Journal of Cellular Biochemistry 98:895–911 (2006)
SUMO Regulates the Cytoplasmonuclear
Transport of its Target Protein Daxx
Angela Chen,1* Ping-Yao Wang,1Yu-Chih Yang,1Yi-Hsin Huang,1Jeng-Jung Yeh,1 Yu-Huai Chou,1Jiin-Tsuey Cheng,2Yi-Ren Hong,3and Steven S.-L. Li1,4
1Center for Nanoscience, Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan
2Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan 3Graduate Institute of Biochemistry, Kaohsiung Medical University, Kaohsiung, Taiwan 4Department of Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan
Abstract It is known that Fas death domain-associated protein (Daxx) possesses both putative nuclear and cytoplasmic functions. However, the nuclear transport mechanism is largely unknown. This study examined the nuclear location signal (NLS) of Daxx and whether the nuclear transport of Daxx was mediated by small ubiquitin-related modifier (SUMO). Two NLS motifs of Daxx, leucine (L)-rich nuclear export signal (NES)-like motif (188IXXLXXLLXL197) and C-terminal lysine (K) rich NLS2(amino acids 627–634) motif, were identified and the K630and K631on the NLS2motif were characterized as the major sumoylation sites of Daxx by in vitro sumoylation analysis. Proteins of inactive SUMO (SUMO-D), a sumoylation-incompetent mutant, and Daxx NLS mutants (Daxx-NESmutand Daxx NLS2mut) were dispersed in cytoplasm. The cytoplasmic dispersed Daxx mutants could be relocalized to nucleus by cotransfection with active SUMO, but not with inactive SUMO-D, demonstrating the role of SUMO on regulating the cytoplasmonuclear transport of Daxx. However, inactive SUMO-D could also be relocalized to nucleus during cotransfection with wild-type Daxx, suggesting that SUMO regulation of the cytoplasmonuclear transport of its target protein Daxx does not need covalent modification. This study shows that cytoplasmic SUMO has a biological role in enhancing the cytoplasmonuclear transport of its target protein Daxx and it may be done through the non-sumoylation interactions. J. Cell. Biochem. 98: 895–911, 2006. ß2006 Wiley-Liss, Inc.
Key words: Daxx; SUMO; cytoplasmonuclear transport; nuclear localization signals; non-sumoylation interactions
Human Fas death domain-associated protein (Daxx) is a 740-amino acids protein mainly localized in nucleus. It functioned as a tran-scriptional repressor when associated with chromatin in nucleus [Hollenbach et al., 1999, 2002]. Daxx-mediated repression of glucocorti-coid receptor (GR) transcriptional activity was enhanced [Muromoto et al., 2004] by interacting with tumor susceptibility gene (TSG) 101. However, its transcription repression can be
inhibited by the nuclear body-associated pro-myelocytic leukemia (PML) protein sequester-ing Daxx to the PML oncogenic domains (PODs) [Ishov et al., 1999; Li et al., 2000a; Lin et al., 2003], or by microspherule protein (MSP) 58 sequestering Daxx to the nucleolus [Lin and Shih, 2002]. Daxx’s functions were terminated as its subnuclear localization changed [Muller et al., 1998; Ecsedy et al., 2003]. In cytoplasm Daxx was associated with cell surface and cytoplasmic molecules, including Fas [Yang et al., 1997] and transforming growth factor beta (TFG-b) [Perlman et al., 2001]. The increased cytoplasmic Daxx was more suscep-tible to apoptosis [Mo et al., 2004]. In addition, Daxx also interacts directly with apoptosis signal regulating kinase 1 (ASK1) and activates ASK1 in cytoplasm [Chang et al., 1998]. Daxx recruited to cytoplasm during overexpression of ASK1 has subsequently induced caspase-independent cell death [Charette et al., 2001]. ß2006 Wiley-Liss, Inc.
Grant sponsor: National Science Council of Taiwan; Grant number: NSC 89-2311-B110-0215.
*Correspondence to: Dr Angela Chen, Center for Nanoscience, Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan.
E-mail: [email protected]
Received 20 August 2005; Accepted 6 October 2005 DOI 10.1002/jcb.20703
Daxx is a shuttle protein participating in biological functions on various subcellular loca-lizations. The suggestion was that it contained both the nuclear localization signal (NLS) [Pluta et al., 1998] and the nuclear export signal (NES) [Song and Lee, 2004] for translocating into and exporting out of nucleus, respectively. The NES motif of Daxx has been identified on amino acid 565–575 residues by Song and Lee [2004], but the NLS motif of Daxx remains unclear. The lysine-rich consensus sequence proposed [Pluta et al., 1998] that Daxx contains two candidate NLS motifs, NLS1(391–395 a.a.) and NLS2(627–633 a.a.). The former, however, has been demonstrated to be non-functional by Daxx deletion mutant (1–625 a.a.) and site-direct mutagenesis [Torii et al., 1999]. Although the C-terminal Daxx (625–740 a.a.) localized in nucleus, its NLS2 motif was regarded as non-functional by site-direct mutagenesis on both lysine 630 and 631 residues [Jang et al., 2002]. These two residues were regarded as the major sumoylation sites on Daxx by sumoylation assays in vivo by Jang et al. [2002].
Small ubiquitin-related modifier (SUMO) played a general role in regulating protein– protein interactions both in cytoplasm and in nucleus. In nucleus the treanscriptional activ-ities of SUMO-1 modified transcription factors, including p53, c-jun, Sp-3, c-Myb, and c/EBP families were reduced [Gill, 2004]. It inhibits RAD51-mediated homologous recombination by interaction with RAD51 [Li et al., 2000b]. In cytoplasm SUMO-1 stabilized IkBa by blocking ubiquitination [Desterro et al., 1998] and modu-lated the partitioning of Ran-GAPase-activat-ing protein (RanGAP1) between the cytosol and nuclear pore complex (NPC) [Matunis et al., 1996; Tatham et al., 2005; Reverter and Lima, 2005]. It represses ASK1 activation through physical interaction and not through covalent modification in cytoplasm [Lee et al., 2005]. Nevertheless, whether SUMO has a role in the cytoplasmonuclear transport is not completely understood.
It is reported in this paper that SUMO enhances the nuclear transport of Daxx. Three sumoylation sites of Daxx were determined by sumoylation assays in vitro and two NLS motifs of Daxx were identified. The cytoplasm mis-localized mutants of Daxx were used to analyze the nuclear transport function of SUMO and the non-sumoylation interactions between SUMO and Daxx are discussed.
MATERIALS AND METHODS Plasmids
Plasmids encoding wild-type pAS2-SUMO, active pAS2-SUMO and inactive pAS2-SUMO-D deletion mutants were subcloned from SUMO cDNA sequences [Mannen et al., 1996] using appropriate oligonucleotides. The inactive SUMO-D were amplified by PCR from wild-type SUMO using forward primer of the wild-type and specific reverse primers to delete the amino acids beyond or including double glycines of the C-terminal of SUMO. The plasmids encoding wild-type pACT2-Daxx, pGEX-KG-p53 (377– 393 a.a.), and pGEX-KG-PML (466–502 a.a.) were constructed by PCR amplification of the human liver cDNA libraries. Wild-type pACT2-Daxx was used as a template to construct Daxx deletion mutants, pACT2-D1 (1–282 a.a.), pACT2-D2 (245–508 a.a.), pACT2-D3 (342–625 a.a.), pACT2-D4 (607–740 a.a.), and pGEX-KG-D1-S (46–75 a.a.). Mutations were created by an overlap extention PCR method [Pan and McEver, 1993].
For sumoylation assays in vitro, SUMO and inactive SUMO-D were subcloned into NH2 -terminal His-fusion pET-28a expression vector. Daxx and its deletion mutants were subcloned into NH2-terminal glutathione-S-transferase (GST)-fusion pGEX-KG vector using appro-priate oligonucleotides. SUMO and inactive SUMO-D deletion mutants were subcloned into NH2-terminal fusion pRED vector and Daxx and its deletion mutants were subcloned into NH2-terminal fusion pEGFPC1/2 vectors using appropriate oligonucleotides for in vivo fluores-cence microscopy assays. All constructs used in this study were restriction mapped and sequenced.
Yeast Transformation and b-Galactosidase Assay Yeast strain CG-1945 was co-transformed with a possible pair of pAS2 and pACT2 cons-tructs and analyzed for the interaction. The b-galactosidase assays have been described previously [Ryu et al., 2000].
Sumoylation Assays In Vitro
Sumoylation assays developed by Tatham et al. [2001] have been applied in this study. Plasmids encoding pGST-SAE1/SAE2 and pGST-Ubc9 were kindly provided by Prof. R.T. Hay (University of St. Andrews, UK). The 10 ml reactions were incubated for 3 h at 378C, then
stopped by adding 1/5 vol 10% SDS, boiled and separated by SDS–PAGE. Sumoylation pro-ducts were discerned by Coomassie blue stain-ing or immunoblot analysis.
Cell Culture and Transfections
HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) containing 3.7 g/L sodium bicarbonate (Amresco), 2 mM L-glutamine (Hyclone),
50 mg/L gentamicin (Invitrogen), and 1% penicillin and streptomycin (Invitrogen), and supplemented with 10% bovine calf serum (Hyclone) in a 5% CO2incubator.Transfection was performed using Superfect transfection reagent (Qiagen) according to the manufac-turer’s instructions. Briefly, 10 ml Superfect was mixed with 2 mg of DNA in a serum free Opti-MEM medium (Invitrogen) and added to approximately 1 106 cells. Two hours after transfection, the medium was removed and cells were cultured in fresh DMEM with 10% bovine calf serum. Transfected cells were used for observing the protein expressions and cellular localizations by fluorescence/confocal micro-scopy, Western blotting, and immunoprecipita-tion assays.
Fluorescent/Confocal Microscopy pEGFP-Daxx, their mutants, as well as pDsRFP-SUMOs, and C-terminal GG deleted mutants were transfected in HeLa cells and incubated for 12 h. Cells were washed with PBS three times and fixed with 4% paraformalde-hyde followed by 0.4% Triton X-100 (ACROS) with 2% FBS (HyClone) in PBS and stain-ing nuclei with 4, 6-diamidino-2-phenylindole (DAPI) (Sigma) for 5 min, washed with PBS three times. Cellular localizations of expressed proteins were examined with the fluorescence/ confocal microscope (AXIOskop2þFL, Zeiss/ FluoView 500, Olympus), and images were processed with Adobe photoshop software. For each experiment, at least 200 cells were examined.
Immunoblot Analysis
Cells were lysed with ice cold mammalian lysis buffer. The cell lysates containing 1,000 mg proteins were immunoprecipitated with mono-clonal mouse anti-GFP (SC-9996) from Santa Cruz, monoclonal mouse living colors-DsRed antibody (8374-2) and polyclonal rabbit living colors-DsRed antibody (632397) from Clontech,
and monoclonal mouse anti-actin antibody (MAB1501) from Chemicon, and subjected to electrophoresis. Proteins were immunoblotted with appropriate antibodies and detected with the Western lightning chemiluminescence reagent plus (PerkinElmer life science).
RESULTS
Yeast Two Hybrid Interactions
Using SUMO as bait in yeast two hybrid assays, the C-terminal Daxx fragment was first identified from human liver cDNA libraries. Four Daxx deletion mutants (D1, D2, D3, and D4) (Fig. 1A) were then constructed for further identification of the interaction domains of Daxx with SUMO. Cotransformants were ana-lysized by in vivo filter assay (Table I). The interaction intensities between active SUMO and Daxx fragments were further examined for the b-galactosidase activities using ONPG as a substrate (Fig. 1B). Interaction between pAS2-1-p53 and pACT2-SV40 was used as a positive control and the empty vectors of pAS2-1and pACT2 were used as a negative control.
Table I shows that active SUMO-1 and SUMO-2 have positive filtered assay (indicated as þ in Table I) with full length Daxx, N-terminal D1 and C-N-terminal D4 fragments, but not D2 and D3. The b-galactosidase activities between SUMO-2 and full length of Daxx at 90%, D1 at 98%, and D4 at 121% (shown in Fig. 1B) were found to be similar to that of SUMO-1 and full length Daxx (taken as 100% interaction). However, it was decreased to 25% between SUMO-1 and D1, but increased to about 200% between SUMO-1 and D4. Distinc-tive b-galactosidase activities suggested that strong interactions between SUMO-2 and D1 as well as SUMO-1 and D4 occurred. Inactive SUMO (SUMO-D), a sumoylation-incompetent mutant, was used to suppress the formation of sumoylation linkages in the in-yeast interac-tions. Positive filter assays (Table I) were obtained from the interactions between SUMO-2-D and full length Daxx or D1 deletion mutant. Similarly, SUMO-1-D also positively interacted with the full length Daxx or D4 mutant. It further demonstrated that strong non-sumoylation binding interactions exist between SUMOs and Daxx, particularly for SUMO-2 and D1 fragment, and for SUMO-1 and D4 fragment. The fact that D1-K60R mutant, a D1 mutant of which the lysine of amino acid SUMO Regulates the Nuclear Transport of Daxx 897
residue 60 (K60) of the N-terminal sumoylation consensus sequence (57YKXE62) has changed to arginine, terminating its interactions with SUMO in yeast further suggests that K60is an important interacting residue for positive yeast two hybrid interactions between SUMO and D1 fragment (Table I). Whether K60residue of Daxx is a sumoylation site for SUMO could not be ascertained from the yeast two-hybrid interactions.
In Vitro Sumoylation Assays
The fact that the N-terminal (D1) and the C-terminal (D4) deletion mutants interact positively with SUMO (Table I) indicates that both fragments may contain sumoylation sites. Sumoylation assays in vitro were applied to manifest sumoylation between SUMO and D1 or D4. Active His-SUMO and inactive His-SUMO-D reacted with the GST-fused His-SUMO-D1 or His-SUMO-D4, and then reaction products were analyzed by Wes-tern blotting with polyclonal GST or anti-D4 antibodies. The in vitro sumoylation com-plexes of SUMO and D1 could not be identified by immunoblots with GST-antibodies. This was due to the fact that non-specific proteins migrated at the same position on the gel after electrophoresis and also due to the fact that the D1-specific antibodies were not available. How-ever, the sumoylation complexes of D4 and active SUMO-2 or SUMO-1 (lanes 4 and 6 of Fig. 2, respectively) were obtained, but not using inactive SUMO-2-D and SUMO-1-D (lanes 5 and 7, Fig. 2, respectively). This indicates that sumoylation sites exist on the D4 fragment. Therefore, three single-site muta-nts (D4-K630A, D4-K 631A, and D4-K634A), a double-site mutant (D4-K630, 631A), and a triple-site mutant (D4-K630, 631, 634A) of GST-fused Daxx C-terminal mutant proteins (constructs shown in Fig. 3A) were applied to identify the sumoylation sites on the C-terminal Daxx. TABLE I. Interaction of SUMOs With Daxx Deletion Fragments by Yeast
Two Hybrid Filter Assays
Yeast two hybrid filter assays
(Amin acids) SUMO-2 SUMO-2-D SUMO-1 SUMO-1-D
Daxx (1–740) þ þ þ þ D1-K60R (1–282) D1 (1–282) þ þ þ D2 (244–508) D3 (342–625) D4 (607–740) þ þ þ
SUMO-2-D, inactive SUMO.
NLS1 (389-394) D1 D2 D3 D4 1-282 244-508 342-625 607-740 1-740 NLS2 (627-633) Daxx WT NES (188-197) YKXE (59-62) 1-282 * D1-K60R
A
200 100 20 40 60 80 SUMO-1 SUMO-2 Daxx D1 D1-K60R D2 D3 D4 0 120 140 160 180 % β se iti vit ca es a di s ot ca la -gB
Fig. 1. The interactions between SUMO and Daxx by yeast two hybrid assays. A: Schematic representation of wild-type Daxx and its deletion fragments, designated D1, D2, D3, and D4, and a site mutation D1 fragment, D1-K60R, are shown. Amino acid sequences of the deletion fragments are in blocks and those of the related functional motifs in brackets. The N-terminal deletion mutant (D1) contains a sumoylation consensus sequence YKXE and a NES-like motif when both D2 and D3 contains a nuclear localization signal NLS1 and the C-terminal fragment (D4)
contains another nuclear localization signal NLS2. B:
b-galactosidase assays of SUMO and Daxx. Plasmids pACT2 encoding Daxx wild-type and deletion mutants, illustrated in Figure 1A, are co-transformed with a possible pair of pAS2-SUMO-2 (or SUMO-1) in yeast strain CG-1945 and analyzed for the interaction. Results of the filter assays are shown on Table I. The b-galactosidase assays were described previously [Ryu et al., 2000]. NES, nuclear export signal; NLS, nuclear localization signal.
Sumoylation complexes of His-SUMO-2 or SUMO-1 with GST-D4-K630A, GST-D4-K631A, GST-D4-K630, 631A, and GST-D4-K630, 631, 634A mutants were shown in Figure 3B. They were analyzed by Western blotting using polyclonal anti-D4 antibodies (panel a and c, Fig. 3B),
polyclonal anti-SUMO-2 antibodies (panel b, Fig. 3B) and monoclonal anti-His antibodies (panel d, Fig. 3B). Comparing with that of GST-D4 proteins (lane 3, Fig. 3B), a decreasing trend of sumoylation products (lanes 4, 5, 7, and 8, Fig. 3B) was observed. This indicates K630and Fig. 2. In vitro SUMO conjugation assays on the C-terminal D4
deletion mutants. In vitro symoylation reactions contained 10 mg GST-Daxx deletion fragments, 10 ml ATP regenerating system (50 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 2 mM ATP, 10 mM creatine
phosphate, 3.5 U/ml creatine kinase and 0.6 U/ml inorganic pyrophosphatase), 5 mg purified His-SUMO and 0.65 mg purified,
recombinant GST-Ubc9 (E2) and 0.12 mg purified, recombinant GST-SAE1/2 (E1). The 10 ml reactions are incubated for 3 h at 378C, then stopped by adding 1/5 vol. 10% SDS, boiled and separated by SDS–PAGE. Sumoylation products are visualized by Western blot with rabbit polyclonal anti-D4 antibody developed by our laboratory.
DSF-K60R D1 D4-K630,631A TM DM 75 46 D4-K630A D4-K631A D4-K634A 630A 631A D4-K630, 631,634A 634A NLS1 (389-394) 1-740 NLS2 (627-633) Daxx WT NES (188-197) YKXE (59-62) D1-K60R * * * * * *** * *
A
Fig. 3. Lysine 60, Lysine 630, and 631 amino acid residues are the sumoylation sites on Daxx. A: Schematic of GST-tagged D4 and GST-tagged D1 mutants are shown with mutated sites indicated by asterisks. YKXE, sumoylation consensus sequence; NES, nuclear export signal like motif; NLS, nuclear localization signal; WT, wild-type; DM, double mutants; TM, triple mutants; DSF, D1 short fragment. B: Methods of sumoylation assay in vitro described in Figure 2. Proteins of His-SUMO-2 (and His-SUMO-1) and various GST-tagged D4 mutants (Fig. 3A) were applied on
sumoylation assay in vitro. Sumoylation complexes separated by SDS–PAGE, blotted with rabbit polyclonal anti-D4 antibody, anti SUMO-2 or anti-His antibody were detected with the Western lightning chemiluminescence reagent. C: His-SUMO-2 (and His-SUMO-1) and GST-tagged DSF and its mutants (Fig. 3A) were applied on sumoylation assay in vitro. Sumoylation complexes were immunobloted with rabbit polyclonal anti-GST antibody when anti-GST-p53F (377-393 a.a.) served as a positive control of sumoylation assay.
K631 are the major SUMO modification sites on the C-terminal Daxx for both SUMO-1 and SUMO-2. Jang et al. [2002] have suggested that K630and K631are the major sumoylation sites of Daxx for SUMO-1 by sumoylation assays in vivo. Current results confirm that the two major sumoylation sites of Daxx are indeed located at the lysine 630 and 631 amino acid residues by in vitro sumoylation assay for not only SUMO-1 but also SUMO-2.
Since D1-K60R mutant has terminated its interactions with SUMO in yeast, indicated as in Table I, it is likely that sumoylation occurs
between SUMO and D1. As previously des-cribed, in vitro sumoylation assays could not identify the sumoylation complexes of D1. Therefore, a shorter peptide fragment of Daxx (DSF), amino acid residues 46–75 (constructs shown in Fig. 3A) was used to avoid the interference from non-specific proteins. Small amount of SUMO-1 and SUMO-2 complexes (lanes 1 and 4, Fig. 3C) were observed on DSF, but not on DSF-K60R mutant (lanes 2 and 5, Fig. 3C). The indication is that K60 is also a sumoylation site. However, the product yields suggest that K60 is a minor sumoylation site, Fig. 3. (Continued )
whereas K630and K631are the major sites for SUMO-1 and SUMO-2 modifications.
Cellular Localization of Daxx Deletion Fragments
Four GFP fused Daxx deletion mutants GFP-D1, GFP-D2, GFP-D3, and GFP-D4, as shown in Figure 1A, were transfected into HeLa cells. Results showed that GFP-D2 (Fig. 4Ac) and GFP-D3 (Fig. 4Ad), both fusion proteins con-tained NLS1(amino acids 389–394), were found to diffuse in cytoplasm. However, the fact that N-terminal GFP-D1 (amino acids 1–282) (Fig. 4Ab) and C-terminal Daxx GFP-D4 (amino acids 607–740) (Fig. 4Ae) were localized in nucleus as that of full length Daxx (amino acids 1–740) (Fig. 4Aa) suggests that these two deletion mutants contain the functional NLS motifs.
Cytoplasmic Mislocalization of Daxx Mutants A consensus sumoylation site K60and a leucine-rich NES-like motif (188IXXLXXLLXL197) found on the N-terminal D1 (Fig. 1A) indicates that they are associated with nuclear translocaliza-tion. Single and triple-site mutants were used to examine whether K60or the NES-like motif mediates the nuclear transport of Daxx. Single-site mutants have amino acid altered on both the sumoylation site K60 (GFP-D1-K60R) and NES-like motif of D1 (GFP-D1-I188A, GFP-D1-L191A, GFP-D1-L194A, GFP-D1-L195A, and GFP-D1-L197A). Triple-site mutants were pre-pared by having leucine of 191, 194, and 195
amino acid residues mutated to alanine on both the D1 (GFP-D1-NESmut) fragments and full length Daxx (GFP-Daxx-NESmut). Similarly, triple-site mutants (K630, 631, 634A) on both the C-terminal D4 (GFP-D4-NLS2mut) and full length Daxx (GFP-Daxx-NLS2mut) were created in order to identify the nuclear translocation function of NLS2 motif (627PPCKKSRK634). The mutants are illustrated schematically in Figure 5A and the GFP-Daxx fusion proteins of transfected cells analyzed by anti-GFP antibody are shown in Figure 5B.
The nuclear localization of the GFP-D1-K60R (shown in Fig. 5Cb) suggests that the N-terminal consensus sumoylation site K60is not associated with the nuclear transport of D1. Except for some of the GFP-D1-L194A proteins (Fig. 5Ce), all D1 single site mutants (Fig. 5Cb– Ch) were localized in nucleus as that of the wild-type Daxx (Fig. 5Ca). In fact, the GFP-D1-L194A proteins mislocalized in cytoplasm (Fig. 5Ce) were only observed in 50% of the transfected cells. Evidence is compelling that the leucine of amino acids residue 194 is involved in the nuclear transport of D1. Triple-site (191, 194, and 195 a.a.) mutant of D1, GFP-D1-NESmut including leucine 194, mislocalized in cyto-plasm on all transfected cells, as shown in Fig. 5Ci, further demonstrates the involvement of NES-like motif on the nuclear transport of D1. Similarly, the triple-site mutant of full length Daxx, GFP-Daxx-NESmut, was misloca-lized in cytoplasm on all transfected cells (Fig. 5Cj). This further supports the association of NES-like motif with nuclear translocalization. Fig. 3. (Continued )
The N-terminal NES-like motif thus appears to have certain functions in mediating the nuclear transport of D1 as well as of Daxx.
On the other hand, proteins of NLS2 triple-site (630, 631, and 634 amino acids) mutants GFP-D4-NLS2mut and GFP- Daxx-NLS2mut, shown in Figure 5Ck and 5Cl, respectively, have dispersed in cytoplasm on all transfected cells. It is, therefore, likely that the C-terminal NLS2 motif of Daxx is responsible for the nuclear localization of the C-terminal D4 and for the full length Daxx.
Nuclear Relocalization of Daxx Mutants It is found that RFP-SUMO-2/-1 fusion proteins localized in nucleus have formed nuclear dots (indicated as N in Table II and Fig. 6Aa,Ac) while the inactive RFP-SUMO-2-D proteins were diffused in cytoplasm (indicated as C in Table II and Fig. 6Ab,Ad). The transfected RFP-SUMO fusion proteins ana-lyzed with anti-RFP or anti-SUMO-2 antibodies are shown in Figure 6B with lane 3 showing
active SUMO-1 and lane 4 the inactive one; lanes 5 and 9 showing active SUMO-2; and lanes 6 and 10 the inactive SUMO-2.
For further examination of the in vivo interactions between SUMO-2 and Daxx, active SUMO-2 (RFP-SUMO-2) were cotrans-fected with GFP-Daxx, D1 as well as D4 in HeLa cells. Results (shown in Table II) are consistent with yeast two hybrid interactions in vivo (shown previously in Table II). Active SUMO-2 (RFP-SUMO-2) colocalized with Daxx in nucleus and formed nuclear dots were also observed (indicated as N in Table II and shown in Fig. 7a). Similar images were obtained on the cotransfection of RFP-SUMO-2 and D1 or D4. As cotransfection of RFP-SUMO-2 with Daxx mutants (GFP-Daxx-NESmut, GFP-Daxx-NLS2mutm, and GFP-DaxxK60 R-NLS2mut), some cytoplasm mislocalized GFP-Daxx mutants were colocalized in nucleus with SUMO-2 as indicated C/N in Table IV and Figure 7b,d, respectively. To investigate if sumoylation was required for the nuclear relocalization of Daxx mutants, inactive Fig. 4. Fluorescence microscopy reveals D1 and D4 deletion
fragments localized in nucleus. The green fluorescent proteins (GFP) fused Daxx and its deletion fragments transiently expressed in HeLa cells after12 h incubation were observed by fluorescence microscopy. The fusion proteins were revealed by the intrinsic green fluorescence of GFP and the nuclei visualized by DAPI
staining. The wild-type Daxx (GFP-Daxx) containing YKXE, NES-like motif, NLS1, and NLS2motifs (a), the N-terminal deletion
mutant D1 (GFP-D1) containing YKXE and NES-like motif (b), GFP-D2 deletion mutant containing NLS1(c), GFP-D3 deletion
mutant also containing NLS1(d), and the C-terminal deletion
D1-I188A 60R D4-NLS2mut NLS1 (389-394) 1-740 NLS2 (627-633) Daxx-WT NES (188-197) YKXE (59-62) D1-K60R *** * D1-L197A D1-L191A D1-L194A D1-L195A D1-NESmut 188A * 191A * 194A * 195A * 197A * 191, 194, 195A*** 630, 631, 634A Daxx-NLS 2mut *** 191, 194, 195A*** Daxx-NESmut 630, 631, 634A
A
Fig. 5. The NES-like motif and NLS2motif are required for the
nuclear localization of Daxx. A: Schematic representations of Daxx mutants for fluorescent localization assays are shown. The mutated sites are indicated by asterisks. WT, wild-type; YKXE, sumoylation consensus sequence; NES, nuclear export signal-like motif; NLS2, nuclear localization signal 2. B: Western blots of
transfected GFP-fusion proteins. Immunoblot of total protein lysates collected from cell populations transfected with plasmids are indicated on the right of Figure 5B. GFP-fused full length Daxx (upper panel) and its deletion mutants (lower panel) were
detected with an antibody specific for GFP. C: The NES-like motif and the NLS2 motif are required for the nuclear localization of Daxx. Plasmids encoding various GFP-fused Daxx mutants, as shown in Figure 5A, were transfected into HeLa cells and analyzed by fluorescence microscopy after 12 h incubation to identify the NLS motifs. The nuclei were visualized by DAPI staining. The wild-type Daxx (a), the single site mutants of D1 (b–h), the triple-site NES-like motif mutants of D1, and full length Daxx (i and j, respectively) and the triple-site NLS2mutants of D4
SUMO-2-D (or SUMO-1-D) was cotransfected with various cytoplasm-mislocalized triple-sites Daxx mutants. RFP-SUMO-D and GFP-Daxx mutants were found to disperse in cytoplasm, as indicated C in Table IV and Figure 7e,f. The indication is that sumoylation was needed for the nuclear traslocalization of Daxx.
Inactive SUMO-2-D is Associated With Daxx in Nucleus
To examine the in vivo interactions of inactive His-SUMO-2D (or His-SUMO-1-D) and GST-Daxx, which give positive interactions by yeast two hybrid assays, the inactive RFP-SUMO-2-D (or SUMO-1-D) and GFP-Daxx were cotrans-Fig. 5. (Continued )
fected in HeLa cells. Figure 8Aa,Ab shows the nuclear colocalization and formation of nuclear dots of these two proteins by fluorescence microscopy assays. They are in good accord with those of yeast two hybrid assays. Moreover, the immunoprecipitation of the cotransfected cell lysates with anti-RFP antibody, then Wes-tern blotting with anti-GFP antibody shows that GFP-Daxx is associated with inactive RFP-SUMO-2-D (or RFP-SUMO-1-D) (upper panel, lane 6 and 3, respectively), as that of active RFP-SUMO-2 (or RFP-SUMO-1) (upper panel, lane 5 and 2, respectively). Similarly, when the cotransfected cell lysates were immunoprecipi-tated with anti-GFP antibody and Western blotted with anti-RFP antibody, the inactive RFP-SUMO-2-D (or RFP-SUMO-1-D) was obser-ved on the lower image of Figure 8B lane 4 and 2, respectively. The positive controls of the asso-ciations between active SUMO-2 (or RFP-SUMO-1) and GFP-Daxx are shown on the lower images of Figure 8B, lane 3 and 1.
DISCUSSION
Yeast Two Hybrid Interactions
Sumoylation assay is a covalent bonding reaction between glycine of the C-terminal SUMO and the lysine of its target protein, while yeast two hybrid assay is the binding interac-tions between SUMO and Daxx molecules, which may involve a wider range of binding interactions including possibly non-covalent interactions and covalent linkages. Sumoyla-tion between SUMO and Daxx may have occurred during yeast two hybrid interactions; however, the positive filter assays or b-galacsi-dase assays between SUMO and Daxx could
also be achieved through the physical inter-action between these two molecules without sumoylation linkages, which is referred to as ‘‘non-sumoylation interactions’’ in this study. There are strong interactions between SUMO-2 and D1 as well as SUMO-1 and D4, as shown on Figure 1B. The interactions were mainly non-covalent illustrated by the positive filter assay when sumoylation-incompetent inactive SUMO-D was used (Table I). However, the reason SUMO-1 and SUMO-2 behave differ-ently in yeast two hybrid interactions with D1 or D4 might due to the effects of their variable N-terminus. It has been suggested in the previous studies by Su and Li that although the function of SUMO’s N-terminal extension is presently unknown, its characteristics, that is being rich in charged amino acids, glycines and prolines make it an excellent candidate for specific protein–protein interactions. [Su and Li, 2002]
Two Functional Motifs are Required for the Nuclear Transport of Daxx
The fact that the NLS1on D2 and D3 of Daxx deletion fragments is not a functional nuclear localization signal (Fig. 3c,d) confirms that NLS1is not a functional motif as proposed by Torii et al. [1999].
Aleucine-richNES-likemotif(188IXXLXXLLXL197), termed conveniently as NES, was found on the N-terminal Daxx. Its conserved sequence of leucine residues was similar to that of p53 (341FXXLXXXLXL350) [Stommel et al., 1999]. This motif was not a conventional lysine-rich NLS, but it has become an NLS candidate due to possible interactions with the nuclear pore complexes (NPCs). That the triple-site mutants of D1 (GFP-D1-NESmut) terminate nuclear TABLE II. Cellular Localization on Transfection of SUMO and Daxx by
Fluorescence Microscopy
Fluorescent assay in vivo
SUMO-2 SUMO-2-D SUMO-1 SUMO-1-D
N C N C
Daxx N N N N N
D1 N N N N C
D4 N N C N N
Daxx-NESmut C C/N C C/N C
Daxx-K60R-NESmut C C/N C C/N C
Daxx-NLS2mut C C/N C C/N C
Daxx-K60R-NLS
2mut C C/N C C/N C
SUMO-2-D, inactive SUMO; N, nucleus; C, cytoplasm; Daxx-NESmut, Daxx-K191, 194, 195A; Daxx-NLS2mut,
Daxx-K630, 631, 634A.
Fig. 6. Inactive SUMO is mislocalized in cytoplasm. A: Plasmids encoding red fluorescent proteins fused SUMO (RFP-SUMO-1/-2) or inactive SUMO (RFP-SUMO-1-D/-2-D) were transfected into HeLa cells. The slide was stained with DAPI and analyzed by fluorescence microscopy. These figures are merged. B: RFP-SUMO fusion proteins were detected with anti-RFP antibody (lanes 3–6) and RFP-RFP-SUMO-2/-2-D were detected with anti-SUMO-2 antibody (lanes 9–10).
transport (Fig. 5Bi) confirm that the NES motif is required in mediating the nuclear transport of D1.
C-terminal Daxx has indeed been suggested to be a nuclear localized protein before [Pluta et al., 1998; Hollenbach et al., 1999; Jang et al., 2002]. The nuclear translocalizations of the C-terminal Daxx could have been due to the function of proposed C-terminal NLS2 (amino acids 627–633). However, it was regarded by Jang et al. [2002] as non-functional (amino acids 627–633) by cotransfection of double-site
mutant Daxx-K630, 631A and PML colocalized in nucleus. The termination of nuclear trans-port of the C-terminal fragment D4 (shown in Fig. 5Bk) by triple-site mutants K630, 631, 634A of D4 (GFP-D4-NLS2mut) has established the nuclear transport function of NLS2 on the C-terminal Daxx.
Dispersion of mutants of full length Daxx (GFP-Daxx-NESmut and GFP-Daxx-NLS2mut) on either N-terminal NES-like motif or C-terminal NLS2 motif in cytoplasm (shown in Fig. 5Bj,Bl) leads to the conclusion that both Fig. 7. Sumoylation enhances the nuclear localization of Daxx mutants. HeLa cells were cotransfected
with GFP-Daxx (a) or various Daxx mutants (b–d) and SUMO-2 (a–d) or inactive SUMO-2-D (e, f), as indicated above the images. The transfected cells were incubated for 12 h before analyzed with fluorescence microscopy. The yellow color in the merged image is due to colocalization of green fluorescent protein fused Daxx and red fluorescent protein fused SUMO.
NES-like motif and NLS2motif are required for the nuclear transport of Daxx.
SUMOs Enhance the Nuclear Transport of its Target Protein
Investigating the role of SUMO-2 on the nuclear transport of Daxx has demonstrated that relocalization of cytoplasm mislocalized Daxx mutants occurs during cotransfection with SUMO-2. It can be seen that almost all GFP-Daxx-NESmutproteins were colocalized in nucleus with SUMO-2 (Fig. 7b), whereas GFP-Daxx-NLS2mut and GFP-DaxxK60R-NLS2mut proteins were partially colocalized in nucleus with SUMO-2 (Fig. 7c,d, respectively). This could be explained by intact major sumoylation sites K630and K631that existed in GFP-Daxx-NESmutproteins but that were lacking in GFP-Daxx-NLS2mut and GFP-DaxxK60R-NLS2mut proteins. Relocalization of GFP-Daxx-NLS2mut proteins has probably occurred through inter-acting with SUMO-2 on minor sumoylation sites K60. A mutated K60 should not have relocalized GFP-Daxx-K60R-NLS2mutproteins; relocalization could then have taken place through non-sumoylation interactions or uni-dentified minor sumoylation sites on the C-terminal of Daxx. Unidentified minor sumoyla-tion sites were indicated by the observasumoyla-tion of sumoylation complexes from in vitro sumoyla-tion of TM (K630, 631, 634A) on D4 (Fig. 3B).
Relocalization of Daxx mutants (Figs. 7e,f) blocked in inactive SUMO-2-D, however, sug-gests sumoylation enhances cytoplasmonuclear translocalization of Daxx.
Interactions between RanGAP1 and NPC enhanced by sumoylation RanGAP1 [Matunis et al., 1996; Tatham et al., 2005; Reverter and Lima, 2005] suggest the role of sumoylation in regulating protein–protein interactions in cyto-plasm. Similar to RanGAP1, Daxx may also have sumoylated in cytoplasm before interact-ing with NPC. That is why GFP-Daxx-NESmut and GFP-Daxx-NLS2mutwere localized in cyto-plasm with sumoylation defective SUMO-2-D (Figs. 7e,f). Nuclear localization of sumoylated Caspase 8 was studied by Besnault-Mascard et al. [2005]. They reported that Caspase 8 was cytoplasm localized while the sumoylated Cas-pase 8 was found only in nucleus. This is similar to Daxx mutants; the nuclear transport of Caspase 8 could have been mediated by sumoy-lation. Proteins entering nucleus facilitated by sumoylation has been demonstrated by SUMO-1 fused NF-kB essential modulator (NEMO) [Huang et al., 2003] that functions similarly to Daxx. They reported that the wild-type NEMO proteins were localized in cytoplasm whereas the SUMO-1 fused NEMO were found in both cytoplasm and nucleus. It is likely that a new biological role of sumoylation is to regulate the cytoplasmonuclear transport of its target proteins.
Fig. 8. Inactive SUMO-D can be relocalized in nucleus by non-sumoylation interactions with Daxx. A: Inactive RFP-SUMO-2-D and GFP-Daxx are colocolized in nucleus. The inactive RFP-SUMO-2-D (or RFP-SUMO-1-D) were cotransfected with GFP-Daxx in HeLa cells. The transfected cells were incubated for 12 h before analysis by fluorescence microscopy. The yellow color in the image is due to colocalization of GFP-fused Daxx and
rRFP-SUMO (Fig. 8Aa,Ab). B: Non-sumoylation interactions occur between inactive SUMO-D (or active SUMO) and Daxx. HeLa cells were cotransfected with RFP-SUMO (or inactive SUMO-D) and GFP-Daxx as indicated. Approximately 1,000 mg of total cell extracts were subjected to IP with anti-RFP antibody followed by WB with anti GFP antibody (upper panel) and vise versa (lower panel).
Non-Sumoylation Interactions are Found Between Inactive SUMO-D and Daxx Forming SUMO–SUMO complexes or SUMO-target protein complexes appears neces-sary for nuclear translocation since inactive SUMO-D, being unable to sumolyate (lane 5 in Fig. 2), was localized in cytoplasm (Fig. 6Ab). Nevertheless, inactive SUMO-D relocalizing to nucleus by its target protein Daxx in fluores-cence microscopy assays (Fig. 8Aa,Ab) and giving positive yeast two hybrid interactions
(Table I) with Daxx were found consistently in this study. The suggestion is that a significant non-sumoylation interaction has occurred between inactive SUMO-D and Daxx. This could have provided a new mechanism, besides sumoylation, that acts on improving the nuclear transport of target proteins by SUMO.
From yeast two hybrid studies and in vivo fluorescent assays, it can be concluded that double glycines on the C-terminus of SUMO and the K60amino acid residue, the NES-like motif as well as the NLS2 motif of Daxx are critical Fig. 8. (Continued )
areas for non-sumoylation interactions between SUMO and Daxx. Considerable non-sumoyla-tion interacnon-sumoyla-tions between SUMO and its target proteins (Fig. 8) may explain why mutating sumoylation sites in Sp100, PML, or p53 did not prevent intranuclear accumulation in transfec-tion experiments [Sternsdorf et al., 1999; Zhong et al., 2000; Lallemand-Breitenbach et al., 2001; Kwek et al., 2001]. Therefore, nuclear localiza-tion of Daxx may have been regulated by SUMO through sumoylation as well as non-sumoyla-tion interacnon-sumoyla-tions in cytoplasm. Several conclu-sions can be drawn from this study: (a) two functional motifs, leucine-rich NES-like motif (188IXXLXXLLXL197) and C-terminal NLS
2 (amino acids 627–633), are required for the nuclear transport of Daxx; (b) SUMO enhance the nuclear transport of its target proteins Daxx; and (c) it may be done through the non-sumoylation interaction.
The cytoplasm mislocalized Daxx mutants (Daxx-NESmut, Daxx-NLS2mut, and Daxx-K60 R-NLS2mut) with different sumoylation conditions available may provide us an opportunity of exploring the necessities of sumoylation on Daxx-involved interactions on the Fas induced apoptotic signals or ASK1 apoptotic signals. Therefore, identifying SUMO’s involvement on Daxx-related apoptosis and its connection to the degradation of Daxx after transferring apopto-tic signals becomes an important task in the future.
ACKNOWLEDGMENTS
We thank Professor R.T. Hay of the Univer-sity of St. Andrews, Scotland, UK for kindly providing the GST-SAE1/2 (E1) and GST-Ubc9 (E2) expression vectors. This research is sup-ported in part by the National Science Council of Taiwan through NSC 89-2311-B110-0215.
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