro transcription and translation system (Promega). The in vitro SUMOylation assay was carried out as reported previously (Pichler et al. 2002). An in vitro SUMOylation analysis of BCL11A K123R, K637R, K833R and K637/833R was carried out with the SUMOylation kit (BIOMOL, Plymouth Meeting, PA).

Immunofluorescence

Cells grown onto 4-well chamber slides (BD Falcon, Franklin Lakes, NJ) were transfected with plasmids for expressing indicated proteins. Twenty-four hours after transfection, cells were washed with PBS and fixed with 3% paraformaldehyde. After permealizing the cell membrane with 0.2% tritonX-100, samples were stained with the indicated primary antibodies followed by Alexa488-, Alexa536 and/or Alexa647-conjugated corresponding secondary antibodies. Nuclei were stained with ToPro3 and mounted in ProlongGold (Invitrogen). Images were captured and analyzed by a confocal microscopy (Leica AS MDW system).


   Acknowledgements
 
This work was supported by Grant-in-Aid for Scientific Research on Priority Areas "Integrative Research Toward the Conquest of Cancer" from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and for Scientific Research (C) from the Japan Society for the Promotion of Science, and Kitasato University Research Grant for Young Researchers. We would like to thank Drs. Issay Kitabayashi and Flank Melchior for providing constructs.


   Footnotes
 
Communicated by: Masayuki Yamamoto (The University of Tokyo)

* Correspondence: takuro-ind{at}umin.net


   References
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 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Received: 26 February 2008
Accepted: 4 June 2008





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