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Human ubiquitin specific protease 31 is a deubiquitinating enzyme implicated in activation of nuclear factor-κB
Cellular Signalling 18 (2006) 83 – 92 www.elsevier.com/locate/cellsig
Human ubiquitin specific protease 31 is a deubiquitinating enzyme implicated in activation of nuclear factor-nB Christos Tzimasa,c, Gianna Michailidoua, Minas Arsenakisa, Elliott Kieff b, George Mosialosc,*, Eudoxia G. Hatzivassilioua,c,* a Department of Biology, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece Infectious Disease Division, Brigham and Women’s Hospital, 181 Longwood Ave, Boston, MA 02115, USA c Institute of Immunology, Biomedical Sciences Research Center Al. Fleming, 34 Al. Fleming Str., Vari 16672, Greece b
Received 4 March 2005; received in revised form 16 March 2005; accepted 16 March 2005 Available online 26 April 2005
Abstract TRAF2 mediates activation of the transcription factors NF-nB and AP1 by TNF. A yeast two-hybrid screen of a human cDNA library identified a ubiquitin specific protease homologue (USP31) as a TRAF2-interacting protein. Two cDNAs encoding for USP31 were identified. One cDNA encodes a 1035-amino acid long isoform of USP31 (USP31, long isoform) and the other a 485-amino acid long isoform of USP31 (USP31S1, short isoform). USP31 and USP31S1 share a common amino terminal region with homology to the catalytic region of known deubiquitinating enzymes. Enzymatic assays demonstrated that USP31 but not USP31S1 possess deubiquitinating activity. Furthermore, it was shown that USP31 has a higher activity towards lysine-63-linked as compared to lysine-48-linked polyubiquitin chains. Overexpression of USP31 in HEK 293T cells inhibited TNFa, CD40, LMP1, TRAF2, TRAF6 and IKKh-mediated NF-nB activation, but did not inhibit Smad-mediated transcription activation. In addition, both USP31 isoforms interact with p65/RelA. Our data support a role for USP31 in the regulation of NF-nB activation by members of the TNF receptor superfamily. D 2005 Elsevier Inc. All rights reserved. Keywords: NF-nB signaling; TNF; TNFR; TRAF; Ubiquitin proteasome system; Ubiquitin specific protease
1. Introduction TRAFs constitute a family of proteins that have been implicated in signal transduction by members of the TNF receptor superfamily including TNFRI, TNFRII, CD40, Abbreviations: AP1, activator protein 1; DTT, dithiotreitol; DUB, deubiquitinating enzyme; EBV, Epstein-Barr virus; IKK, InB kinase; JNK, Jun-kinase; LMP1, latent membrane protein 1; LThR, lymphotoxin h receptor; MAPK, mitogen activated protein kinase; NP-40, Nonidet P-40; NF-nB, nuclear factor-kappaB; TAK1, transforming growth factor-beta activated protein kinase; TNF, tumor necrosis factor; TNFR, TNF receptor; TRAF, tumor necrosis factor receptor associated factor; Ub, Ubiquitin; USP, ubiquitin specific protease. * Corresponding authors. Institute of Immunology, Biomedical Sciences Research Center Al. Fleming, 34 Al. Fleming Str., Vari 16672, Greece. Tel.: +30 2109656703; fax: +30 2109653934. E-mail addresses: [email protected] (G. Mosialos), [email protected] (E.G. Hatzivassiliou). 0898-6568/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2005.03.017
CD30, CD27, LThR and the EBV oncoprotein LMP1 [1]. Six mammalian TRAF molecules have been identified so far. Among them, TRAF2, TRAF5 and TRAF6 have the ability to activate the NF-nB and AP1 signaling pathways, upon overexpression [1]. TRAF-mediated activation of the NF-nB pathway is dependent on the activation of the IKK complex, whereas AP1 activation by TRAFs is mediated by MAPKs including JNK and p38 [2,3]. TRAFs are characterized by the presence of a conserved carboxyl terminal region known as the TRAF domain, which interacts with the cytoplasmic domains of TNF receptors. The amino terminal region of TRAFs is rich in cysteine and histidine residues arranged in putative zinc-finger motifs. With the exception of TRAF1, TRAFs contain an amino terminal RING finger motif, which has been implicated in the activation of NFnB and MAPKs.
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TRAF2 and TRAF6 have a ubiquitin ligase (E3) activity, which is dependent on the integrity of their RING finger domain and has been associated with the ability to activate the NF-nB and AP1 signaling pathways [4,5]. Protein modification by ubiquitination has been primarily associated with degradation by the proteasome. However, protein ubiquitination has also been implicated in other cellular functions, including transcription regulation, receptor endocytosis, DNA repair, ribosomal function, stress response and mitochondrial inheritance [6]. TRAF2 and TRAF6 mediate polyubiquitination by catalyzing the formation of an isopeptide bond between the carboxyl terminus of one ubiquitin moiety and the e amino group of lysine 63 of another ubiquitin molecule. Lysine-63-linked polyubiquitin chains have been associated primarily with non-proteolytic processes, whereas lysine-48-linked polyubiquitin chains usually mark proteins for proteasomal degradation [6]. Both types of ubiquitination (lysine-48 or lysine-63) play critical role in NF-nB activation by TNF family members and other cytokines. TRAF2 and TRAF6 mediated lysine-63 ubiquitination is essential for NF-nB activation. Signal transduction by TRAF6 is initiated upon its oligomerization. Oligomerization of TRAF6 triggers its polyubiquitination, which is essential for the subsequent activation of protein kinase TAK1 [5]. It is believed that TRAF6 polyubiquitination facilitates the assembly and activation of multisubunit protein complexes [7]. TAK1 can activate both the NF-nB and AP1 pathways [5,8]. Activation of the IKK complex mediates the phosphorylation of the NF-nB inhibitor InB. Phosphorylation of InB by IKK causes its lysine-48 ubiquitination and its subsequent degradation by the ubiquitin –proteasome pathway. This results in nuclear translocation and activation of NF-nB [9]. Postinduction repression and termination of NF-nB activity depends on NF-nB regulated resynthesis of InBa which dissociates NF-nB from DNA and exports it to the cytoplasm. NF-nB regulated resynthesis of InBa provides a strong negative feedback and a fast down-regulation of the NF-nB response [10,11]. Additionally, it has been found that after activation p65/RelA is degraded by the proteasome in the nucleus and in a DNA binding-dependent manner [12]. If proteasome activity is blocked, NF-nB is not promptly removed from some target genes in spite of InBa resynthesis and sustained transcription occurs. These results indicate that lysine-48 ubiquitination and proteasomal degradation of p65/RelA does not merely regulate its stability and abundance, but also actively promotes transcriptional termination. In order to elucidate further the TRAF2 signaling mechanism and its regulation, we have performed a yeast two-hybrid screen of a human cDNA library with TRAF2 as bait and identified a ubiquitin specific protease homologue (USP31). Our findings support a role for USP31 in NF-nB activation by members of the TNFR superfamily.
2. Materials and methods 2.1. Yeast two-hybrid assay The yeast two-hybrid screen was performed as previously described [13]. The bait consisted of amino acids 1– 358 of TRAF2 fused to the amino terminus of GAL4 (pTRAF2(1 – 358)D151). 2.2. Plasmid construction pTRAF2(1 – 358)D151 was constructed by inserting the TRAF2 encoding fragment that resulted from digestion of pSG5FLAGTRAF2 [14] with EcoRI and T4 polymerasemediated blunting and BglII in D151 (a gift by Dr. R. Brazas, University of California, San Francisco), digested with HindIII and T4 polymerase-mediated blunting and Bam HI. pTRAF2(1 – 358)C34/37AD151 which is pTRAF2(1 –358)D151 with TRAF2 cysteines 34 and 37 mutated to alanines was constructed by replacing the KpnI– AgeI fragment of pTRAF2(1 –358)D151 with the corresponding mutagenized fragment that was generated by PCR. The expressing constructs for the Flag-tagged long isoform USP31 (pcDNA3FlagUSP31), the point mutant USP31C98S (pcDNA3FlagUSP31C98S) and the short isoform USP31S1 (pcDNA3FlagUSP31S1) were constructed in several steps. The USP31 encoding EcoRI fragment of a cDNA clone spanning amino acids 1 to 634 (USP31(1 – 634)) isolated from a human B lymphoma library [15] was subcloned into pBluescript KS+ (Stratagene) digested with EcoRI to generate pBluescriptUSP31(1 –634). The NcoI– EcoRI USP31(1 –634) encoding fragment of pBluescriptUSP31(1 –634) was blunted at the NcoI site and subcloned into pcDNA3Flag [16] digested with HpaI and EcoRI to generate pCDNA3FlagUSP31(1 – 634). The USP31S1 encoding BstEII –EcoRI fragment of a human EST clone (IMAGE:259632) was subcloned into pcDNA3FlagUSP31(1 – 634) digested with BstEII and EcoRI to generate pcDNA3FlagUSP31S1. The complete sequence of USP31 was determined from overlapping cDNA clones isolated from a human B lymphoma library [15] and human EST clones. Flag-tagged full-length USP31 expressing construct pcDNA3FlagUSP31 was constructed in several steps. Human leukocyte marathon cDNA was used as a template DNA in PCR with the primers 1041F6 (GCTGAAAGCAGCAGTAAAGGGCAGCGATGG) and 1041R7 (GCTCTAGAGCGGCCGCCTTTTCTATGTCGCATACTTCG). A band of approximately 1 kbp was amplified, isolated and reamplified. The resulting PCR product was digested with restriction enzymes NheI and XbaI, and it was cloned into pcDNA3FlagUSP31(1 – 634) digested with NheI and XbaI to generate the clone pcDNA3FlagUSP31(1 –938). HEK 293T cells cDNA was used as template DNA in PCR using the primers 1041FL (AAGCCTACAAGATGCAGCCCTGTG) and 1041R8 ( G C T C TA G A G C G G C C G C C C TA A C AT G T C A A -
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ACTCCTCTTCC). A band of approximately 800 bp was amplified, and digested with restriction enzymes NdeI and NotI, and it was cloned into pcDNA3FlagUSP31(1 – 938) digested with NdeI and NotI. The resulting construct was pcDNA3FlagUSP31(799 – 1035) that contained the 3¶ end of USP31 and it also had a deletion of approximately 3 kbp at the 5¶ end of USP31 spanning amino acids 1 to 798. This construct was digested with BspEI and NotI to generate a fragment of approximately 400 bp that contained the 3¶ end of USP31 (amino acids 881 to 1035). This fragment was ligated into pcDNA3FlagUSP31(1 – 938) digested with BspEI and NotI to generate the full-length pcDNA3FlagUSP31. FLAG-tagged full-length USP31C98S was generated by PCR-mediated site directed mutagenesis. To generate this clone pcDNA3FlagUSP31S1 was digested with BspD1, HpaI and ligated to a PCR fragment, produced with three rounds of PCR, using the mutagenic primers C98SF(forward) (GGAGCCACTTCTTATGTCAACACATTTCTTC) and C98SR(reverse) (GTT-GACATAAGAAGTGGCTCCAAGGTTAGTC). In the first PCR round the C98SF was combined to T7 and the template plasmid was pBluescriptUSP31(1 – 634). In the second PCR round the C98SF was combined to pGEXF1 and the template plasmid was pGEXUSP31(1 –634), which was constructed by inserting the USP31(1 –634) fragment that resulted from the digestion of pBluescriptUSP31(1 – 634) with NcoI and T4 polymerase mediated blunting and EcoRI into pGEX2TK (Pharmacia) digested with SmaI and EcoRI. The two PCR products were then combined and PCR-amplified with the primers T7 and GEXF1 and the resulting product was digested with BspD1 and NcoI that was blunted and cloned in pcDNA3Flag USP31S1 digested with BspD1 and HpaI. This procedure led to the construction of the mutated plasmid pcDNA3FlagUSP31S1C98S. The full-length pcDNA3FlagUSP31C98S was constructed by inserting the BstEII, NotI fragment of pcDNA3FlagUSP31(1 – 938) into pcDNA3Flag-USP31S1C98S digested with BstEII and NotI to generate pcDNA3FlagUSP31(1 – 938)C98S. Finally, the BspEI, NotI 400 bp fragment of pcDNA3Flag-USP31(799– 1035) was ligated into pcDNA3FlagUSP31(1– 938)C98S digested with BspEI and NotI to generate the full-length pcDNA3FlagUSP31C98S.
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2.4. Cell lines, transfections and reporter assays The HEK 293T cell line and the HeLa cell line were maintained in Dulbecco’s Modified Eagle’s Medium (Gibco Invitrogen) supplemented with 10% heat inactivated fetal bovine serum (Gibco Invitrogen), 2 mM l-glutamine (Biochrom AG) and 50 units/ml penicillin and 50 Ag/ml streptomycin (Gibco Invitrogen), at 37 -C, in a humidified incubator with 5% CO2. Transfections were performed on HEK 293T cells with ExGen500 (Fermentas) and on HeLa cells with Lipofectamine 2000 (Invitrogen) transfection reagents according to the instructions of the manufacturer, unless otherwise indicated. TNF was added for 5 h before harvesting, at a concentration of human TNFa, 10 ng/ml. The h-galactosidase and luciferase reporter activities were assayed 24 h post-transfection. The h-galactosidase activity was measured using the Galacton-Plus substrate system (TROPIX, Inc) according to the manufacturer’s instructions. For luciferase-reporter assays, cells from a 12-well plate were lysed in 100 Al reporter lysis buffer (Promega) and luciferase activity was assayed with the luciferase assay system (Promega) according to the manufacturer’s instructions. Luciferase activities were normalized by dividing the luciferase activity values by the corresponding h-galactosidase activity values. Relative luciferase activities were expressed as folds of activation relative to the activity of the luciferase reporter alone. This was calculated by dividing every normalized luciferase activity value with the normalized luciferase activity value of the reporter alone. Finally, relative luciferase activities (%) were
2.3. Northern blots Northern blots were performed by using human multiple tissue Northern blots that were purchased from Clontech. A cDNA fragment encoding most of the catalytic region of USP31 was used as probe. The cDNA probe was labeled with 32P using the Multiprime DNA Labeling System (Amersham Biosciences). Hybridization was performed by standard procedures according to the manufacturers’ protocol. Hybridized membranes were exposed to X-ray film (X-OMAT AR) for 2 days at 70 -C.
Fig. 1. USP31 expression pattern in normal tissues and cancer cell lines. PolyA+ RNA from various human tissues (A) and cancer cell lines (B) was analyzed by Northern blot for the expression of USP31 mRNAs. USP31 was expressed in all normal tissues and cancer cell lines examined. The cancer cell lines were derived from the following tumors: promyelocytic leukemia (HL60), cervical carcinoma (HeLa), chronic myelogenous leukemia (K-562), lymphoblastic leukemia (MOLT-4), Burkitt’s lymphoma (Raji), colorectal adenocarcinoma (SW480), lung carcinoma (A549) and melanoma (G361).
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calculated as % of the fold of activation in transfected cells which express the effector USP31, relative to the corresponding transfected cells that do not express USP31. Results were analyzed statistically with a Student’s t-test (Microsoft Excel). The parameters used for the comparison of values were two-tailed, unpaired arrays. The difference between two samples was considered statistically significant when the p-value <0.01. 2.5. In vitro deubiquitination assays Wild type or mutated Flag-tagged USP31 proteins (FUSP31, FUSP31C98S and FUSP31S1) were immunoprecipitated from HEK 293T cell lysates prepared with NP-40
lysis buffer (25 mM Tris – HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 10% (v/v) glycerol, 0.5% (v/v) NP-40, 1 mM PMSF and 1 mM DTT). The immunoprecipitated proteins were incubated with 1.25 Ag of tetraubiquitin (Affinity Research Products) or 1.5 Ag of either lysine-48- or lysine63-linked polyubiquitin substrates (Boston Biochem, Inc.) in 15 Al of 50 mM Tris – HCl buffer (pH 7.2) containing 1 mM DTT for 2 h at 37 -C. Reaction mixtures were filtered and the flow through fraction that contained the cleaved substrates was analyzed by tricine SDS PAGE followed by silver staining. The beads containing the immunoprecipitated Flag-tagged proteins were analyzed by SDS-PAGE followed by immunoblotting with a mouse monoclonal antiFlag (M5) antibody.
Fig. 2. Deubiquitinating activity and specificity of USP31 towards tetraubiquitin and polyubiquitin substrates. Flag-tagged USP31 (FUSP31), USP31C98S (FUSP31C98S) and USP31S1 (FUSP31S1) were immunoprecipitated with the M5 anti-FLAG antibody from lysates of transfected HEK 293T cells and tested for their ability to cleave lysine-48-linked tetraubiquitin (K-48-linked tetraUb, A) and lysine-48-linked (K-48-linked polyUb, B) or lysine-63-linked (K-63linked polyUb, C) polyubiquitin chains. The reaction mixture was analyzed by tricine SDS-PAGE and silver straining (A, B, C, left panels). Arrows indicate the positions of tetrameric (Ub4), trimeric (Ub3), dimeric (Ub2) and monomeric (Ub1) ubiquitin. The amount of immunoprecipitated Flag-tagged proteins (shown by arrows) was determined by immunoblotting with the M5 antibody (A, B, C, right panels). In B and C the procedures for silver staining or immunoblotting were performed at the same time under identical and therefore absolutely comparable conditions.
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2.6. Immunofluorescense, immunoprecipitations and immunoblotting Indirect immunofluorescense was performed on HeLa cells as previously described [17]. Immunoprecipitations were performed in NP-40 lysis buffer (25 mM Tris –HCl pH 7.5, 250 mM NaCl, 5 mM EDTA, 10% (v/v) glycerol, 0.5% (v/v) NP-40, 1 mM PMSF and 1 mM DTT) or RIPA lysis buffer (50 mM Tris – HCl pH 8.0, 250 mM NaCl, 0.1% (w/ v) SDS, 0.1% (w/v) sodium deoxycholate, 1% (v/v) TritonX 100, 1 mM PMSF). The lysis buffer was supplemented with complete protease inhibitor cocktail (Roche) according to the manufacturer_s instructions. Cells were lysed for 30 min on ice in 500 Al NP-40 lysis buffer for TRAF2 immunoprecipitations shown in Fig. 3 or RIPA lysis buffer for p65 immunoprecipitations shown in Fig. 7. Immunoprecipitations were carried out as previously described [18]. Immunoblots were developed with the ECL-plus reagent (Amersham Biosciences) according to the manufacturer’s instructions. Images were obtained using the STORM 860 system (Molecular Dynamics). Antibodies were obtained from Santa Cruz Biotechnology with the exception of M2 and M5 antibodies against Flag, which were obtained from Sigma. The secondary antibodies were sheep – anti-mouse and donkey – anti-rabbit conjugated to horseradish peroxidase and they were purchased from Amersham Pharmacia and Santa Cruz Biotechnology, respectively.
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01440). USP31S1 has in addition to the amino terminal 483 residues a glycine (G) and a leucine (L) residue at its carboxyl terminus. USP31 and USP31S1 contain all eight characteristic motifs of known deubiquitinases [22]. The rat ortholog of USP31 (synUSP) was recently identified in Rat neurons [23]. Full-length human USP31 has not been analyzed for deubiquitinating activity. A cDNA encoding full-length USP31 was assembled from partial cDNA clones and used for further characterization. 3.2. Northern blot analysis Northern blot analysis of normal tissues and cancer cell lines revealed two major mRNA species, which are likely to be derived from alternative mRNA processing and may code for USP31 and USP31S1 (Fig. 1). USP31 was expressed in all normal tissues and cancer cell lines tested,
3. Results 3.1. Identification of USP31 cDNAs TRAF2 amino acids 1 – 358, containing the RING finger domain, were fused to GAL4 DNA-binding domain and used as bait in a yeast two-hybrid screening of a cDNAlibrary prepared from human EBV-transformed B lymphocytes [19]. Sixteen positive cDNA clones were studied further because they demonstrated a strong positive interaction with wild type TRAF2(1 – 358), but very weak or no interaction with TRAF2(1 –358), which contained a mutated RING finger domain. One of these clones encodes a protein with homology to deubiquitinating enzymes, which turned out to be identical to the product of a recently identified cDNA (USP31 [20], which is also known as USP48 [21]). Comparison of the USP31 cDNA sequence with human ESTs revealed that more than one USP31 cDNAs exist. Two cDNAs were identified and studied further in this report. One cDNA encodes for a longer 1035-amino acid long polypeptide (USP31, long isoform) [20], whereas the other encodes for a shorter 485-amino acid long polypeptide (short isoform, USP31S1). The two isoforms of USP31 share a common amino terminal region of 483 amino acids with significant similarity to known deubiquitinases (29% identity and 45% homology to a known S. pombe deubiquitinase with GenBank accession number AAF-
Fig. 3. USP31 interacts with TRAF2 in mammalian cells. HEK 293T cells were transfected with expression vectors for TRAF2 (A and B) or a TRAF2 deletion lacking the RING Finger (TRAF2DRF, C) in the presence or absence of constructs expressing Flag-tagged USP31 (A, C) or USP31S1 (B), as indicated. Cell lysates were subjected to immunoprecipitation with an antibody (M5) against Flag (IP: anti-Flag). The immunoprecipitated material and an aliquot of the whole cell extract (WCE) were subjected to immunoblotting (WB: anti-TRAF2 or WB: anti-Flag) with a rabbit polyclonal antibody against TRAF2 (A, B and C, top) or a mouse monoclonal antibody (M5) against Flag (A, B and C, bottom). The positions of TRAF2, TRAF2DRF, FUSP31 and FUSP31S1 are shown by arrows.
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indicating a ubiquitous role for this protein, in accordance with the potential functional relationship of USP31 with the broadly expressed TRAF2. Interestingly, in several cancer cell lines tested, the ratio of the lower to the higher molecular weight USP31 mRNA was significantly elevated (Fig. 1B), compared to the normal tissues (Fig. 1A). This may reflect a regulatory role of short USP31 isoform on the function of USP31 in cancer cells. 3.3. Biochemical characterization of USP31 The extensive similarity of USP31 to deubiquitinating enzymes led to the examination of its ability to act as a ubiquitin carboxyl terminal hydrolase. In addition to wild type USP31, a point mutant containing a serine instead of cysteine at position 98 (USP31C98S) was constructed and tested for its enzymatic activity. Cysteine 98 corresponds to a highly conserved cysteine of the catalytic domain of deubiquitinating enzymes (Cys box) [22]. Flag-tagged USP31, USP31C98S and USP31S1 were transiently expressed in HEK 293T cells upon transfection of the appropriate expression vectors. Cells were lysed and flagtagged proteins were immunoprecipitated using anti-Flag (M5) antibodies and tested for their ability to cleave
tetraubiquitin. Wild type full-length USP31 readily cleaved the tetrameric substrate to its monomer, dimer and trimer (Fig. 2A, left panel). However, the point mutant USP31C98S and the short isoform USP31S1 were enzymatically inactive (Fig. 2A, left panel), despite the fact that they were expressed in similar levels with USP31 (Fig. 2A, right panel). These results indicate that cysteine 98 and the carboxyl-terminal region of USP31 are essential for the deubiquitinating activity of the protein. These data are consistent with the findings of Tian et al. [23], who have demonstrated that full-length synUSP has deubiquitinating activity in bacteria, which is abolished by a smaller than 250-amino acid carboxyl-terminal truncation. Furthermore, USP31 was tested for its deubiquitinating specificity towards lysine-48- or lysine-63-linked polyubiquitin chains. For this purpose, Flag-tagged wild type USP31, the point mutant USP31C98S and the short isoform USP31S1 were transiently expressed in HEK 293T cells, immunoprecipitated and tested for their ability to cleave lysine-48- or lysine-63-linked polyubiquitin substrates. Wild type USP31 cleaved almost completely lysine-63-linked polyubiquitin to its monomer, whereas the catalytically inactive point mutant USP31C98S or the short isoform USP31S1 did not cleave the substrate (Fig. 2C, left panel). Wild type USP31 also
Fig. 4. USP31 inhibits NF-nB activation by TNFa, CD40 and LMP1. HEK 293T cells were transfected with 0.2 Ag of the 3xnBLuc NF-nB-dependent luciferase reporter plasmid and 0.2 Ag of pGK-h-galactosidase reporter plasmid, to normalize luciferase activities. Relative luciferase activities (%) are calculated in the presence or absence of 0.5 Ag of expression vector for Flag-tagged USP31 (FUSP31) and in the absence (A) or presence of 10 ng/ml of human TNFa (B), or 0.25 Ag of expression vector for CD40 (C), or 0.05 Ag of expression vector for LMP1 (D), as indicated. Twenty-four hours post-transfection cell extracts were prepared and the levels of luciferase and h-galactosidase activity were determined. Results shown in the left panels are the mean T S.D. of relative luciferase activity (%) from three independent experiments. Statistical analysis was performed as described in Materials and methods and the p-value is indicated. Representative Western blots of whole cell lysates from equal number of HEK 293T cells subjected to transfection are shown in the right panels. FUSP31 was detected with a mouse anti-Flag monoclonal antibody (M5). CD40 was detected with a rabbit polyclonal antibody and LMP1 with a mouse monoclonal antibody (S12), respectively. The positions of FUSP31, CD40 and LMP1 are shown by arrows.
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Fig. 6. USP31 does not affect Smad-dependent transcription activation. HEK 293T cells were transfected with 0.2 Ag of the p(CAGA)12-luc Smad-dependent luciferase reporter plasmid (CAGA) and 0.2 Ag of pGKh-galactosidase reporter plasmid, to normalize luciferase activities. Relative luciferase activities (%) are calculated in the presence or absence of 0.5 Ag of expression vector for Flag-tagged USP31 (FUSP31) and in the absence (A) or presence of 0.3 Ag of expression vectors for myctagged Smad3 and Smad4 (mSmad3, mSmad4, B) as indicated. Twentyfour hours post-transfection cell extracts were prepared and the levels of luciferase and h-galactosidase activity were determined. Results shown in the left panels are the mean T S.D. of relative luciferase activity (%) from three independent experiments. Statistical analysis was performed as described in Materials and methods and the p-value is indicated. Representative Western blots of whole cell lysates from equal number of HEK 293T cells subjected to transfection are shown in the right panels. FUSP31 was detected with an anti-Flag mouse monoclonal antibody (M5) and myc-tagged Smad3 and Smad4 were detected with a mouse anti-myc monoclonal antibody. The positions of FUSP31, mSmad3 and mSmad4 are shown by arrows. Fig. 5. USP31 inhibits NF-nB activation by TRAF2, TRAF6 and IKKh. HEK 293T cells were transfected with 0.2 Ag of the 3xnBLuc NF-nBdependent luciferase reporter plasmid and 0.2 Ag of pGK-h-galactosidase reporter plasmid, to normalize luciferase activities. Relative luciferase activities (%) are calculated in the presence or absence of 0.5 Ag of expression vector for Flag-tagged USP31 (FUSP31) and in the presence of 0.25 Ag of expression vector for TRAF2 (A), or Flag-tagged TRAF6 (FTRAF6, B), or Flag-tagged IKKh (FIKKh, C), as indicated. Twenty-four hours post-transfection cell extracts were prepared and the levels of luciferase and h-galactosidase activity were determined. Results shown in the left panels are the mean T S.D. of relative luciferase activity (%) from three independent experiments. Statistical analysis was performed as described in Materials and methods and the p-value is indicated. Representative Western blots of whole cell lysates from equal number of HEK 293T cells subjected to transfection are shown in the right panels. Flag-tagged USP31, TRAF6 and IKKh were detected with a mouse antiFlag monoclonal antibody (M5). TRAF2 was detected with a rabbit polyclonal antibody. The positions of TRAF2, FUSP31, FTRAF6 and FIKKh are shown by arrows.
partially cleaved lysine-48-linked polyubiquitin to its monomer, whereas USP31C98S or USP31S1 did not (Fig. 2B, left panel). The expression levels of Flag-tagged USP31, USP31C98S and USP31S1 were similar as indicated by Western blot analysis (Fig. 2B,C, right panels). These results demonstrate that USP31 is a deubiquitinating enzyme that cleaves preferentially lysine-63-linked polyubiquitin chains. USP31 can also cleave lysine-48-linked polyubiquitin chains albeit to a lesser extent. 3.4. USP31 –TRAF2 interaction in mammalian cells and subcellular localization of USP31 The ability of USP31 to interact biochemically with TRAF2 in mammalian cells was tested by co-immunopre-
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cipitation of Flag-epitope-tagged USP31 (FUSP31) or USP31S1 (FUSP31S1) and TRAF2 from whole cell extracts of HEK 293T cells. For this purpose, HEK 293T cells were co-transfected with either FUSP31 or FUSP31S1 and TRAF2-expression constructs. Flag-tagged proteins were immunoprecipitated using the M5 antibody against Flag. Immunoprecipitated samples and whole cell extracts were subjected to immunoblotting with an antibody against TRAF2 and, subsequently, an antibody against Flag. TRAF2 was co-immunoprecipitated with FUSP31 or FUSP31S1 (Fig. 3A,B). To confirm that the interaction between TRAF2 and USP31 in mammalian cells occurs via the RING finger domain of TRAF2, as indicated by our two-hybrid results, a mutated form of TRAF2 with a RING finger deletion (TRAF2DRF) was tested for its ability to interact with FUSP31. For this purpose, FUSP31 and TRAF2DRF were co-expressed in HEK 293T cells. Co-immunoprecipitation experiments indicated that USP31 does not interact with a mutated TRAF2 that lacks the RING finger domain (Fig. 3C). These results show that USP31 and TRAF2 interact specifically in mammalian cells and their interaction requires an intact RING finger domain of TRAF2 and the amino terminal 483 residues of USP31. Indirect immunofluorescense showed that USP31 and USP31S1 were localized in the nucleus and to some extent in the cytoplasm (data not shown). 3.5. The role of USP31 in NF-jB activation Oligomerization of TRAF2 by overexpression or receptor activation induces the activation of the transcription factor NF-nB. To determine whether USP31 is implicated in NF-nB activation co transfection experiments were performed in HEK 293T cells in order to overexpress transiently USP31 along with NF-nB luciferase reporter plasmid in the absence or presence of TNFa or various effector plasmids. The effector plasmids were expression vectors for CD40, LMP1, TRAF2, TRAF6 and IKKh. USP31 overexpression inhibited NF-nB activation mediated by TNFa and two members of the TNFR family, CD40 and LMP1 (Fig. 4B,C,D), whereas it had no effect on the basal NF-nB activity (Fig. 4A). USP31 also inhibited NF-nB activation mediated by two TRAF proteins (TRAF2 and TRAF6), as well as by the IKKh kinase (Fig. 5). The inhibition of NF-nB activation by USP31 was not due to a reduction of effector protein expression, since Western blot analysis revealed that the levels of all effector proteins were unaffected by overexpression of USP31 (Figs. 4 and 5, right panels). These experiments indicate that USP31 is implicated in the process of NF-nB activation and that at least some of its targets are located downstream of IKKh, and possibly in the nucleus. The latter is consistent with the nuclear localization of USP31. To determine whether the effects of USP31 on transcription activation were general, its ability to affect Smad-dependent transcription activation of a
Fig. 7. USP31 can interact with p65/RelA. HEK 293T cells were transfected with expression vectors for p65 (A and B) in the presence or absence of constructs expressing Flag-tagged USP31 (FUSP31, A) or USP31S1 (FUSP31S1, B) as indicated. Cell lysates were subjected to immunoprecipitation with an antibody (M5) against Flag (IP: anti-Flag). The immunoprecipitated materials and an aliquot of the whole cell extract (WCE) were subjected to immunoblotting (WB: anti-p65 or WB: anti Flag) with a rabbit polyclonal antibody against p65 (A and B, top panels) or a mouse monoclonal antibody (M5) against Flag (A and B, bottom panels). The positions of p65, FUSP31 and FUSP31S1 are shown by arrows.
Smad-responsive luciferase reporter construct was tested [24]. Overexpression of USP31 did not affect Smad3- and Smad4-mediated transcription activation, indicating that USP31 is not a general regulator of transcription (Fig. 6). These results reinforce the notion that USP31 has a specific role in NF-nB activation induced by TRAFs and members of the TNFR family. 3.6. USP31-p65/RelA interaction The nuclear localization of USP31 and its implication in NF-nB activation prompted us to investigate potential target molecules of USP31 in the nucleus. p65/RelA is a possible target of USP31, since it is a major component of NF-nB and it is ubiquitinated and degraded by the proteasome in the nucleus [12]. For this purpose, the ability of p65 to interact with USP31 was tested using co-immunoprecipitation experiments. HEK 293T cells were co-transfected with constructs expressing either FUSP31 or FUSP31S1 and p65. Flag-tagged proteins were immunoprecipitated using the M5 antibody against Flag. Immunoprecipitated samples and whole cell extracts were subjected to immunoblotting with an antibody against p65 and subsequently an antibody against Flag. Our results indicate that both USP31 and USP31S1 have the ability to interact with p65/RelA in mammalian cells (Fig. 7).
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4. Discussion Our results indicate that USP31 is involved in the regulation of NF-nB activation by members of the TNFR superfamily. USP31 may remove ubiquitin from TRAF2, TRAF6 or another essential intermediate that lies downstream of TRAFs in these pathways. For example, TRAF2and TRAF6-mediated ubiquitination leads to the activation of downstream kinases through a proteasome-independent mechanism. For this purpose, K-63-linked polyubiquitin chains are conjugated to signaling proteins including TRAF2, TRAF6, RIP and NEMO [25]. K-63 polyubiquitination leads to the recruitment and activation of the TAK1 kinase and ultimately to the activation of the NF-nB pathway [5]. Our data indicate that USP31 could modulate the activity of TRAF2 and/or TRAF6 by reversing their ubiquitination. The presence of USP31 in the nucleus raises the possibility that it may also affect NF-nB signal transduction by targeting ubiquitin modifications of nuclear factors implicated in transcription regulation. This hypothesis is consistent with our finding that USP31 affects NF-nB activation downstream from the IKKh. A candidate target of USP31 could be p65/RelA, since there is a physical interaction between USP31 and p65 as indicated by the immunoprecipitation experiments. In addition, it has been shown that p65 is ubiquitinated in the nucleus in a DNA binding-dependent manner. The ubiquitination and proteasomal degradation of p65 is an important mechanism not only to control its stability and abundance, but to promote transcriptional termination of target genes as well [12]. At the same time, p65 ubiquitination may exert non-proteolytic activities that stimulate transcription. The interplay between ubiquitin, proteasome and transcriptional regulation is extremely complex. A model compatible with many observations is that ubiquitination regulates transcriptional activation by serving as a dual signal for activation and activator destruction. It has been speculated that nonubiquitinated transcription factors are stable but inactive and that ubiquitination simultaneously activates transcription factors and primes them for destruction by the proteasome [26]. Ubiquitin ligases as well as deubiquitinases play a key role in this procedure. USP31 could control the transactivating activity and degradation of p65/RelA. Furthermore, USP31 could influence transcription of NF-nB target genes, by regulating in space or time the association of the NF-nB family of transcription factors with partner proteins such as co-activators or co-repressors [27]. Finally, it should be mentioned that TRAF2 has been reported to enter the nucleus under conditions of stress and therefore USP31 might affect the activity of TRAF2 in the nucleus [28,29]. Another nuclear target of USP31 might be histones. Histone ubiquitination affects chromatin structure. It has been evident also for more than two decades that histones H2A and H2B are monoubiquitinated, but this post-trans-
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lational modification does not lead to enhanced proteolytic degradation. In contrast it has been related with both transcriptional repression and maintenance of telomeric gene silencing [30]. It also seems that histone H2B ubiquitination influences other chromatin modifications such as acetylation and methylation [31] to control transcription. Other deubiquitinating enzymes that have been reported to act on chromatin may also interact, either directly or indirectly, with specific histones. Yeast Ubp-3 associates with several proteins known as silent information regulators (SIRs 2, 3 and 4) that are both essential for gene silencing and interact with the amino terminal segments of histones H3 and H4 [32]. In conclusion, USP31 is a deubiquitinating enzyme that is implicated in NF-nB activation. While all NF-nB activation pathways share a central and critical upstream proteasome-mediated step that leads to the degradation of inhibitory proteins and the release of the DNA-binding subunits there is evidence for a downstream level of NF-nB regulation that employs several mechanisms. These mechanisms include promoter specific exchange of dimers as well as phosphorylation, acetylation, ubiquitination and prolyl isomerization of the transactivating p65/RelA subunit [33]. USP31 could participate in the regulation of NF-nB in the nucleus in addition to a possible role in the cytoplasmic events that lead to NF-nB activation. Further characterization of the role of USP31 in TRAF- and TNFR-mediated signal transduction will require the identification of the optimal substrate for USP31 and the inactivation of the usp31 gene in mice.
Acknowledgements This work was supported by the Greek Secretariat of Research and Technology (Career Development Award for Greek-speaking scientists returning to Greece to EH, program PENED 1999 to George Mosialos), an International Scholarship from the Howard Hughes Medical Institute (to George Mosialos), a Human Frontier Science Program grant (to George Mosialos), a graduate student stipend from State Scholarship Foundation of Greece (to CT) and US National Institute of Health grant CA47006 (to EK). George Mosialos is a Leukemia and Lymphoma Society of America Scholar.
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Human ubiquitin specific protease 31 is a deubiquitinating enzyme implicated in activation of nuclear factor-nB Christos Tzimasa,c, Gianna Michailidoua, Minas Arsenakisa, Elliott Kieff b, George Mosialosc,*, Eudoxia G. Hatzivassilioua,c,* a Department of Biology, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece Infectious Disease Division, Brigham and Women’s Hospital, 181 Longwood Ave, Boston, MA 02115, USA c Institute of Immunology, Biomedical Sciences Research Center Al. Fleming, 34 Al. Fleming Str., Vari 16672, Greece b
Received 4 March 2005; received in revised form 16 March 2005; accepted 16 March 2005 Available online 26 April 2005
Abstract TRAF2 mediates activation of the transcription factors NF-nB and AP1 by TNF. A yeast two-hybrid screen of a human cDNA library identified a ubiquitin specific protease homologue (USP31) as a TRAF2-interacting protein. Two cDNAs encoding for USP31 were identified. One cDNA encodes a 1035-amino acid long isoform of USP31 (USP31, long isoform) and the other a 485-amino acid long isoform of USP31 (USP31S1, short isoform). USP31 and USP31S1 share a common amino terminal region with homology to the catalytic region of known deubiquitinating enzymes. Enzymatic assays demonstrated that USP31 but not USP31S1 possess deubiquitinating activity. Furthermore, it was shown that USP31 has a higher activity towards lysine-63-linked as compared to lysine-48-linked polyubiquitin chains. Overexpression of USP31 in HEK 293T cells inhibited TNFa, CD40, LMP1, TRAF2, TRAF6 and IKKh-mediated NF-nB activation, but did not inhibit Smad-mediated transcription activation. In addition, both USP31 isoforms interact with p65/RelA. Our data support a role for USP31 in the regulation of NF-nB activation by members of the TNF receptor superfamily. D 2005 Elsevier Inc. All rights reserved. Keywords: NF-nB signaling; TNF; TNFR; TRAF; Ubiquitin proteasome system; Ubiquitin specific protease
1. Introduction TRAFs constitute a family of proteins that have been implicated in signal transduction by members of the TNF receptor superfamily including TNFRI, TNFRII, CD40, Abbreviations: AP1, activator protein 1; DTT, dithiotreitol; DUB, deubiquitinating enzyme; EBV, Epstein-Barr virus; IKK, InB kinase; JNK, Jun-kinase; LMP1, latent membrane protein 1; LThR, lymphotoxin h receptor; MAPK, mitogen activated protein kinase; NP-40, Nonidet P-40; NF-nB, nuclear factor-kappaB; TAK1, transforming growth factor-beta activated protein kinase; TNF, tumor necrosis factor; TNFR, TNF receptor; TRAF, tumor necrosis factor receptor associated factor; Ub, Ubiquitin; USP, ubiquitin specific protease. * Corresponding authors. Institute of Immunology, Biomedical Sciences Research Center Al. Fleming, 34 Al. Fleming Str., Vari 16672, Greece. Tel.: +30 2109656703; fax: +30 2109653934. E-mail addresses: [email protected] (G. Mosialos), [email protected] (E.G. Hatzivassiliou). 0898-6568/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2005.03.017
CD30, CD27, LThR and the EBV oncoprotein LMP1 [1]. Six mammalian TRAF molecules have been identified so far. Among them, TRAF2, TRAF5 and TRAF6 have the ability to activate the NF-nB and AP1 signaling pathways, upon overexpression [1]. TRAF-mediated activation of the NF-nB pathway is dependent on the activation of the IKK complex, whereas AP1 activation by TRAFs is mediated by MAPKs including JNK and p38 [2,3]. TRAFs are characterized by the presence of a conserved carboxyl terminal region known as the TRAF domain, which interacts with the cytoplasmic domains of TNF receptors. The amino terminal region of TRAFs is rich in cysteine and histidine residues arranged in putative zinc-finger motifs. With the exception of TRAF1, TRAFs contain an amino terminal RING finger motif, which has been implicated in the activation of NFnB and MAPKs.
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TRAF2 and TRAF6 have a ubiquitin ligase (E3) activity, which is dependent on the integrity of their RING finger domain and has been associated with the ability to activate the NF-nB and AP1 signaling pathways [4,5]. Protein modification by ubiquitination has been primarily associated with degradation by the proteasome. However, protein ubiquitination has also been implicated in other cellular functions, including transcription regulation, receptor endocytosis, DNA repair, ribosomal function, stress response and mitochondrial inheritance [6]. TRAF2 and TRAF6 mediate polyubiquitination by catalyzing the formation of an isopeptide bond between the carboxyl terminus of one ubiquitin moiety and the e amino group of lysine 63 of another ubiquitin molecule. Lysine-63-linked polyubiquitin chains have been associated primarily with non-proteolytic processes, whereas lysine-48-linked polyubiquitin chains usually mark proteins for proteasomal degradation [6]. Both types of ubiquitination (lysine-48 or lysine-63) play critical role in NF-nB activation by TNF family members and other cytokines. TRAF2 and TRAF6 mediated lysine-63 ubiquitination is essential for NF-nB activation. Signal transduction by TRAF6 is initiated upon its oligomerization. Oligomerization of TRAF6 triggers its polyubiquitination, which is essential for the subsequent activation of protein kinase TAK1 [5]. It is believed that TRAF6 polyubiquitination facilitates the assembly and activation of multisubunit protein complexes [7]. TAK1 can activate both the NF-nB and AP1 pathways [5,8]. Activation of the IKK complex mediates the phosphorylation of the NF-nB inhibitor InB. Phosphorylation of InB by IKK causes its lysine-48 ubiquitination and its subsequent degradation by the ubiquitin –proteasome pathway. This results in nuclear translocation and activation of NF-nB [9]. Postinduction repression and termination of NF-nB activity depends on NF-nB regulated resynthesis of InBa which dissociates NF-nB from DNA and exports it to the cytoplasm. NF-nB regulated resynthesis of InBa provides a strong negative feedback and a fast down-regulation of the NF-nB response [10,11]. Additionally, it has been found that after activation p65/RelA is degraded by the proteasome in the nucleus and in a DNA binding-dependent manner [12]. If proteasome activity is blocked, NF-nB is not promptly removed from some target genes in spite of InBa resynthesis and sustained transcription occurs. These results indicate that lysine-48 ubiquitination and proteasomal degradation of p65/RelA does not merely regulate its stability and abundance, but also actively promotes transcriptional termination. In order to elucidate further the TRAF2 signaling mechanism and its regulation, we have performed a yeast two-hybrid screen of a human cDNA library with TRAF2 as bait and identified a ubiquitin specific protease homologue (USP31). Our findings support a role for USP31 in NF-nB activation by members of the TNFR superfamily.
2. Materials and methods 2.1. Yeast two-hybrid assay The yeast two-hybrid screen was performed as previously described [13]. The bait consisted of amino acids 1– 358 of TRAF2 fused to the amino terminus of GAL4 (pTRAF2(1 – 358)D151). 2.2. Plasmid construction pTRAF2(1 – 358)D151 was constructed by inserting the TRAF2 encoding fragment that resulted from digestion of pSG5FLAGTRAF2 [14] with EcoRI and T4 polymerasemediated blunting and BglII in D151 (a gift by Dr. R. Brazas, University of California, San Francisco), digested with HindIII and T4 polymerase-mediated blunting and Bam HI. pTRAF2(1 – 358)C34/37AD151 which is pTRAF2(1 –358)D151 with TRAF2 cysteines 34 and 37 mutated to alanines was constructed by replacing the KpnI– AgeI fragment of pTRAF2(1 –358)D151 with the corresponding mutagenized fragment that was generated by PCR. The expressing constructs for the Flag-tagged long isoform USP31 (pcDNA3FlagUSP31), the point mutant USP31C98S (pcDNA3FlagUSP31C98S) and the short isoform USP31S1 (pcDNA3FlagUSP31S1) were constructed in several steps. The USP31 encoding EcoRI fragment of a cDNA clone spanning amino acids 1 to 634 (USP31(1 – 634)) isolated from a human B lymphoma library [15] was subcloned into pBluescript KS+ (Stratagene) digested with EcoRI to generate pBluescriptUSP31(1 –634). The NcoI– EcoRI USP31(1 –634) encoding fragment of pBluescriptUSP31(1 –634) was blunted at the NcoI site and subcloned into pcDNA3Flag [16] digested with HpaI and EcoRI to generate pCDNA3FlagUSP31(1 – 634). The USP31S1 encoding BstEII –EcoRI fragment of a human EST clone (IMAGE:259632) was subcloned into pcDNA3FlagUSP31(1 – 634) digested with BstEII and EcoRI to generate pcDNA3FlagUSP31S1. The complete sequence of USP31 was determined from overlapping cDNA clones isolated from a human B lymphoma library [15] and human EST clones. Flag-tagged full-length USP31 expressing construct pcDNA3FlagUSP31 was constructed in several steps. Human leukocyte marathon cDNA was used as a template DNA in PCR with the primers 1041F6 (GCTGAAAGCAGCAGTAAAGGGCAGCGATGG) and 1041R7 (GCTCTAGAGCGGCCGCCTTTTCTATGTCGCATACTTCG). A band of approximately 1 kbp was amplified, isolated and reamplified. The resulting PCR product was digested with restriction enzymes NheI and XbaI, and it was cloned into pcDNA3FlagUSP31(1 – 634) digested with NheI and XbaI to generate the clone pcDNA3FlagUSP31(1 –938). HEK 293T cells cDNA was used as template DNA in PCR using the primers 1041FL (AAGCCTACAAGATGCAGCCCTGTG) and 1041R8 ( G C T C TA G A G C G G C C G C C C TA A C AT G T C A A -
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ACTCCTCTTCC). A band of approximately 800 bp was amplified, and digested with restriction enzymes NdeI and NotI, and it was cloned into pcDNA3FlagUSP31(1 – 938) digested with NdeI and NotI. The resulting construct was pcDNA3FlagUSP31(799 – 1035) that contained the 3¶ end of USP31 and it also had a deletion of approximately 3 kbp at the 5¶ end of USP31 spanning amino acids 1 to 798. This construct was digested with BspEI and NotI to generate a fragment of approximately 400 bp that contained the 3¶ end of USP31 (amino acids 881 to 1035). This fragment was ligated into pcDNA3FlagUSP31(1 – 938) digested with BspEI and NotI to generate the full-length pcDNA3FlagUSP31. FLAG-tagged full-length USP31C98S was generated by PCR-mediated site directed mutagenesis. To generate this clone pcDNA3FlagUSP31S1 was digested with BspD1, HpaI and ligated to a PCR fragment, produced with three rounds of PCR, using the mutagenic primers C98SF(forward) (GGAGCCACTTCTTATGTCAACACATTTCTTC) and C98SR(reverse) (GTT-GACATAAGAAGTGGCTCCAAGGTTAGTC). In the first PCR round the C98SF was combined to T7 and the template plasmid was pBluescriptUSP31(1 – 634). In the second PCR round the C98SF was combined to pGEXF1 and the template plasmid was pGEXUSP31(1 –634), which was constructed by inserting the USP31(1 –634) fragment that resulted from the digestion of pBluescriptUSP31(1 – 634) with NcoI and T4 polymerase mediated blunting and EcoRI into pGEX2TK (Pharmacia) digested with SmaI and EcoRI. The two PCR products were then combined and PCR-amplified with the primers T7 and GEXF1 and the resulting product was digested with BspD1 and NcoI that was blunted and cloned in pcDNA3Flag USP31S1 digested with BspD1 and HpaI. This procedure led to the construction of the mutated plasmid pcDNA3FlagUSP31S1C98S. The full-length pcDNA3FlagUSP31C98S was constructed by inserting the BstEII, NotI fragment of pcDNA3FlagUSP31(1 – 938) into pcDNA3Flag-USP31S1C98S digested with BstEII and NotI to generate pcDNA3FlagUSP31(1 – 938)C98S. Finally, the BspEI, NotI 400 bp fragment of pcDNA3Flag-USP31(799– 1035) was ligated into pcDNA3FlagUSP31(1– 938)C98S digested with BspEI and NotI to generate the full-length pcDNA3FlagUSP31C98S.
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2.4. Cell lines, transfections and reporter assays The HEK 293T cell line and the HeLa cell line were maintained in Dulbecco’s Modified Eagle’s Medium (Gibco Invitrogen) supplemented with 10% heat inactivated fetal bovine serum (Gibco Invitrogen), 2 mM l-glutamine (Biochrom AG) and 50 units/ml penicillin and 50 Ag/ml streptomycin (Gibco Invitrogen), at 37 -C, in a humidified incubator with 5% CO2. Transfections were performed on HEK 293T cells with ExGen500 (Fermentas) and on HeLa cells with Lipofectamine 2000 (Invitrogen) transfection reagents according to the instructions of the manufacturer, unless otherwise indicated. TNF was added for 5 h before harvesting, at a concentration of human TNFa, 10 ng/ml. The h-galactosidase and luciferase reporter activities were assayed 24 h post-transfection. The h-galactosidase activity was measured using the Galacton-Plus substrate system (TROPIX, Inc) according to the manufacturer’s instructions. For luciferase-reporter assays, cells from a 12-well plate were lysed in 100 Al reporter lysis buffer (Promega) and luciferase activity was assayed with the luciferase assay system (Promega) according to the manufacturer’s instructions. Luciferase activities were normalized by dividing the luciferase activity values by the corresponding h-galactosidase activity values. Relative luciferase activities were expressed as folds of activation relative to the activity of the luciferase reporter alone. This was calculated by dividing every normalized luciferase activity value with the normalized luciferase activity value of the reporter alone. Finally, relative luciferase activities (%) were
2.3. Northern blots Northern blots were performed by using human multiple tissue Northern blots that were purchased from Clontech. A cDNA fragment encoding most of the catalytic region of USP31 was used as probe. The cDNA probe was labeled with 32P using the Multiprime DNA Labeling System (Amersham Biosciences). Hybridization was performed by standard procedures according to the manufacturers’ protocol. Hybridized membranes were exposed to X-ray film (X-OMAT AR) for 2 days at 70 -C.
Fig. 1. USP31 expression pattern in normal tissues and cancer cell lines. PolyA+ RNA from various human tissues (A) and cancer cell lines (B) was analyzed by Northern blot for the expression of USP31 mRNAs. USP31 was expressed in all normal tissues and cancer cell lines examined. The cancer cell lines were derived from the following tumors: promyelocytic leukemia (HL60), cervical carcinoma (HeLa), chronic myelogenous leukemia (K-562), lymphoblastic leukemia (MOLT-4), Burkitt’s lymphoma (Raji), colorectal adenocarcinoma (SW480), lung carcinoma (A549) and melanoma (G361).
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calculated as % of the fold of activation in transfected cells which express the effector USP31, relative to the corresponding transfected cells that do not express USP31. Results were analyzed statistically with a Student’s t-test (Microsoft Excel). The parameters used for the comparison of values were two-tailed, unpaired arrays. The difference between two samples was considered statistically significant when the p-value <0.01. 2.5. In vitro deubiquitination assays Wild type or mutated Flag-tagged USP31 proteins (FUSP31, FUSP31C98S and FUSP31S1) were immunoprecipitated from HEK 293T cell lysates prepared with NP-40
lysis buffer (25 mM Tris – HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 10% (v/v) glycerol, 0.5% (v/v) NP-40, 1 mM PMSF and 1 mM DTT). The immunoprecipitated proteins were incubated with 1.25 Ag of tetraubiquitin (Affinity Research Products) or 1.5 Ag of either lysine-48- or lysine63-linked polyubiquitin substrates (Boston Biochem, Inc.) in 15 Al of 50 mM Tris – HCl buffer (pH 7.2) containing 1 mM DTT for 2 h at 37 -C. Reaction mixtures were filtered and the flow through fraction that contained the cleaved substrates was analyzed by tricine SDS PAGE followed by silver staining. The beads containing the immunoprecipitated Flag-tagged proteins were analyzed by SDS-PAGE followed by immunoblotting with a mouse monoclonal antiFlag (M5) antibody.
Fig. 2. Deubiquitinating activity and specificity of USP31 towards tetraubiquitin and polyubiquitin substrates. Flag-tagged USP31 (FUSP31), USP31C98S (FUSP31C98S) and USP31S1 (FUSP31S1) were immunoprecipitated with the M5 anti-FLAG antibody from lysates of transfected HEK 293T cells and tested for their ability to cleave lysine-48-linked tetraubiquitin (K-48-linked tetraUb, A) and lysine-48-linked (K-48-linked polyUb, B) or lysine-63-linked (K-63linked polyUb, C) polyubiquitin chains. The reaction mixture was analyzed by tricine SDS-PAGE and silver straining (A, B, C, left panels). Arrows indicate the positions of tetrameric (Ub4), trimeric (Ub3), dimeric (Ub2) and monomeric (Ub1) ubiquitin. The amount of immunoprecipitated Flag-tagged proteins (shown by arrows) was determined by immunoblotting with the M5 antibody (A, B, C, right panels). In B and C the procedures for silver staining or immunoblotting were performed at the same time under identical and therefore absolutely comparable conditions.
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2.6. Immunofluorescense, immunoprecipitations and immunoblotting Indirect immunofluorescense was performed on HeLa cells as previously described [17]. Immunoprecipitations were performed in NP-40 lysis buffer (25 mM Tris –HCl pH 7.5, 250 mM NaCl, 5 mM EDTA, 10% (v/v) glycerol, 0.5% (v/v) NP-40, 1 mM PMSF and 1 mM DTT) or RIPA lysis buffer (50 mM Tris – HCl pH 8.0, 250 mM NaCl, 0.1% (w/ v) SDS, 0.1% (w/v) sodium deoxycholate, 1% (v/v) TritonX 100, 1 mM PMSF). The lysis buffer was supplemented with complete protease inhibitor cocktail (Roche) according to the manufacturer_s instructions. Cells were lysed for 30 min on ice in 500 Al NP-40 lysis buffer for TRAF2 immunoprecipitations shown in Fig. 3 or RIPA lysis buffer for p65 immunoprecipitations shown in Fig. 7. Immunoprecipitations were carried out as previously described [18]. Immunoblots were developed with the ECL-plus reagent (Amersham Biosciences) according to the manufacturer’s instructions. Images were obtained using the STORM 860 system (Molecular Dynamics). Antibodies were obtained from Santa Cruz Biotechnology with the exception of M2 and M5 antibodies against Flag, which were obtained from Sigma. The secondary antibodies were sheep – anti-mouse and donkey – anti-rabbit conjugated to horseradish peroxidase and they were purchased from Amersham Pharmacia and Santa Cruz Biotechnology, respectively.
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01440). USP31S1 has in addition to the amino terminal 483 residues a glycine (G) and a leucine (L) residue at its carboxyl terminus. USP31 and USP31S1 contain all eight characteristic motifs of known deubiquitinases [22]. The rat ortholog of USP31 (synUSP) was recently identified in Rat neurons [23]. Full-length human USP31 has not been analyzed for deubiquitinating activity. A cDNA encoding full-length USP31 was assembled from partial cDNA clones and used for further characterization. 3.2. Northern blot analysis Northern blot analysis of normal tissues and cancer cell lines revealed two major mRNA species, which are likely to be derived from alternative mRNA processing and may code for USP31 and USP31S1 (Fig. 1). USP31 was expressed in all normal tissues and cancer cell lines tested,
3. Results 3.1. Identification of USP31 cDNAs TRAF2 amino acids 1 – 358, containing the RING finger domain, were fused to GAL4 DNA-binding domain and used as bait in a yeast two-hybrid screening of a cDNAlibrary prepared from human EBV-transformed B lymphocytes [19]. Sixteen positive cDNA clones were studied further because they demonstrated a strong positive interaction with wild type TRAF2(1 – 358), but very weak or no interaction with TRAF2(1 –358), which contained a mutated RING finger domain. One of these clones encodes a protein with homology to deubiquitinating enzymes, which turned out to be identical to the product of a recently identified cDNA (USP31 [20], which is also known as USP48 [21]). Comparison of the USP31 cDNA sequence with human ESTs revealed that more than one USP31 cDNAs exist. Two cDNAs were identified and studied further in this report. One cDNA encodes for a longer 1035-amino acid long polypeptide (USP31, long isoform) [20], whereas the other encodes for a shorter 485-amino acid long polypeptide (short isoform, USP31S1). The two isoforms of USP31 share a common amino terminal region of 483 amino acids with significant similarity to known deubiquitinases (29% identity and 45% homology to a known S. pombe deubiquitinase with GenBank accession number AAF-
Fig. 3. USP31 interacts with TRAF2 in mammalian cells. HEK 293T cells were transfected with expression vectors for TRAF2 (A and B) or a TRAF2 deletion lacking the RING Finger (TRAF2DRF, C) in the presence or absence of constructs expressing Flag-tagged USP31 (A, C) or USP31S1 (B), as indicated. Cell lysates were subjected to immunoprecipitation with an antibody (M5) against Flag (IP: anti-Flag). The immunoprecipitated material and an aliquot of the whole cell extract (WCE) were subjected to immunoblotting (WB: anti-TRAF2 or WB: anti-Flag) with a rabbit polyclonal antibody against TRAF2 (A, B and C, top) or a mouse monoclonal antibody (M5) against Flag (A, B and C, bottom). The positions of TRAF2, TRAF2DRF, FUSP31 and FUSP31S1 are shown by arrows.
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indicating a ubiquitous role for this protein, in accordance with the potential functional relationship of USP31 with the broadly expressed TRAF2. Interestingly, in several cancer cell lines tested, the ratio of the lower to the higher molecular weight USP31 mRNA was significantly elevated (Fig. 1B), compared to the normal tissues (Fig. 1A). This may reflect a regulatory role of short USP31 isoform on the function of USP31 in cancer cells. 3.3. Biochemical characterization of USP31 The extensive similarity of USP31 to deubiquitinating enzymes led to the examination of its ability to act as a ubiquitin carboxyl terminal hydrolase. In addition to wild type USP31, a point mutant containing a serine instead of cysteine at position 98 (USP31C98S) was constructed and tested for its enzymatic activity. Cysteine 98 corresponds to a highly conserved cysteine of the catalytic domain of deubiquitinating enzymes (Cys box) [22]. Flag-tagged USP31, USP31C98S and USP31S1 were transiently expressed in HEK 293T cells upon transfection of the appropriate expression vectors. Cells were lysed and flagtagged proteins were immunoprecipitated using anti-Flag (M5) antibodies and tested for their ability to cleave
tetraubiquitin. Wild type full-length USP31 readily cleaved the tetrameric substrate to its monomer, dimer and trimer (Fig. 2A, left panel). However, the point mutant USP31C98S and the short isoform USP31S1 were enzymatically inactive (Fig. 2A, left panel), despite the fact that they were expressed in similar levels with USP31 (Fig. 2A, right panel). These results indicate that cysteine 98 and the carboxyl-terminal region of USP31 are essential for the deubiquitinating activity of the protein. These data are consistent with the findings of Tian et al. [23], who have demonstrated that full-length synUSP has deubiquitinating activity in bacteria, which is abolished by a smaller than 250-amino acid carboxyl-terminal truncation. Furthermore, USP31 was tested for its deubiquitinating specificity towards lysine-48- or lysine-63-linked polyubiquitin chains. For this purpose, Flag-tagged wild type USP31, the point mutant USP31C98S and the short isoform USP31S1 were transiently expressed in HEK 293T cells, immunoprecipitated and tested for their ability to cleave lysine-48- or lysine-63-linked polyubiquitin substrates. Wild type USP31 cleaved almost completely lysine-63-linked polyubiquitin to its monomer, whereas the catalytically inactive point mutant USP31C98S or the short isoform USP31S1 did not cleave the substrate (Fig. 2C, left panel). Wild type USP31 also
Fig. 4. USP31 inhibits NF-nB activation by TNFa, CD40 and LMP1. HEK 293T cells were transfected with 0.2 Ag of the 3xnBLuc NF-nB-dependent luciferase reporter plasmid and 0.2 Ag of pGK-h-galactosidase reporter plasmid, to normalize luciferase activities. Relative luciferase activities (%) are calculated in the presence or absence of 0.5 Ag of expression vector for Flag-tagged USP31 (FUSP31) and in the absence (A) or presence of 10 ng/ml of human TNFa (B), or 0.25 Ag of expression vector for CD40 (C), or 0.05 Ag of expression vector for LMP1 (D), as indicated. Twenty-four hours post-transfection cell extracts were prepared and the levels of luciferase and h-galactosidase activity were determined. Results shown in the left panels are the mean T S.D. of relative luciferase activity (%) from three independent experiments. Statistical analysis was performed as described in Materials and methods and the p-value is indicated. Representative Western blots of whole cell lysates from equal number of HEK 293T cells subjected to transfection are shown in the right panels. FUSP31 was detected with a mouse anti-Flag monoclonal antibody (M5). CD40 was detected with a rabbit polyclonal antibody and LMP1 with a mouse monoclonal antibody (S12), respectively. The positions of FUSP31, CD40 and LMP1 are shown by arrows.
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Fig. 6. USP31 does not affect Smad-dependent transcription activation. HEK 293T cells were transfected with 0.2 Ag of the p(CAGA)12-luc Smad-dependent luciferase reporter plasmid (CAGA) and 0.2 Ag of pGKh-galactosidase reporter plasmid, to normalize luciferase activities. Relative luciferase activities (%) are calculated in the presence or absence of 0.5 Ag of expression vector for Flag-tagged USP31 (FUSP31) and in the absence (A) or presence of 0.3 Ag of expression vectors for myctagged Smad3 and Smad4 (mSmad3, mSmad4, B) as indicated. Twentyfour hours post-transfection cell extracts were prepared and the levels of luciferase and h-galactosidase activity were determined. Results shown in the left panels are the mean T S.D. of relative luciferase activity (%) from three independent experiments. Statistical analysis was performed as described in Materials and methods and the p-value is indicated. Representative Western blots of whole cell lysates from equal number of HEK 293T cells subjected to transfection are shown in the right panels. FUSP31 was detected with an anti-Flag mouse monoclonal antibody (M5) and myc-tagged Smad3 and Smad4 were detected with a mouse anti-myc monoclonal antibody. The positions of FUSP31, mSmad3 and mSmad4 are shown by arrows. Fig. 5. USP31 inhibits NF-nB activation by TRAF2, TRAF6 and IKKh. HEK 293T cells were transfected with 0.2 Ag of the 3xnBLuc NF-nBdependent luciferase reporter plasmid and 0.2 Ag of pGK-h-galactosidase reporter plasmid, to normalize luciferase activities. Relative luciferase activities (%) are calculated in the presence or absence of 0.5 Ag of expression vector for Flag-tagged USP31 (FUSP31) and in the presence of 0.25 Ag of expression vector for TRAF2 (A), or Flag-tagged TRAF6 (FTRAF6, B), or Flag-tagged IKKh (FIKKh, C), as indicated. Twenty-four hours post-transfection cell extracts were prepared and the levels of luciferase and h-galactosidase activity were determined. Results shown in the left panels are the mean T S.D. of relative luciferase activity (%) from three independent experiments. Statistical analysis was performed as described in Materials and methods and the p-value is indicated. Representative Western blots of whole cell lysates from equal number of HEK 293T cells subjected to transfection are shown in the right panels. Flag-tagged USP31, TRAF6 and IKKh were detected with a mouse antiFlag monoclonal antibody (M5). TRAF2 was detected with a rabbit polyclonal antibody. The positions of TRAF2, FUSP31, FTRAF6 and FIKKh are shown by arrows.
partially cleaved lysine-48-linked polyubiquitin to its monomer, whereas USP31C98S or USP31S1 did not (Fig. 2B, left panel). The expression levels of Flag-tagged USP31, USP31C98S and USP31S1 were similar as indicated by Western blot analysis (Fig. 2B,C, right panels). These results demonstrate that USP31 is a deubiquitinating enzyme that cleaves preferentially lysine-63-linked polyubiquitin chains. USP31 can also cleave lysine-48-linked polyubiquitin chains albeit to a lesser extent. 3.4. USP31 –TRAF2 interaction in mammalian cells and subcellular localization of USP31 The ability of USP31 to interact biochemically with TRAF2 in mammalian cells was tested by co-immunopre-
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cipitation of Flag-epitope-tagged USP31 (FUSP31) or USP31S1 (FUSP31S1) and TRAF2 from whole cell extracts of HEK 293T cells. For this purpose, HEK 293T cells were co-transfected with either FUSP31 or FUSP31S1 and TRAF2-expression constructs. Flag-tagged proteins were immunoprecipitated using the M5 antibody against Flag. Immunoprecipitated samples and whole cell extracts were subjected to immunoblotting with an antibody against TRAF2 and, subsequently, an antibody against Flag. TRAF2 was co-immunoprecipitated with FUSP31 or FUSP31S1 (Fig. 3A,B). To confirm that the interaction between TRAF2 and USP31 in mammalian cells occurs via the RING finger domain of TRAF2, as indicated by our two-hybrid results, a mutated form of TRAF2 with a RING finger deletion (TRAF2DRF) was tested for its ability to interact with FUSP31. For this purpose, FUSP31 and TRAF2DRF were co-expressed in HEK 293T cells. Co-immunoprecipitation experiments indicated that USP31 does not interact with a mutated TRAF2 that lacks the RING finger domain (Fig. 3C). These results show that USP31 and TRAF2 interact specifically in mammalian cells and their interaction requires an intact RING finger domain of TRAF2 and the amino terminal 483 residues of USP31. Indirect immunofluorescense showed that USP31 and USP31S1 were localized in the nucleus and to some extent in the cytoplasm (data not shown). 3.5. The role of USP31 in NF-jB activation Oligomerization of TRAF2 by overexpression or receptor activation induces the activation of the transcription factor NF-nB. To determine whether USP31 is implicated in NF-nB activation co transfection experiments were performed in HEK 293T cells in order to overexpress transiently USP31 along with NF-nB luciferase reporter plasmid in the absence or presence of TNFa or various effector plasmids. The effector plasmids were expression vectors for CD40, LMP1, TRAF2, TRAF6 and IKKh. USP31 overexpression inhibited NF-nB activation mediated by TNFa and two members of the TNFR family, CD40 and LMP1 (Fig. 4B,C,D), whereas it had no effect on the basal NF-nB activity (Fig. 4A). USP31 also inhibited NF-nB activation mediated by two TRAF proteins (TRAF2 and TRAF6), as well as by the IKKh kinase (Fig. 5). The inhibition of NF-nB activation by USP31 was not due to a reduction of effector protein expression, since Western blot analysis revealed that the levels of all effector proteins were unaffected by overexpression of USP31 (Figs. 4 and 5, right panels). These experiments indicate that USP31 is implicated in the process of NF-nB activation and that at least some of its targets are located downstream of IKKh, and possibly in the nucleus. The latter is consistent with the nuclear localization of USP31. To determine whether the effects of USP31 on transcription activation were general, its ability to affect Smad-dependent transcription activation of a
Fig. 7. USP31 can interact with p65/RelA. HEK 293T cells were transfected with expression vectors for p65 (A and B) in the presence or absence of constructs expressing Flag-tagged USP31 (FUSP31, A) or USP31S1 (FUSP31S1, B) as indicated. Cell lysates were subjected to immunoprecipitation with an antibody (M5) against Flag (IP: anti-Flag). The immunoprecipitated materials and an aliquot of the whole cell extract (WCE) were subjected to immunoblotting (WB: anti-p65 or WB: anti Flag) with a rabbit polyclonal antibody against p65 (A and B, top panels) or a mouse monoclonal antibody (M5) against Flag (A and B, bottom panels). The positions of p65, FUSP31 and FUSP31S1 are shown by arrows.
Smad-responsive luciferase reporter construct was tested [24]. Overexpression of USP31 did not affect Smad3- and Smad4-mediated transcription activation, indicating that USP31 is not a general regulator of transcription (Fig. 6). These results reinforce the notion that USP31 has a specific role in NF-nB activation induced by TRAFs and members of the TNFR family. 3.6. USP31-p65/RelA interaction The nuclear localization of USP31 and its implication in NF-nB activation prompted us to investigate potential target molecules of USP31 in the nucleus. p65/RelA is a possible target of USP31, since it is a major component of NF-nB and it is ubiquitinated and degraded by the proteasome in the nucleus [12]. For this purpose, the ability of p65 to interact with USP31 was tested using co-immunoprecipitation experiments. HEK 293T cells were co-transfected with constructs expressing either FUSP31 or FUSP31S1 and p65. Flag-tagged proteins were immunoprecipitated using the M5 antibody against Flag. Immunoprecipitated samples and whole cell extracts were subjected to immunoblotting with an antibody against p65 and subsequently an antibody against Flag. Our results indicate that both USP31 and USP31S1 have the ability to interact with p65/RelA in mammalian cells (Fig. 7).
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4. Discussion Our results indicate that USP31 is involved in the regulation of NF-nB activation by members of the TNFR superfamily. USP31 may remove ubiquitin from TRAF2, TRAF6 or another essential intermediate that lies downstream of TRAFs in these pathways. For example, TRAF2and TRAF6-mediated ubiquitination leads to the activation of downstream kinases through a proteasome-independent mechanism. For this purpose, K-63-linked polyubiquitin chains are conjugated to signaling proteins including TRAF2, TRAF6, RIP and NEMO [25]. K-63 polyubiquitination leads to the recruitment and activation of the TAK1 kinase and ultimately to the activation of the NF-nB pathway [5]. Our data indicate that USP31 could modulate the activity of TRAF2 and/or TRAF6 by reversing their ubiquitination. The presence of USP31 in the nucleus raises the possibility that it may also affect NF-nB signal transduction by targeting ubiquitin modifications of nuclear factors implicated in transcription regulation. This hypothesis is consistent with our finding that USP31 affects NF-nB activation downstream from the IKKh. A candidate target of USP31 could be p65/RelA, since there is a physical interaction between USP31 and p65 as indicated by the immunoprecipitation experiments. In addition, it has been shown that p65 is ubiquitinated in the nucleus in a DNA binding-dependent manner. The ubiquitination and proteasomal degradation of p65 is an important mechanism not only to control its stability and abundance, but to promote transcriptional termination of target genes as well [12]. At the same time, p65 ubiquitination may exert non-proteolytic activities that stimulate transcription. The interplay between ubiquitin, proteasome and transcriptional regulation is extremely complex. A model compatible with many observations is that ubiquitination regulates transcriptional activation by serving as a dual signal for activation and activator destruction. It has been speculated that nonubiquitinated transcription factors are stable but inactive and that ubiquitination simultaneously activates transcription factors and primes them for destruction by the proteasome [26]. Ubiquitin ligases as well as deubiquitinases play a key role in this procedure. USP31 could control the transactivating activity and degradation of p65/RelA. Furthermore, USP31 could influence transcription of NF-nB target genes, by regulating in space or time the association of the NF-nB family of transcription factors with partner proteins such as co-activators or co-repressors [27]. Finally, it should be mentioned that TRAF2 has been reported to enter the nucleus under conditions of stress and therefore USP31 might affect the activity of TRAF2 in the nucleus [28,29]. Another nuclear target of USP31 might be histones. Histone ubiquitination affects chromatin structure. It has been evident also for more than two decades that histones H2A and H2B are monoubiquitinated, but this post-trans-
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lational modification does not lead to enhanced proteolytic degradation. In contrast it has been related with both transcriptional repression and maintenance of telomeric gene silencing [30]. It also seems that histone H2B ubiquitination influences other chromatin modifications such as acetylation and methylation [31] to control transcription. Other deubiquitinating enzymes that have been reported to act on chromatin may also interact, either directly or indirectly, with specific histones. Yeast Ubp-3 associates with several proteins known as silent information regulators (SIRs 2, 3 and 4) that are both essential for gene silencing and interact with the amino terminal segments of histones H3 and H4 [32]. In conclusion, USP31 is a deubiquitinating enzyme that is implicated in NF-nB activation. While all NF-nB activation pathways share a central and critical upstream proteasome-mediated step that leads to the degradation of inhibitory proteins and the release of the DNA-binding subunits there is evidence for a downstream level of NF-nB regulation that employs several mechanisms. These mechanisms include promoter specific exchange of dimers as well as phosphorylation, acetylation, ubiquitination and prolyl isomerization of the transactivating p65/RelA subunit [33]. USP31 could participate in the regulation of NF-nB in the nucleus in addition to a possible role in the cytoplasmic events that lead to NF-nB activation. Further characterization of the role of USP31 in TRAF- and TNFR-mediated signal transduction will require the identification of the optimal substrate for USP31 and the inactivation of the usp31 gene in mice.
Acknowledgements This work was supported by the Greek Secretariat of Research and Technology (Career Development Award for Greek-speaking scientists returning to Greece to EH, program PENED 1999 to George Mosialos), an International Scholarship from the Howard Hughes Medical Institute (to George Mosialos), a Human Frontier Science Program grant (to George Mosialos), a graduate student stipend from State Scholarship Foundation of Greece (to CT) and US National Institute of Health grant CA47006 (to EK). George Mosialos is a Leukemia and Lymphoma Society of America Scholar.
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