Báo cáo khoa học: KCTD5, a putative substrate adaptor for cullin3 ubiquitin ligases docx

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KCTD5, a putative substrate adaptor for cullin3ubiquitin ligasesYolanda Bayo´n1, Antonio G. Trinidad1, Marı´aL. de la Puerta1, Marı´adel Carmen Rodrı´guez1,Jori Bogetz2, Ana Rojas3, Jose´M. De Pereda4, Souad Rahmouni5, Scott Williams2,Shu-ichi Matsuzawa6, John C. Reed6, Mariano Sa´nchez Crespo1, Tomas Mustelin2andAndre´s Alonso11 Instituto de Biologı´a y Gene´tica Molecular, CSIC-Universidad de Valladolid, Spain2 Program of Inflammation, Inflammatory and Infectious Disease Center, and Program of Signal Transduction, Burnham Institute for MedicalResearch, La Jolla, CA, USA3 Structural Bioinformatics Group, Centro Nacional de Investigaciones Oncolo´gicas, Madrid, Spain4 Centro de Investigacio´n del Ca´ncer, CSIC-Universidad de Salamanca, Spain5 Department of Pathology B-35, University of Lie`ge, CHU of Lie`ge, Belgium6 Program of Apoptosis and Cell Death, Burnham Institute for Medical Research, La Jolla, CA, USAThe BTB (bric-a-brac, tramtrak and broad com-plex) ⁄ POZ (poxvirus zinc finger) domain is a protein–protein interaction domain first described in severalproteins of Drosophila melanogaster and poxvirus [1,2].BTB ⁄ POZ domain-containing proteins constitute adiverse group of proteins involved in transcriptionalrepression, cytoskeletal regulation, and ion channelfunction [3]. More recently, some BTB proteins havebeen characterized as substrate-specific adaptors forcullin(CUL)3-based E3 ligases [4–7]. The BTB domainof these substrate-specific adaptors binds to CUL3,whereas additional domains in these polypeptides, suchas zinc fingers, meprin and traf homology (MATH)domain, and Kelch repeats, work as substrate recogni-tion domains. The first protein shown to be regulatedby a CUL3 ligase was MEI-1 in Caenorhaditis elegans.This protein is part of the katanin-like microtubulesevering complex [5,6] and is recruited to CUL3 by theKeywordsBTB; cullin; E3 ligases; KCTD; ubiquitinCorrespondenceA. Alonso, Instituto de Biologı´a y Gene´ticaMolecular, CSIC-Universidad de Valladolid,c ⁄ Sanz y Fore´ss⁄ n, 47003 Valladolid, SpainFax: +34 983 184800Tel: +34 983 184839E-mail: andres@ibgm.uva.es(Received 7 April 2008, revised 30 May2008, accepted 3 June 2008)doi:10.1111/j.1742-4658.2008.06537.xPotassium channel tetramerization domain (KCTD) proteins contain abric-a-brac, tramtrak and broad complex (BTB) domain that is most simi-lar to the tetramerization domain (T1) of voltage-gated potassium chan-nels. Some BTB-domain-containing proteins have been shown recently toparticipate as substrate-specific adaptors in multimeric cullin E3 ligase reac-tions by recruiting proteins for ubiquitination and subsequent degradationby the proteasome. Twenty-two KCTD proteins have been found in thehuman genome, but their functions are largely unknown. In this study, wehave characterized KCTD5, a new KCTD protein found in the cytosol ofcultured cell lines. The expression of KCTD5 was upregulated post-trans-criptionally in peripheral blood lymphocytes stimulated through the T-cellreceptor. KCTD5 interacted specifically with cullin3, bound ubiquitinatedproteins, and formed oligomers through its BTB domain. Analysis of theinteraction with cullin3 showed that, in addition to the BTB domain, someamino acids in the N-terminus of KCTD5 are required for binding tocullin3. These findings suggest that KCTD5 is a substrate-specific adaptorfor cullin3-based E3 ligases.AbbreviationsAU, arbitrary unit; BTB, bric-a-brac, tramtrak and broad complex; CT, cycle threshold; CUL, cullin; GFP, green fluorescent protein; GST,glutathione S-transferase; HA, hemagglutinin; IL-2, interleukin-2; KCTD, potassium channel tetramerization domain; MATH, meprin and trafhomology; PBL, peripheral blood lymphocyte; PHA, phytohemagglutinin; PMA, 4b-phorbol 12-myristate 13-acetate; POZ, poxvirus zinc finger;Ub, ubiquitin.3900 FEBS Journal 275 (2008) 3900–3910 ª 2008 The Authors Journal compilation ª 2008 FEBSBTB protein MEL-26. In mammalian cells, a few otherBTB proteins, e.g. SPOP, a BTB-MATH protein, andKEAP1, a BTB-KELCH protein, have been describedas adaptors of CUL3-based E3 ligases [8]. CUL3 isone of the seven cullins found in the human genome(CUL1, CUL2, CUL3, CUL4A, CUL4B, CUL5 andCUL7), and most of them bind to adaptors throughtheir BTB domains, which, in turn, bind to additionalproteins that work as substrate-specific adaptors. Thus,in SKP1–CUL1–F-box, the archetypical cullin E3ligase, CUL1 binds on the N-terminus to the adaptorSkp1 that associates with an F-box protein working assubstrate-specific adaptor, and on the C-terminus tothe RING domain-containing protein Roc1 ⁄ Rbx ⁄ Hrt[9]. Cullin E3 ligases are multimeric RING E3 ligasesthat participate in protein ubiquitination, a processmediated by a three-step enzymatic cascade. Ubiquitin(Ub) is initially activated by the Ub-activating enzyme(E1) and then transferred to a Ub-conjugating enzyme(E2), which associates with a third protein, the Ubligase (E3), involved in recruiting the substrates forubiquitination and, therefore, providing specificity tothis process [10]. Ubiquitination is involved in a widerange of cellular functions, such as cell proliferation,differentiation, and apoptosis, mainly by targeting pro-teins for degradation by the 26S proteasome, but it isalso involved in protein transport and signalingthrough additional mechanisms [10,11].Although the human genome might include about400 BTB proteins [8], only a few have been shown towork as substrate-binding proteins for CUL3 E3 ligases.In this connection, potassium channel tetramerizationdomain (KCTD) proteins form a group of proteinscontaining a BTB domain, the function of which is lar-gely unknown. Herein, we report the characterizationof KCTD5, a new POZ ⁄ BTB protein that is a putativenew substrate-specific adaptor for CUL3-based E3ligases.Results and DiscussionKCTD5 was identified in a yeast two-hybrid screeningfor the dual-specificity phosphatase VHR while look-ing for adaptors that help us to understand how thisphosphatase targets its substrates, Erk and Jnk. Theclone obtained in this assay contained a cDNAsequence present in public databases with the Genbankaccession number NM_018992. Next, we tested VHRinteraction with KCTD5 in mammalian cells, andcould not find evidence for this. Nevertheless, we con-tinued the study of this new protein. First, we studiedthe expression of this gene, finding that its mRNA wasexpressed in all the tissues and cell lines tested(Fig. 1A). On the contrary, protein expression wasonly observed in transformed cells and was absentfrom primary cells, such as peripheral blood leukocytes(PBLs), mouse brain cells or human brain cells(Fig. 1B, lanes 6, 9 and 10), thus suggesting that itsexpression was upregulated post-transcriptionally.Prompted by these results, especially by the differencesobserved between the expression of mRNA and pro-tein in PBLs, we hypothesized that KCTD5 might beinduced by mitogens such as as phytohemagglutinin(PHA) and interleukin-2 (IL-2) in these cells. Usingthese stimuli, we observed a 2.5-fold increase (Fig. 1D,lane 5) in mRNA expression and an 84.7-fold increasein protein expression at 48 h (Fig. 1C, lane 7) in PBLsstimulated with PHA. To investigate whether otherstimuli known to induce T-cell proliferation increaseKCTD5 protein, 4b-phorbol 12-myristate 13-acetate(PMA) plus ionomycin and a combination of antibod-ies for the T-cell and CD28 receptors, which mimicantigen stimulation, were used. As shown in Fig. 1E,these stimuli increased KCTD5 protein to an extentsimilar to that observed for PHA. As RT-PCR assayslack enough sensitivity to detect changes in the amountof mRNA, quantitative PCR assays were conducted inorder to detect subtle changes in KCTD5 mRNA. Asshown in Fig. 1F, there was only a slight decrease ofKCTD5 mRNA after PHA stimulation of PBLs. Thesedata suggested that these stimuli regulated KCTD5 ata post-transcriptional level, by increasing either thetranslation or the stability of KCTD5 protein. In thelatter case, this would imply that KCTD5 is an unsta-ble protein in the absence of stimuli. In this regard,treatment of resting PBLs with MG132, a proteasomeinhibitor, has no effect on KCTD5 protein (data notshown), meaning that stimulus-dependent translation isinvolved in increasing the quantity of KCTD5 proteinin PBLs. Altogether, these data suggest that KCTD5expression is mainly regulated by a post-transcriptionalmechanism in PBLs, possibly at the translational level.Several databases were searched to find homologs ofKCTD5, using as query its BTB domain. Although theBTB domain is present in proteins from all eukaryoticgroups, when the query included the KCTD5 C-termi-nal region in addition to the BTB domain, homologswere only found among the metazoans. However, noprotein was found with a BTB domain followed by theC-terminus of KCTD5 in plants and fungi. An align-ment of KCTD5 orthologs in several species is shownin Fig. 2A. Among BTB proteins, KCTD5 is groupedwith potassium channels. The similarity with potas-sium channels is restricted to the T1 domain, which isa BTB domain. Whereas cullins are present in alleukaryotes, KCTD5-like proteins appeared later inY. Bayo´n et al. KCTD5, a new substrate-specific adaptor for Cul3FEBS Journal 275 (2008) 3900–3910 ª 2008 The Authors Journal compilation ª 2008 FEBS 3901evolution in multicellular organisms, most likely to ful-fil a new function, which is at the present timeunknown. Searches for human paralogs, using as querythe BTB domain of KCTD5 to generate a phylogenetictree (Fig. 2B), gave 22 sequences. Some of thesehuman paralogs are found in highly similar groupswith conserved sequences out of the BTB domain usedfor this analysis, e.g. the group constituted by KCTD5,KCTD2, and KCTD17. Elements recently cloned havebeen included in two groups: (a) the group formed bypolymerase d and proliferating cell nuclear antigen-interacting proteins, tumor necrosis factor-a-inducedprotein 1 [12], KCTD13 product polymerase delta-interacting protein 1 [12], and KCTD10 [13]; and (b)the group formed by the leftover-related proteinsKCTD8, Pfetin (predominantly fetal expressed T1domain) (KCTD12), and KCTD16, which are involvedin development [14]. For the remaining sequences thereare clear paralogy relationships, which indicate closerelationships within the sequences, as in the case ofKCTD3 and Q8TBC3, a human homolog of mouseseta-binding protein-1 [15], KCTD1 and KCTD15,and KCTD21 and KCTD6. Most of these sequencesremain uncharacterized. This analysis of KCTDsequences shows that they form a group clearly differ-entiated from the voltage-gated potassium channels,not only by the absence of transmembrane domains,but also on the basis of the differences in BTBsequences.To determine the subcellular localization of KCTD5,green fluorescent protein (GFP)–KCTD5 was trans-fected and detected by confocal microscopy (Fig. 3A).Whereas GFP alone is found in the nucleus as well asin the cytosol, fusion of KCTD5 to GFP restricts theexpression of the fusion protein, GFP–KCTD5, to thecytosol. Furthermore, HEK293 cells were transfectedwith a plasmid expressing myc–KCTD5, and thisprotein was detected by immunocytochemistry in thecytosol (Fig. 3B). As it has been recently reported thatdeletion of the C-terminus of KCTD5 [16] changes itslocation to the nucleus, cells were transfected withdifferent deletion mutants of KCTD5. Immunocyto-chemistry of these cells showed that these constructswere again detected in the cytosol (Fig. 3B). Therefore,in our hands, KCTD5 is detected only in the cytosol.As we had a specific antibody for KCTD5, we triedseveral times to reveal the endogenous protein withthis antibody, but we could not see any specific bind-ing, so we consider that this antibody is not suitablefor immunocytochemistry.Although it has been proposed that all the proteinscontaining a BTB domain are substrate-specificFig. 1. KCTD5 expression. (A) RNA from different tissues and cell lines was analyzed by RT-PCR using specific primers for KCTD5. A plas-mid encoding KCTD5 was used as a positive control (lane 1) for the RT-PCR. (B) Expression of KCTD5 in different cell types detected byimmunoblot with antibody to KCTD5 (upper panel). b-Actin was detected by immunoblot on the same membrane as an internal control ofprotein loading (lower panel). (C) Time course of the expression of KCTD5 protein in PBLs stimulated with PHA. (D) Time course of theexpression of KCTD5 mRNA in PBLs stimulated with PHA, where numbers indicate hours of stimulation. (E) Expression of KCTD5 protein inPBLs subjected to various stimuli. (F) Levels of KCTD5 mRNA assayed by quantitative PCR in PBLs stimulated with PHA. TCR+CD28 indi-cates antibodies specific for T-cell receptor plus CD28.KCTD5, a new substrate-specific adaptor for Cul3 Y. Bayo´n et al.3902 FEBS Journal 275 (2008) 3900–3910 ª 2008 The Authors Journal compilation ª 2008 FEBSadaptors for cullin ubiquitin ligases [5,6], in the case ofCUL3, most of the adaptors described so far belong tothe Kelch group. Thus, we investigated whetherKCTD5 could interact with CUL3. To test this inter-action, HEK293 cells were transfected with plasmidsencoding CUL1, CUL2, CUL3, CUL4A and CUL4BABFig. 2. Analysis of KCTD5 homologs. (A) Multiple protein sequence alignment of various KCTD5 orthologous sequences from different spe-cies. (B) Phylogenetic tree built from human paralogs of KCTD5 using the BTB domain of 23 peptides. The BTB domain (T1 domain) of thevoltage-gated potassium channel KCNC1 protein is included in the analysis to root the tree.Y. Bayo´n et al. KCTD5, a new substrate-specific adaptor for Cul3FEBS Journal 275 (2008) 3900–3910 ª 2008 The Authors Journal compilation ª 2008 FEBS 3903along with KCTD5. Total lysates were prepared fromthese cells and used for immunoprecipitation assays. Aspecific interaction of KCTD5 with CUL3 wasobserved (Fig. 4A, lane 6), but not with the other cul-lins (Fig. 4A, lane 5 for CUL1 and data not shown).This interaction was confirmed in primary cells by car-rying out immunoprecipitation assays in lysates fromPBLs stimulated with PHA for 2 days. Under theseconditions, CUL3 was detected by immunoblot inKCTD5 precipitates (Fig. 4B, lane 2), but not whenthe immunoprecipitation was carried out with an irrel-evant antibody (Fig. 4B, lane 1). Then, the ability toform a functional E3 ligase complex with CUL3 andRbx1 was assayed. Expression vectors for these pro-teins were transfected into HEK293 cells, and celllysates were subjected to immunoprecipitation withantibody to myc. As KCTD5 was precipitated whenCUL3 was present in the lysate (Fig. 4C, lane 5), thisresult indicates that KCTD5 is part of a canonical cul-lin-based E3 ligase complex. A faint band is also seenin Fig. 4C (lane 2) that is probably due to the interac-tion of Rbx1 with endogenous CUL3.We also addressed whether KCTD5 could be ubiqui-tinated, based on the fact that other BTB adaptor pro-teins have been shown to be substrates of E3 ligases.To do this, we transfected cells with expression vectorsfor myc–Ub and hemagglutinin (HA)–KCTD5, andcell lysates were immunoprecipitated with an antibodyspecific for HA. The precipitates showed the presenceof ubiquitinated proteins (Fig. 4D) by immunoblotting.To distinguish between covalent and noncovalent Ubbinding to KCTD5, we repeated this experiment, lys-ing the cells with a highly denaturing buffer containing8 m urea. Under these conditions, no smear wasdetected in the KCTD5 immunoprecipitation and norwas a KCTD5 ladder observed in Ub precipitates,which is typical of ubiquitinated proteins (data notshown). In addition, we could not detect KCTD5ubiquitination in in vitro assays (data not shown).Thus, unlike to what has been described for otherBTB proteins that work as substrate-specific adaptors,KCTD5 is not ubiquitinated.The interaction between BTB proteins and CUL3 isconsidered to be mediated by the BTB domain andthe N-terminal region of CUL3 [8], mainly on thebasis of assays in which deletion of the BTB domainand the N-terminal region of CUL3 is accompaniedby loss of binding. To analyze in detail KCTD5 bind-ing to CUL3, pull-down and immunoprecipitationassays with a series of deletion mutants of KCTD5and CUL3 were carried out (Fig. 5A,C). These exper-iments showed that the C-terminal region of KCTD5was dispensable for CUL3 interaction, whereas theBAFig. 3. Subcellular localization of KCTD5. (A) Left panels: fluores-cence images of HEK293 cells transfected with either GFP or GFP–KCTD5. Right panels: phase contrast images of the same cells. (B)Immunofluorescence staining of HEK293 cells transfected withplasmids encoding KCTD5 and several deletion mutants with mAbto myc followed by a secondary antibody labeled with AlexaFluor 594.KCTD5, a new substrate-specific adaptor for Cul3 Y. Bayo´n et al.3904 FEBS Journal 275 (2008) 3900–3910 ª 2008 The Authors Journal compilation ª 2008 FEBSBTB domain alone (45–145 amino acids), althoughessential for this interaction, was not sufficient(Fig. 5B). In fact, it required additional amino acids(40–45) on the N-terminus, outside of the BTB fold,as the 40–145 amino acid peptide is the smallest moi-ety able to interact with CUL3. Studies with otherBTB proteins, e.g. SPOP [17]or the BTB proteinAt1g21780 from Arabidopsis thaliana [18], have alsoshown that other parts of their sequence, in additionto the BTB domain, are involved in the associationwith CUL3. On the other hand, the CUL3 regioninvolved in this interaction was the N-terminus, asdescribed for other BTB proteins, because a deletionof 75 amino acids in the N-terminus of CUL3 com-pletely abrogated the binding of KCTD5 to CUL3(Fig. 5D, lane 8). Therefore, this detailed study onthe interaction of KCTD5 with CUL3 shows that thesole BTB domain of KCTD5 does not support thisFig. 4. KCTD5 interacts with CUL3 and ubiquitinated proteins. (A) HEK293 cells were transfected with plasmids encoding myc–KCTD5, HA–CUL1, and HA–CUL3, as indicated. Cell lysates were subjected to immunoprecipitation (IP) with antibody to myc followed by immunoblottingwith antibodies to HA and myc. Expression of the tagged proteins is shown in the lower panels as WCL (whole cell lysate). (B) Lysates fromPBLs treated with PHA for 2 days (upper panel) were immunoprecipitated with either KCTD5 or an irrelevant IgG antibody and then blottedwith antibodies to CUL3 (upper panel) and KCTD5. The panels marked WCL show the expression levels of KCTD5 and CUL3 in the PBLwhole cell lysates. (C) HEK293 cells were transfected with plasmids encoding myc–Rbx1, HA–CUL3 and GST–KCTD5, lysates from thesecells were processed for pull-down with GST beads, and the presence of KCTD5 in the precipitates was checked by western blotting withantibody to GST, followed by anti-HA and anti-myc blots to detect HA–CUL3 and myc–Rbx1. WCLs were immunoblotted with antibodies toGST, HA and myc to assess the expression of the tagged proteins. (D) HEK293 cells were transfected with plasmids encoding forHA–KCTD5 and myc–ubiquitin. Cell lysates were immunoprecipitated with antibody to HA, and ubiquitinated proteins that interact withKCTD5 were detected with antibody to myc. WCLs were immunoblotted with the antibodies to HA and myc to assess the expression ofthe tagged proteins.Y. Bayo´n et al. KCTD5, a new substrate-specific adaptor for Cul3FEBS Journal 275 (2008) 3900–3910 ª 2008 The Authors Journal compilation ª 2008 FEBS 3905association and requires additional amino acids in theN-terminus of this domain.As the BTB domain is responsible for homo-oligo-merization in BTB proteins [3], we addressed whetherKCTD5 might form homo-oligomers. For this purpose,HEK293 cells were transfected with different constructsof KCTD5 to show this association by either immuno-precipitation or pull-down assays (Fig. 6A,B). We foundthe BTB domain to be essential for KCTD5 oligomeri-zation, as peptides expressing the KCTD5 N-terminalregion (N55) or the C-terminal sequence (POZCO,amino acids 145–234) could not interact with themselves(Fig. 6B). As the POZ ⁄ BTB domain of KCTD5 is dis-tantly related to the T1 domain of voltage-gated potas-sium channels, this fact was taken as a hint that KCTD5could also tetramerize. To address this issue, gel exclu-sion chromatography was run with recombinantKCTD5 protein and KCTD5 was collected in fractionsconsistent with the estimated molecular mass of an oct-amer (Fig. 6C), which in turn can be explained by theformation of two tetramers.Taken together, our results show that the BTBdomain of KCTD5 is not able to bind alone to CUL3,indicating that although it is critical for this associa-tion, other sequences contribute to the binding of sub-strate-specific adaptors to CUL3, namely, five aminoacids in the N-terminus of the BTB domain. In addi-tion to the BTB fold, KCTD5 presents two otherregions: 40 amino acids in the N-terminal sequence,which include a low-complexity region (12–33 aminoFig. 5. Mapping the interaction of KCTD5 with CUL3. (A) Schematic diagram of the several KCTD5 deletion mutants used in this study. (B)Plasmids for KCTD5 and different deletion mutants expressed as GST fusion proteins were transfected, along with HA–CUL3, in HEK293cells. Lysates were subjected to pull-down assays with glutathione–Sepharose beads, and the presence of CUL-3 in the precipitates wasdetected by immunoblot with antibody to HA, followed by antibody to GST. The expression of the proteins was checked in the whole celllysate (WCL) by western blot with antibodies to HA and CUL3. (C) Schematic diagram of the CUL3 deletion mutants used in this study. (D)myc–KCTD5 was expressed in HEK293 cells along with different deletion mutants of HA–CUL3. The presence of the different CUL3 pep-tides was checked in the myc immunoprecipitates by western blot with an antibody to HA. myc–KCTD5 was detected in the immunoprecipi-tates by immunoblot with antibody to myc. The same antibodies were used to show the expression in the WCL (lower panels).KCTD5, a new substrate-specific adaptor for Cul3 Y. Bayo´n et al.3906 FEBS Journal 275 (2008) 3900–3910 ª 2008 The Authors Journal compilation ª 2008 FEBSacids), and 88 amino acids in the C-terminus (PO-ZCO). Taking into account that KCTD5 could be anadaptor of CUL3 E3 ligases, we favor the hypothesisthat the POZCO region could participate in substraterecognition, and that this could be a new protein inter-action domain conserved through evolution, as seen inorthologs. The fact that KCTD5 can form octamersand the recent description of heterodimerization ofCUL3 [19] would indicate that complexes of higherorder could be formed among CUL3 and BTBsubstrate adaptors, implying the recruitment of a greatnumber of substrates by these E3 ligases.Although scarce, the information available aboutKCTD proteins suggests that these proteins might beinvolved in development and cellular differentiation.For example, in zebra fish, three members of this group– lov (leftover), ron (righton), and dex (dexter) – areexpressed asymmetrically in the left and right zebrafishdiencephalons [14]. Pfetin, a human ortholog of lov andron genes, encoded by human gene KCTD12, is detectedas mRNA preferentially expressed in fetal organs [20],with the highest expression levels in the cochlea.Another KCTD protein, KCTD11 ⁄ REN, is also regu-lated developmentally in the nervous system [21], and ithas been implicated in the regulation of the Hedgehogpathway [22]. The information presented in this articlewould indicate that KCTD proteins might function byrecruiting specific substrates involved in developmentand cellular differentiation for ubiquitination by CUL3Ub ligases and degradation by the proteasome. Asregards KCTD5, there is another report that shows itsability to interact with two viral regulatory proteins,Rep68 and Rep78, of the adeno-associated virus type 2,which are essential for viral DNA replication and geneexpression [16], although no relationship was establishedwith CUL3.In summary, in this study we present evidence thatKCTD5 is a new substrate-specific adaptor for CUL3-based Ub ligases. Our data indicate that a relevantmechanism underlying the physiological role of KCTDproteins includes recruitment of proteins to CUL3-based E3 Ub ligases for degradation in the protea-some. As identification of substrates recruited to theproteasome would be very valuable for understandingthe function of these proteins, we are pursuingthe identification of KCTD5-interacting proteins,especially those that are ubiquitinated.Experimental proceduresAntibodies and reagentsTissue culture reagents were from Cambrex (Verviers,Belgium). The 12CA5 mAb against HA was from Roche(Indianapolis, IN, USA), anti-HA clone HA.11 was fromCovance (Berkely, CA, USA), anti-glutathione S-transferase(GST) and mAb against myc (9E10) were from Santa CruzFig. 6. KCTD5 oligomerization. (A) HEK293 cells were transfectedwith plasmids encoding for GST–KCTD5 and different deletionmutants of KCTD5, and cell lysates were subjected to pull-downwith Glutathione–Sepharose beads and immunoblotted with a spe-cific antibody to FLAG followed by antibody to GST. (B) HEK293cells were transfected with HA–KCTD5 and several plasmids thatexpressed different deletion mutants of KCTD5 as GST-fusion pro-teins. Anti-HA immunoprecipitates of the cell lysates were analyzedby immunoblot with antibody to GST followed by antibody to HA.(C) Gel filtration chromatography of KCTD5 recombinant proteinproduced in bacteria. The presence of KCTD5 in the fractions wasanalyzed by immunoblot with antibody to KCTD5. Numbers underthe arrows indicate the chromatography fractions in which mole-cular mass markers are eluted.Y. Bayo´n et al. KCTD5, a new substrate-specific adaptor for Cul3FEBS Journal 275 (2008) 3900–3910 ª 2008 The Authors Journal compilation ª 2008 FEBS 3907Biotechnology Inc. (Santa Cruz, CA, USA), anti-cullin 3 wasfrom Abcam (Cambridge, UK), and mAbs against b-actin,PHA, FLAG M2 mAb and PMA were from Sigma ChemicalCo. (St Louis, MO, USA). Antibodies against CD3(UCHT1) and CD28 (clone CD28.2) were from BD Pharm-ingen (Franklin Lakes, NJ, USA). MG-132 was from Calbio-chem (Darmstadt, Germany). IL-2 was from PreprotechEC(Rocky Hill, NJ, USA). Goat anti-(mouse IgG) conjugatedwith Alexa FluorÒ 594 was from Molecular Probes (Eugene,OR, USA). A mouse mAb was raised against recombinantfull-length KCTD5. Human MTC panel II was fromClontech (Mountain View, CA, USA).Plasmids and mutagenesisStandard molecular biology techniques were used to gener-ate the different constructs used in this study. All constructswere verified by nucleotide sequencing. KCTD5 from aJurkat cDNA library obtained from Origene (Rockville,MD, USA) was cloned in the pEF plasmid and served as atemplate for the different KCTD5 plasmids used in thisstudy. HA–cullin1 and HA–cullin3 expression plasmidswere a kind gift of C. Geisen (Department of MedicalOncology, Dana-Farber Cancer Institute, Boston, MA,USA). Cullin4A and cullin4B were generously provided byK. Tanaka (Department of Molecular Oncology, TokyoMetropolitan Institute of Medical Science, Japan) [23].Cell culture and transfectionsPBLs were isolated from buffy coats of healthy donors bycentrifugation at 700 g for 30 min on Ficoll–Hypaque (GEHealthcare) cushions. Monocytes ⁄ macrophages were elimi-nated by adherence to plastic for 1 h at 37 °C. Proliferationwas induced by PHA and IL-2, which was added after 48 hwith PHA, antibodies to CD3 plus antibodies to CD28, orPMA plus ionomycin. Jurkat T-leukemia cells were kept atlogarithmic growth in RPMI-1640 medium supplementedwith 10% fetal bovine serum, 2 mml-glutamine, 1 mmsodium pyruvate, nonessential amino acids, 100 UÆmL)1penicillin G, and 100 lgÆmL)1streptomycin. Transfectionof Jurkat T cells was performed as described previously[24]. HEK293 cells were maintained at 37 °C in DMEMsupplemented with 10% fetal bovine serum, 2 mml-gluta-mine, 100 UÆmL)1penicillin G, and 100 lgÆmL)1strepto-mycin. For transient transfection, HEK293 cells weretransfected using the calcium phosphate precipitationmethod [25].Immunoprecipitation, GST pull-down,SDS⁄PAGE, and immunoblottingThese procedures were performed done as reported previ-ously [24]. Briefly, cells were lysed in 20 mm Tris ⁄ HCl,pH 7.5, 150 mm NaCl, 5 mm EDTA containing 1% NP-40,1mm Na3VO4,10lgÆmL)1aprotinin and leupeptin, and1mm phenylmethanesulfonyl fluoride, and clarified bycentrifugation at 16 000 g for 10 min. The clarified lysateswere preabsorbed on protein G-Sepharose and then incu-bated with antibody for 2 h; this was followed by overnightincubation with protein G-Sepharose beads. Immune com-plexes were washed three times in lysis buffer and resus-pended in SDS sample buffer. Proteins resolved bySDS ⁄ PAGE were transferred to a nitrocellulose membrane,and immunoblotted with optimal dilutions of specific anti-bodies followed by the appropriate anti-IgG–peroxidaseconjugate. Blots were developed by the enhanced chemilu-minescence technique (ECL kit; GE Healthcare) accordingto the manufacturer’s instructions. Pull-down of GSTfusion proteins was performed with glutathione–Sepharosebeads (GE Healthcare) incubated with the clarified lysatesfor 2 h. The complexes were then washed and processed asexplained above for the immunoprecipitation. Some blots,after being developed by chemiluminescence, were visual-ized with a Bio-Rad VersaDoc chemiluminescence imager.In this case, quantitation was carried out using quantityone software from Bio-Rad.RT-PCRTotal cellular RNA was extracted by the TRIzol method(Life Technologies, Grand Island, NY, USA). The condi-tions for cDNA first-strand synthesis and PCR reactionswere as described previously [26]. To address more exactlythe expression of KCTD5 mRNA, real-time RT-PCR wascarried out in RNA samples treated with DNase (Turbo-DNA freeTM; Ambion, Austin, TX, USA). The resultingcDNA was amplified in a PTC-200 apparatus equippedwith a Chromo4 detector (BioRad Laboratories), usingSYBR Green I mix containing HotStart polymerase(ABgene, Epsom, UK). b-Actin was used as a housekeepinggene to assess the relative abundance of KCTD5 mRNA,using the comparative cycle threshold (CT) method forrelative expression. This method allows the relativeexpression for a given cDNA using the formula: 2)DCT,where DCT¼ DCKCTD5TÀ DCbÀactinT[27]. Therefore, one arbi-trary unit (AU) corresponds to the expression of b-actin.Indirect immunofluorescence and confocalmicroscopyHEK293 cells were cultured on coverslips and transientlytransfected with the indicated plasmids. Cells transfectedwith GFP plasmids were fixed with 3.7% paraformaldehydeand mounted on microscope slides, and GFP was then visu-alized on an MRC-1024 confocal laser scanning microscope(Bio-Rad). Phase contrast images were also taken. Immuno-fluorescence staining of transfected KCTD5 was performedKCTD5, a new substrate-specific adaptor for Cul3 Y. Bayo´n et al.3908 FEBS Journal 275 (2008) 3900–3910 ª 2008 The Authors Journal compilation ª 2008 FEBSas described previously [24]. HEK293 cells were washed inNaCl ⁄ Pi, fixed in 3.7% formaldehyde, permeabilized with0.1% saponin in NaCl ⁄ Pi, and blocked in the same mediumsupplemented with 2.5% normal goat serum for 30 min atroom temperature. Primary and secondary antibodies werediluted in the same buffer and incubated with the cells for 1 heach at room temperature. After three washes with NaCl ⁄ Pi,the cells were mounted onto glass slides and viewed under aconfocal laser scanning microscope.Gel filtration chromatographyFor gel filtration chromatography, we used recombinantKCTD5 produced in bacteria as His6-KCTD5 afterremoval of the His-tag with thrombin. The protein solutionwas fractionated through a Superdex 200 fast protein liquidchromatography column (GE Healthcare), and collected infractions of 500 lL. Protein was precipitated with 10% tri-chloroacetic acid and washed with acetone before additionof SDS sample buffer and analysis by 10% SDS ⁄ PAGE.Sequence analysis and alignmentsFor sequence retrieval, the BTB domain of human KCTD5was used as query to retrieve the orthologs from theUniPROT (http://www.ebi.uniprot.org/index.shtml) data-base using the blast algorithm [28]. psi-blast [29] searchesretrieved 22 human paralogs. Multiple sequence alignmentsof the BTB domain were conducted using muscle [30] andprobcons [31] in both the orthologs and the paralogs. Togenerate reliable phylogenetic trees, Bayesian inferenceusing mrbayes v3.1.2 software was applied [32]. Multiplealignments were done in two independent runs, with fourindependent Markov chains in each run. One thousand fivehundred samples were used to estimate the posterior proba-bility distribution. The amino acid model is a fixed ratemodel using a mixture of fixed models. To compute a con-sensus tree, we sampled 2502 from a total of 3002 trees intwo independent files (thus discarding 16% of the initialsamples prior to convergence). To root the tree, thesequence of the BTB domain (T1) of the voltage potassiumchannel KCNC1_HUM is included in the analysis.AcknowledgementsWe are grateful to Dr Keiji Tanaka for the CUL4Aand CULB cDNAs, to Dr Cristoff Geisen for theCUL1 and CUL3 plasmids, and to Dr Joan Conawayfor the myc–Rbx1 plasmid. We thank the staff ofCentro de Hemoterapia y Hemodonacio´n de Castilla yLeo´n for its help with the separation of leukocytes.This work was supported by a grant from ProgramaNacional de Biologı´a Fundamental (Grant BFU2006-01203 ⁄ BMC), Red Cardiovascular from Instituto deSalud Carlos III. Y. 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