The Translation Initiation Factor 3 Subunit Eif3k Interacts with PML and Associates with PML Nuclear Bodies

EXPERIMENTAL CELL RESEARCH 319 (2013) 2554– 2565

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Research Article The translation initiation factor 3 subunit eIF3K interacts with PML and associates with PML nuclear bodies

Jayme Salsmana, Jordan Pindera, Brenda Tsea, Dale Corkeryb, Graham Dellairea,b,n aDepartment of Pathology, Dalhousie University, P.O. Box 15000, Halifax, Nova Scotia, Canada B3H 4R2 bDepartment of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada article information abstract

Article Chronology: The promyelocytic leukemia protein (PML) is a tumor suppressor protein that regulates a variety Received 11 December 2012 of important cellular processes, including gene expression, DNA repair and cell fate decisions. Received in revised form Integral to its function is the ability of PML to form nuclear bodies (NBs) that serve as hubs for the 8 August 2013 interaction and modification of over 90 cellular proteins. There are seven canonical isoforms of Accepted 2 September 2013 PML, which encode diverse C-termini generated by alternative pre-mRNA splicing. Recruitment of Available online 11 September 2013 specific cellular proteins to PML NBs is mediated by protein–protein interactions with individual Keywords: PML isoforms. Using a yeast two-hybrid screen employing peptide sequences unique to PML fi PML isoform I (PML-I), we identi ed an interaction with the eukaryotic initiation factor 3 subunit K fi eIF3K (eIF3K), and in the process identi ed a novel eIF3K isoform, which we term eIF3K-2. We further Nuclear structure demonstrate that eIF3K and PML interact both in vitro via pull-down assays, as well as in vivo fl Light microscopy within human cells by co-immunoprecipitation and co-immuno uorescence. In addition, eIF3K Yeast two-hybrid isoform 2 (eIF3K-2) colocalizes to PML bodies, particularly those enriched in PML-I, while eIF3K isoform 1 associates poorly with PML NBs. Thus, we report eIF3K as the first known subunit of the eIF3 translation pre-initiation complex to interact directly with the PML protein, and provide data implicating alternative splicing of both PML and eIF3K as a possible regulatory mechanism for eIF3K localization at PML NBs. & 2013 Elsevier Inc. All rights reserved.

Introduction including tumor suppression, DNA repair, apoptosis, antiviral response, cellular senescence, and transcriptional regulation The human promyelocytic leukemia (PML) protein is an important [4,13,37,45,60]. While PML is the major structural component of tumor suppressor protein, and mice in which the PML gene is the PML NB, more than 90 different proteins can associate with disrupted are more susceptible to carcinogen-induced cancers PML NBs, either transiently or constitutively [15,40,54] and many [5,13,56]. The PML protein acts as the molecular scaffold for the of these proteins themselves are intimately linked to the various formation of promyelocytic leukemia nuclear bodies (PML NBs), functions ascribed to PML NBs. One of the prominent functions which are discrete, dynamic subnuclear structures found within associated with PML and PML NBs is tumor suppression, since loss mammalian cells [3]. These heterogeneous multi-protein com- of the PML protein and/or PML NBs has been associated with plexes are implicated in a wide spectrum of cellular functions acute promyelocytic leukemia [12,32] and various human solid

nCorresponding author at: Dalhousie University, Departments of Pathology, P.O. Box 15000, Halifax, Nova Scotia, Canada B3H 4R2. Fax: þ1 902 494 2519. E-mail address: [email protected] (G. Dellaire).

tumors including carcinomas of the colon, lung, breast and manner or in response to various stimuli [10]. Thus, it is likely that prostate [25,55]. the diversity of cellular functions attributed to the PML NB arise, The PML gene transcript is generated from nine exons, of which at least in part, via equally diverse protein–protein interactions exons 6–9 are spliced alternatively to yield at least seven PML mediated by the various PML isoforms. isoforms with unique C-terminal sequences [20,29]. Exons 1–3 are In this study, we used a yeast two-hybrid screen to identify a conserved in all PML isoforms and encode a tripartite motif novel protein–protein interaction between peptide sequences (TRIM) containing a RING finger, two B-boxes and an α-helical unique to PML-I, encoded by exon 9 of the PML gene, and subunit coiled-coil motif [44] which are involved in mediating homo- and K of the eukaryotic translation initiation factor 3 (eIF3K). In hetero-association between PML isoforms [6,22]. Several motifs addition, we show that eIF3K also interacts with the PML protein have also been identified in the C-terminus of PML, including a in an exon 9-independent manner, and localizes to PML NBs. nuclear localization signal in exon 6 [30] and a nuclear export Furthermore, the recruitment of eIF3K to PML NBs appears to be signal in exon 9 of PML isoform I (PML-I) [26,29]. Relative to the regulated by alternative splicing, as eIF3K isoform 2, which we other PML isoforms, PML-I is more highly conserved between term eIF3K-2, is preferentially recruited to PML NBs and is further mouse and humans, and has the highest expression level [10], enriched at bodies by overexpression of PML-I. This is the first suggesting that this isoform is a significant contributor to PML NB subunit of the eIF3 translation pre-initiation complex shown to functions. It is becoming increasingly clear that isoform-specific interact directly with PML and, in addition to eIF3E, is the second PML protein–protein interactions help regulate PML biology. For subunit of eIF3 known to associate with PML nuclear bodies. example, PML-IV has been shown to interact with p53 [21,24], whereas PML-II has recently been implicated in expression of the MHC class II gene cluster through specific recruitment of CIITA Results [53]. Additionally, PML-I interacts with acute myeloid leukemia 1 (AML1) [41] in an isoform-specific manner and PML-III interacts eIF3K interacts with a peptide encoded by exon 9 of PML-I with TRF1 to facilitate the alternative lengthening of telomeres (ALT) in U2OS cells [58]. Collectively, these examples suggest that In order to identify proteins that specifically interact with PML-I and individual isoforms through their protein–protein interactions can not with other PML isoforms we performed a yeast two-hybrid alter PML NB function, which could occur in a tissue-specific screen using the unique exon 9 region of human PML-I [20,29],

Fig. 1 – Domain mapping of the interaction of PML-I with eIF3K. (A) Schematic of a yeast two-hybrid protein interaction assay, in which the protein fragments indicated in (C) are fused to either the yeast Gal4 DNA-binding domain (Gal4DBD) or the Gal4 transcriptional activation domain (Gal4AD) and expressed in a yeast strain encoding the ADE2 reporter gene. (B) Diagram of eIF3K indicating the locations of the sequence encoded by exon 2 (absent in eIF3K isoform 2), the HAM and WH domains and the aa positions of the truncation points for the deletion series used in (C). (C) Yeast expressing the indicated fusion proteins were patched onto solid YPDA medium and incubated for 7 days at 28 1C. Yeast lacking detectable expression of the ADE2 reporter gene accumulate a dark red pigment (, darker shades), while yeast expressing ADE2 appear pink or light pink (þ or þþ, lighter shades). 2556 EXPERIMENTAL CELL RESEARCH 319 (2013) 2554– 2565

encoding amino acids 604–882 as a bait to screen a human fetal which lacks the exonuclease III-like domain, for interaction with brain cDNA library [16] (Fig. 1A). One of the interacting peptides eIF3K using a yeast two-hybrid assay. This fragment of PML-I was identified was an N-terminal fragment of subunit K of the eukaryotic found to interact with eIF3K, indicating that the PML exonuclease translation initiation factor 3, (eIF3K, aa 1–97) (Supplementary III-like domain was not required for interaction with eIF3K information Fig. 1). The human eIF3 complex contains at least 13 (Supplementary information Fig. 1). subunits, of which eIF3K is non-essential, and may therefore play a In order to map the region of eIF3K required for interaction role in regulating the translation initiation of specifictranscripts with amino acids 750–882 of PML-I, several eIF3K deletion [2,7,27,36,38,42]. There are three well-defined domains within the mutants were also generated, and tested for interaction with eIF3K sequence (Fig. 1B). The first is the N-terminal HEAT (Hunting- PML-I (aa 750–882) in a yeast two-hybrid assay (Fig. 1C). The ton, Elongation factor 3, A subunit of protein phosphatase 2A, smallest fragment of eIF3K found to interact with PML-I contained Target-of-rapamycin) repeat-like HAM (HEAT analogous motif) (aa residues 57–97 that correspond to a region within the HAM 24–121) [57]. HEAT domains often mediate protein–protein interac- domain of eIF3K. The C-terminal region of eIF3K (aa 97–218) tions [1]. The second domain is the winged-helix-like (WH) domain absent in the sequence identified from the yeast two-hybrid in the C-terminus (aa 132–191), which could mediate protein–RNA screen did not interact with PML-I (aa 750–882), suggesting that interactions [57]. In addition, the HAM and WH domains form residues 57–97 may contain the only region required for interac- elements of the PCI (26S proteasome lid, COP9 signalosome, tion with this PML-I fragment. eukaryotic initiation factor 3) domain (aa 60–218). The fragment During the cloning of the full-length human eIF3K cDNA from of eIF3K identified in the yeast two-hybrid screen (aa 1–97) encodes HeLa total mRNA, we isolated two different forms of eIF3K, one 75% of the HAM domain (summarized in Fig. 1B). matching the previously reported isoform ([52]; NCBI Reference To further characterize the interaction between PML-I and ID: NM_013234), as well as an unreported isoform (Supplemen- eIF3K, yeast two-hybrid interaction assays were performed using tary information Fig. 2). The novel eIF3K isoform (eIF3K isoform 2; various truncated peptides encoding fragments of PML-I and GenBank ID: JX870647) is similar to a human (EAW56803) and a eIF3K. The amino acid sequence encoded by exon 9 of PML-I mouse EST (AAH27638), suggesting that this isoform is conserved includes a predicted exonuclease III-like fold (aa 604–750) in mammals. Isoform 2 of eIF3K (eIF3K-2) contains an in-frame required for targeting PML-I to nucleolar fibrillar centers during deletion of exon 2 (encoding aa 21–53) resulting in deletion of stress or senescence [9]. To determine whether the exonuclease part of the HAM domain. Removing the residues encoded by exon III-like domain of PML-I was required for interaction with eIF3K, 2 from the fragment of eIF3K isolated in the yeast two-hybrid we tested a fragment encoded by exon 9 of PML-I (aa 750–882), screen (clone 1–20, 54–97) appeared to weaken but did not

Fig. 2 – In vitro interaction between PML-I and eIF3K. MBP, MBP-PML-I-E9Δexo (750–882 aa), GST and GST-eIF3K proteins were expressed in E. coli and puriﬁed with amylose (MBP) or glutathione Sepharose (GST) beads. (A) GST or GST-eIF3K proteins were bound to GST beads, followed by incubation with MBP–PML-I-E9Δexo. Recovery of MBP–PML-I-E9Δexo with GST-eIF3K was detected by SDS-PAGE and Western blotting. (B) MBP or MBP–PML-I-E9Δexo proteins were bound to amylose beads, followed by incubation with GST-eIF3K. Recovery of GST-eIF3K with MBP–PML-I-E9Δexo was detected by SDS-PAGE and Western blotting. For (A) and (B) lanes 1–3 were loaded with the equivalent of 10% of the sample used for pull-downs. Lanes 4 and 5 show the eluted proteins after puriﬁcation. EXPERIMENTAL CELL RESEARCH 319 (2013) 2554– 2565 2557

abolish interaction with PML-I (aa 750–882), suggesting both isoforms of eIF3K interact with this isoform of PML (Fig. 1C). This weaker two-hybrid interaction could reﬂect a preference of PML-I for isoform 1 over isoform 2 of eIF3K. We also observed that the eIF3K-2 fragment was not as highly expressed in yeast as the isoform 1 fragment (Supplementary information Fig. 3), thus expression level differences could also contribute to the differences observed in the reporter assay.

PML and eIF3K interact in vitro and in vivo

To test whether the interaction between PML-I and eIF3K observed in our yeast assay was direct, we performed in vitro protein interaction assays using affinity-purified recombinant glutathione-S-transferase (GST)-tagged eIF3K-2 and maltose binding protein (MBP)-tagged PML-I (aa 750–882), which lacks the exonuclease III domain. This shorter exon 9 fragment was used in these assays due to the propensity of the full-length peptide encoded by exon 9 to form insoluble protein aggregates when expressed in E. coli (data not shown). In vitro pull-down assays revealed that MBP–PML-I750–882 bound to GST-eIF3K but not GST alone (Fig. 2A). The reciprocal MBP co-purification assay demonstrated that GST-eIF3K bound to MBP–PML-I750–882 but not MBP alone (Fig. 2B). Collectively these data support the interaction of PML-I directly with eIF3K in vitro, which is consistent with the observed yeast two-hybrid interaction assay (Fig. 1C). To address the possibility that eIF3K could interact with other regions of PML-I, hexahistadine-taggedfulllengthPML-IandPML-IV were expressed in insect cells and assessed for the ability to co-purify with GST-tagged eIF3K-1 or eIF3K-2 (Fig. 3A). PML-IV contains the same exon arrangement as PML-I, but lacks exon 9 and instead contains a short sequence of 13 aa at the C-terminus that are unique to PML-IV [10,29].AfterGST-purification, both PML-I and PML-IV were recovered with eIF3K isoforms 1 and 2, but not with GST alone (Fig. 3A), suggesting that both isoforms of eIF3K also interact with PML independent of the sequence encoded by exon 9. To determine whether PML and eIF3K interact in human cells we performed co-immunoprecipitation assays. Consistent with data obtained from the yeast two-hybrid and in vitro protein interaction assays, endogenous PML was recovered when endogenous eIF3K was immunoprecipitated from human HEK-293T cells (Fig. 3B), indicating that these two proteins can also associate in vivo. Fig. 3 – eIF3K interacts with PML in vitro and in vivo. (A) GST or eIF3K colocalizes with PML NBs GST-eIF3K proteins (eIF3K-1 and eIF3K-2) were bound to GST beads, followed by incubation with 6HIS-tagged PML-I or Although several reports indicate that eIF3K is primarily localized PML-IV from insect cell lysates. Recovery of PML-I or PML-IV to the cytoplasm, the precise subcellular localization of eIF3K is following GST purification was detected by SDS-PAGE and not well established. Different reports indicate eIF3K can be found Western blotting. (B) HEK-293 cell lysates were used for associated with intermediate filaments [35] or sites of cell–cell co-immunoprecipitation of endogenous PML and eIF3K contact [31] in epithelial cells as well as adopting either peri- proteins with mouse anti-eIF3K monoclonal antibody. nuclear [31] or pan-cytoplasmic [35] localization in other cell Immunoprecipitated (IP) proteins were separated by SDS-PAGE types. Cytoplasmic localization is also consistent with the ability and the co-immunoprecipitated PML protein was assessed by of eIF3K to associate with the translation pre-initiation complex. Western blotting analysis with rabbit anti-PML polyclonal However, other translation initiation factors, such as eIF3E and antibodies. Input lanes were loaded with the equivalent of 10% eIF4E, also have nuclear functions and have been reported to of the sample used for immunoprecipitation. associate with PML and PML NBs [17,33]. Similarly, although PML is primarily a nuclear localized protein, at least one isoform (PML- VIIb) lacks the nuclear localization signal [29] and is localized to cytoplasm in early G1 [14,19]. In addition, PML-I contains a the cytoplasm where it appears to play a role in TGF-β signaling predicted nuclear export signal (NES), potentially allowing shut- [34]. PML isoforms also localize to both the nucleus and tling to and from the nucleus, although the precise regulation of 2558 EXPERIMENTAL CELL RESEARCH 319 (2013) 2554– 2565

this shuttling and its functional consequences have yet to be (Fig. 4A). Endogenous eIF3K is localized primarily to the cytoplasm determined [26]. Therefore, we wanted to determine the cellular in perinuclear structures consistent with a previous report [31]. compartment in which PML and eIF3K interact, which may then The specificity of the antibody used in our studies for eIF3K-1 and affect the interpretation of the possible functional consequences of eIF3K-2 was confirmed by the co-detection of ectopically their interaction. expressed epitope-tagged eIF3K isoforms by immunofluroesence To determine the cellular localization of PML and eIF3K, U2OS and Western blotting techniques (Supplementary information cells were immunostained for endogenous eIF3K and PML Fig. 4). Conversely, PML was primarily found in the nucleus of

Fig. 4 – eIF3K colocalizes with PML-I and PML-IV in PML NBs. (A) U2OS cells were ﬁxed and immunostained for endogenous eIF3K (green) and endogenous PML (red). (B) U2OS cells stably expressing GFP-tagged PML-I or PML-IV (green) were ﬁxed and immunostained for endogenous eIF3K (red). Examples of colocalization (merge) between eIF3K and PML nuclear bodies are indicated with arrows. DNA is visualized with DAPI (blue, MergeþDAPI). Scale bars¼10 μm. (C) Bar graph indicating the percentage of cells displaying either strong (black) or partial (gray) colocalization between eIF3K and PML in PML-I- or PML-IV-expressing cells as indicated. (D) Western blot of 293A, U2OS and HeLa cell lysates with anti-eIF3K antibody. Arrow indicates a protein of 20 kDa in U2OS and HeLa lysates observed with longer exposure (Long Exp) that is of similar molecular weight as eIF3K-2. (E) U2OS cells were transfected with empty vector, eIF3K-1 or eIF3K-2 expression plasmids and cell lysates were analyzed by Western blot with anti-eIF3K antibodies. Untransfected GFP–PML-I- and GFP–PML-IV-expressing U2OS cells were similarly prepared and analyzed. EXPERIMENTAL CELL RESEARCH 319 (2013) 2554– 2565 2559

cells within punctate nuclear domains known as PML nuclear IV expressing U2OS cells (Fig. 4E) was similar to untreated U2OS bodies (NBs). Thus, PML and eIF3K do not appear to colocalize to (Fig. 4D) or vector-transfected U2OS cells (Fig. 4E), suggesting that the same discrete structures under normal growth conditions in eIF3K-1 is the predominant isoform in these cells and that U2OS cells. However, with the exception of a small number of expression of PML-I or PML-IV does not significantly alter the constitutively associated PML NB components such as SP100, eIF3K isoform distribution. Given that only a portion of the SUMO1 and the PML isoforms [10,51] the majority of PML endogenous eIF3K immnuofluorescence signal is associated with NB-associated proteins interact with PML NBs in a transient PML-NBs (Fig. 4B) it remained unclear which eIF3K isoform(s) was fashion either during the cell cycle or in response to diverse being detected at the PML NB. It is possible that only eIF3K-2, a cellular stimuli and cell stress [5,13]. Often these interactions can minor component of eIF3K in the cell, is specifically recruited to be enhanced by increased expression of PML, the putative PML NBs. Alternatively, only a small portion of total eIF3K-1 may interacting protein, or both. In order to enhance the probability be localized to bodies. Finally, since both isoforms of eIF3K of observing eIF3K at PML NBs, we used U2OS cell lines that interact with PML in vitro, it is possible that both isoforms of express N-terminally GFP-tagged PML-I or PML-IV [18]. PML NBs eIF3K associate with PML NBs. typically contain all nuclear PML isoforms, with PML-I being the In order to more directly test which isoform(s) of eIF3K can predominant isoform [10]. In these cell lines, PML NBs increase in associate with PML bodies, C-terminally FLAG-tagged eIF3K isoforms number and are enriched in the PML isoform that is over- 1 and 2 were expressed in U2OS cells stably expressing GFP–PML-I expressed (Fig. 4B, Supplementary information Fig. 5). or GFP–PML-IV, and the ability to associate with PML NBs was In contrast to untransfected U2OS cells, endogenous eIF3K assessed (Fig. 5A). Isoform 1 of eIF3K showed a pan-cellular associated with PML NBs in a subset of cells expressed GFP- localization pattern and was generally not observed to associate tagged PML-I or PML-IV (Fig. 4B). The percentage of cells in which with PML NBs in either the PML-I- or PML-IV-expressing cells. endogenous eIF3K colocalized with PML nuclear bodies was Isoform 2 of eIF3K adopted a similar pan-cellular localization quantified (Fig. 4C). Colocalization was scored as either strong, pattern, however in contrast to isoform 1, it could additionally be partial or negative (Supplementary information Fig. 6). In cells found at PML NBs (Fig. 5A). In contrast to endogenous eIF3K, this with strong colocalization every PML nuclear body had an obvious association was more apparent in PML-I-enriched bodies than eIF3K punctum associated with it (Supplementary information PML-IV-enriched bodies (Fig. 5A). Similar localization experiments Fig. 6). Partial colocalization was defined as a cell having either performed with untagged or HA-tagged eIF3K-1 and eIF3K-2 yielded some, but not all PML bodies associated with eIF3K or having similar results, with eIF3K-2 showing increased association with poorly defined eIF3K-PML body colocalization (Supplementary PML-I-enriched PML NBs (Supplementary information Figs. 7 and 8). information Fig. 6). In U2OS cells expressing GFP–PML-I, 71% of The percentage of transfected cells showing strong or partial cells (29% strong, 43% partial colocalization) showed some degree colocalization with PML NBs was determined (Fig. 5B) using the of endogenous eIF3K localized to PML-I enriched NBs (Fig. 4C). same scoring criteria as in Fig. 4 (Supplementary information Fig. 9). Similarly, in U2OS cells expressing GFP–PML-IV, 91% of cells (47% When FLAG-tagged eIF3K-1 was expressed in PML-I- or PML-IV- strong, 44% partial colocalization) showed some degree of eIF3K expressing U2OS cells, there was no colocalization between eIF3K-1 association with PML-IV enriched NBs (Fig. 4C). These results and PML NBs. (Fig. 5B). In contrast, FLAG-tagged eIF3K-2 was indicate that endogenous eIF3K isoforms can associate with PML partially (20.5%, gray bars) or strongly (0.7%, black bars) associated NBs enriched in either PML-I or PML-IV, which is consistent with with PML-IV-enriched NBs in 21.2% of the eIF3K-2-transfected cells our ability to co-purify full-length recombinant PML-I and PML-IV (Fig. 5B). In PML-I-expressing U2OS cells, eIF3K-2 was partially (35%) with eIF3K in vitro (Fig. 3A). or strongly (26.1%) associated with nuclear bodies in 61.1% of While the data presented in Fig. 4B indicate that endogenous transfected cells (Fig. 5B). A similar trend was observed with eIF3K interacts with PML-I- or PML-IV-enriched PML NBs, it is untagged and HA-tagged eIF3K (Fig. 5B).These results suggest that unclear which eIF3K isoform(s) might be involved in the associa- eIF3K-2, but not eIF3K-1, associates with PML NBs enriched in PML-I tion. To begin to identify which isoform(s) of eIF3K might be to a greater degree than those bodies enriched in PML-IV. Unlike the associated with PML NBs we first analyzed cell lysates from U2OS, localization pattern observed for endogenous eIF3K (Fig. 4B), in 293 and HeLa cells by Western blotting (Fig. 4D). In all three cell which the isoform(s) of eIF3K being detected are uncertain, these lysates, a protein of 24 kDa was detected with the anti-eIF3K results identify eIF3K-2 as a specific isoform of eIF3K that antibody (Novus Biologicals, NB100-93304, Fig. 4D). There is some interacts with PML NBs. Furthermore, these results implicate the discrepancy about the molecular weight of eIF3K, with various exon 9 encoded peptide, found only in PML-I, as an important reports detecting eIF3K at between 24 and 28 kDa [31,48]. Since determinant of eIF3K-2 interaction with PML NBs in cells. Similar this antibody was confirmed to detect epitope-tagged eIF3K-1 and colocalization of eIF3K-2 and PML-I was also observed when eIF3K-2 by Western blotting (Supplementary information Fig. 4C) HA-tagged eIF3K-2 and FLAG-tagged PML-I were expressed in we also tested the ability to detect untagged eIF3K-1 and eIF3K-2 U2OS cells (Supplementary information Fig. 10). The association of by expressing these proteins in U2OS cells (Fig. 4E). Compared to eIF3K-2 and PML-I was also confirmed by co-immunoprecipitation vector-transfected cells, eIF3K-1-transfected cells showed a sub- from cellular lysates from cells expressing FLAG-tagged PML-I and stantial increase in the 24 kDa band, suggesting that eIF3K-1 is HA-tagged eIF3K-2 (eIF3K-2-HA) in transfected HEK-293A cells the predominant isoform in the cell lines we tested (Fig. 4E). (Fig. 5C). eIF3K-2-HA was not recovered from cell lysates when Expression of untagged eIF3K-2 revealed a protein of 20 kDa FLAG-PML-I was not present or in the rabbit IgG control IP samples (Fig4E) which is similar in size to proteins identified in U2OS and (Fig. 5C), indicating that eIF3K-2-HA was specifically recovered with HeLa lysates upon longer exposure (Fig. 4C, arrow), suggesting PML-I. Taken with the experiments in Fig. 3B, these data also that eIF3K-2 may comprise only a small fraction of total eIF3K in indicate that eIF3K-2 and PML can be reciprocally immunoprecipi- cells. In addition, detection of eIF3K in GFP–PML-I and GFP–PML- tated, further supporting the specificity of this interaction in cells. 2560 EXPERIMENTAL CELL RESEARCH 319 (2013) 2554– 2565

Fig. 5 – eIF3K isoform 2 colocalizes with PML-I-containing nuclear bodies. (A) FLAG-tagged eIF3K was expressed in U2OS cells stably expressing GFP–PML-I or GFP–PML-IV. Cells were ﬁxed and immunostained for eIF3K (anti-FLAG). Examples of colocalization between eIF3K and PML is indicated by arrows. DNA is visualized with DAPI (blue, merge with DAPI). Scale bars¼10 μm. (B) Bar graph indicating the percentage of transfected cells displaying either strong (black bars) or partial (gray bars) colocalization between eIF3K and PML in GFP–PML-I- or GFP–PML-IV-expressing U2OS cells. For each cell line, eIF3K-1 or eIF3K-2 was expressed with either no epitope tag () or with an N-terminal FLAG (F) or HA (H) tag as indicated. Values represent the mean þ/ standard error, n¼3–5. (C) FLAG-PML-I and eIF3K-2-HA expression plasmids were transiently expressed, alone or in combination, in 293A cells. Following immunoprecipitation (IP) with anti-FLAG resin or mouse IgG control beads, recovered proteins were identiﬁed by Western blotting (WB) for PML-I-FLAG and eIF3K-2-HA as indicated. Input lanes were loaded with the equivalent of 10% of the sample used for immunoprecipitation and 80% of the post-IP elution was loaded in the IP lanes. EXPERIMENTAL CELL RESEARCH 319 (2013) 2554– 2565 2561

eIF3K isoform is detected at PML NBs. However, when specific Discussion isoforms of eIF3K were introduced in these cells, only eIF3K-2 was found to be strongly associated with PML NBs, and eIF3K-2 was Loss of the PML protein is associated with genomic instability and associated with PML-I-enriched NBs to a greater degree than cancer of various histological origins [25,59]. There are multiple PML-IV-enriched NBs. Our in vitro data also indicated that eIF3K isoforms of the PML protein that localize to PML NBs [10,29] yet isoforms can interact with PML-I through the unique exon 9 how each individual PML isoform contributes to the various encoded C-terminus, and through another domain that might be functions attributed to PML NBs is poorly understood. The distinct common between all PML isoforms. However, in cells PML-I is C-terminal domains of PML isoforms can coordinate specific more efficient at recruiting eIF3K-2 to PML NBs. The discrepancy protein–protein interactions and contribute to the regulation of between the in vitro and cell line data may reflect the different PML NB function. Although more than 90 proteins are confirmed secondary and tertiary structure of PML within nuclear bodies in to associate with PML NBs [15], the majority of interactions are cells versus soluble PML used for in vitro studies. PML NBs form transitory in nature [13], and only a few isoform-specific protein– through SUMO-dependent heteromultimerization of nuclear body protein interactions have been reported. Furthermore, the major- associated proteins like DAXX and SP100 that are SUMOylated ity of PML NB interactions reported to date have been described in with several PML protein isoforms via their SUMO interaction relation to PML-IV. In this study, we have identified the eukaryotic motif (SIM) domains encoded in exon 7 of isoforms I to VI translation initiation factor 3 subunit K (eIF3K) as a novel [28,47,50]. In addition, multimerization of PML protein isoforms interacting protein of PML. At least two other translation factors, is driven by interactions between the RING finger B-box coiled eIF3E and eIF4E [17,33] have been reported to colocalize with PML coil (RBCC) domain found in all PML isoforms [29]. The highly NBs; however, no PML isoform-specific interactions have been multimeric state of PML within PML NBs is one factor in their described for these proteins nor have specific isoforms of transla- resistance to solubilization, which is in contrast to recombinant tion factors been shown to preferentially interact with PML. soluble PML protein which is either monomeric or in a reduced The interaction between PML and eIF3K appears to be multi- multimeric state, and is not associated with other proteins such as factorial with one interaction domain unique to PML-I (aa 750– DAXX and SP100. Thus, PML protein within PML NBs is likely to 882, encoded by exon 9) and at least one other interaction domain form tertiary structures that may partly occlude protein interac- found in exons 1–8, which are conserved between PML -I and tions that might occur between eIF3K isoforms and the common PML-IV. Yeast two-hybrid assays indicated that the association of N-terminus (encoded by exons 1–7) that encodes the RBCC and eIF3K with exon 9 of PML-I requires a portion of the HAM domain SIM domains found in PML isoforms I to VI. As such, the unique of eIF3K. The HAM domain of eIF3K mediates other protein C-terminus of PML-I may serve as a more accessible docking site interactions, such as with cytoplasmic keratin 18 and the 5-HT7 for eIF3K isoforms in cells. Nonetheless, it remains unclear why receptor [11,35]. Although the PCI domain of eIF3K may also only eIF3K-2, but not eIF3K-1, colocalizes to PML bodies since both mediate protein-protein interactions, as has been seen for other isoforms of eIF3K were able to interact with PML exon 9 in vitro. PCI domain-containing proteins [43], this domain was not It is possible that the inclusion of exon 2 in eIF3K-1 could facilitate required for association with aa 750–882 of PML-I in a yeast an interaction with a cellular factor, not present in our in vitro two-hybrid assay (Fig. 1B and C). interactions studies, that excludes it from interacting with PML In the course of our experiments we also identified a novel bodies. Alternately, secondary structure changes catalyzed by human isoform of eIF3K, which we have designated as isoform 2 exclusion of peptide sequences encoded by exon 2 within eIF3K- (eIF3K-2). eIF3K-2 is encoded by an alternatively spliced mRNA 2 may facilitate better interaction with PML-I within nuclear that results in an in-frame deletion of exon 2, and which can also bodies. Another eIF3 subunit, eIF3E, also colocalizes with PML NBs be identified in cDNA libraries of humans and mice (Supplemen- indirectly via interaction with the Ret finger protein in an tary information Fig. 2). In addition, analysis of expression tagged isoform-independent manner [39]. Therefore, a third possibility, sequences (ESTs) within Genbank encoding eIF3K suggests there although less likely given our in vitro data demonstrating direct may be several other isoforms of eIF3K that could vary in their interaction between eIF3K and the C-terminus of PML-I, is that an ability to interact with PML. adapter protein might interact with eIF3K-2, but not eIF3K-1, to We have shown by cell-free and tissue culture methods that facilitate association with PML-NBs. Alternatively, while eIF3K-2 eIF3K and PML can interact with each other. In vitro, eIF3K-1 and clearly showed stronger association with PML NBs than eIF3K- eIF3K-2 both interact with the C-terminal domain of PML-I (exon 1when these proteins were ectopically expressed (Fig. 5), it is 9, Fig. 1) and with full-length recombinant PML-I and PML-IV possible that endogenous eIF3K-1 (the more abundant eIF3K (Fig. 3). The interaction between aa 750–882 of PML-I and eIF3K is isoform) might also have an affinity for PML NBs when it is likely directly based on our in vitro pull-down assays using expressed at endogenous levels (Fig. 4B). However, since only a purified recombinant proteins (Fig. 2). In addition, we demon- small proportion of total eIF3K is at PML bodies, it is possible that strate that eIF3K and PML form protein complexes in vivo that can eIF3K-2, which represents a small proportion of total eIF3K, is still be co-immunoprecipitated from human HEK-293 cell lysates the major endogenous eIF3K isoform at PML bodies. This latter (Figs. 3 and 5C). interpretation would be consistent with the observation that Although both eIF3K isoforms studied here can interact with eIF3K-2 is more strongly associated with PML NBs when ectopi- PML in vitro, we observed differential abilities to associate with cally expressed. PML NBs in cells. Expression of GFP-tagged PML-I or PML-IV in Finally, in response to various cellular stresses (e.g. DNA U2OS cells promoted an association of endogenous eIF3K with damage) many proteins transiently interact with PML bodies PML NBs (Fig. 4). Although eIF3K-1 appears to be the predominant and post-translational modifications often regulate or result from isoform in these cells (Fig. 4E) it is not clear which endogenous these PML body interactions [5,13]. In the case of another eIF3 2562 EXPERIMENTAL CELL RESEARCH 319 (2013) 2554– 2565

subunit, eIF3F, its localization to the nucleus is regulated via was created by cloning the sequence encoding PML-I-E9 lacking the phosphorylation by the cyclin dependent kinase 11 (CDK11p46) exonuclease III domain (750–882 aa) into pMal-p4G vector (New [49]. Although our in vitro data suggests that eIF3K interacts with England Biolabs). PML in the absence of post-translational modifications, we cannot exclude the possibility that the association of eIF3K isoforms with Yeast strains and media PML NBs in cells may be regulated by post-translational modification of eIF3K and/or the PML protein itself. Nonetheless, given Yeast strains and media—Standard methods were used for growth our novel finding that eIF3K-2 localizes to PML NBs in an isoform- and manipulation of yeast and bacteria [8,46]. Yeast were cultured dependent manner, it is tempting to speculate that alternative in YPDA (1% yeast extract (BioShop Canada), 2% Peptone (BioShop splicing of eIF3K transcripts may regulate the localization of eIF3K Canada), 0.003% adenine, 2% glucose), synthetic defined (0.67% at PML NBs, for example between different cell types or under yeast nitrogen base without amino acids (BioShop Canada), 2% various physiological conditions that promote the alternative glucose, 0.003% adenine, 0.002% uracil, and all required amino acids). splicing of eIF3K. Yeast were transformed by the LiAc/SS-DNA/PEG method [23]. The significance of the association of eIF3K with PML NBs is unclear since the biological role(s) of eIF3K remain poorly Cell culture characterized. In the cytoplasm, the main function of eIF3K is presumably the regulation of translation initiation; however, HEK-293A, U2OS and HeLa cell lines used were cultured in eIF3K does not appear to be an essential subunit of the eIF3 Dulbecco's modified Eagle's medium (Sigma) supplemented with complex [36] and general translation does not appear to be 10% fetal calf serum, 1% penicillin/streptomycin at 37 1C with 5% affected by eIF3K silencing [35]. As well, eIF3K, along with the CO2. Human osteosarcoma (U2OS) cell lines stably expressing eIF3E and eIF3F subunits, are not conserved in budding yeast green fluorescent protein (GFP)–PML-I and GFP–PML-IV [18] were [2,38,42]. Even in the cytoplasm, eIF3K has been reported to play maintained as above with the addition of 800 (PML-I) or 1600 other roles beyond translation, including regulation of apoptosis, (PML-IV) mg/ml of G418. through an interaction with keratin 18 [35], and 5-HT7 receptor trafficking and stability [11]. In addition, eIF3K has been shown to Yeast two-hybrid assay colocalize with cyclin D3 in the cytoplasm [48] and dynein at sites of cell–cell contact in epithelial cells [31]. Our study is the first to Yeast two-hybrid screens were performed according to instruc- describe eIF3K-2 as a novel isoform of eIF3K, and eIF3K-2 is the tions described in the Matchmaker Gold yeast two-hybrid manual predominant isoform associated with PML NBs within cells. (Clontech). PML-I-E9 bait plasmid (pGBKT7) was introduced into Previous studies attributing various cellular functions to eIF3K Saccharomyces cerevisiae PJ696 yeast two-hybrid reporter strain have investigated isoform 1 [11,48], an isoform that we have and mated with an isogenic yeast strain transformed with a demonstrated to be weakly associated with PML NBs in cells, or human adult brain cDNA library cloned into pACT2 (Clontech). endogenous eIF3K, where the isoform(s) being studied are For domain mapping, plasmids directing expression of the indi- unclear [31,35]. Therefore, it will be important to determine what cated Gal4AD-tagged fragments of eIF3K and Gal4DBD-tagged PML-I functions are shared between eIF3K isoforms, and what specific (aa 750–882) were transformed into yeast strains Y2HGold and functions can be attributed to eIF3K-2, before the functional Y187, respectively. Transformants were mated, and diploids were consequences of the localization of this isoform to PML NBs can isolated on selective medium lacking leucine and tryptophan and be fully explored. subsequently patched to solid rich medium containing a limiting amount of adenine (YPDA).

Materials and methods: Expression and puriﬁcation of recombinant eIF3K and PML

Plasmid construction GST-eIF3K and MBP–PML-I (aa 750–882) recombinant proteins were expressed in Escherichia coli Rosetta (DE3) cells (Novagen),

The human PML isoform I, exon 9 (PML-I-E9) sequence encoding while His6-tagged full-length PML-I and PML-IV were expressed amino acids 604–882 (GenBank Accession NM_033238.2) and a in insect cells. smaller fragment of the human PML-I-E9 sequence lacking the For expression of GST-tagged eIF3K in E. coli, the coding exonuclease III domain (amino acids (aa) 750–882) was cloned sequence for eIF3K (isoform 1 or 2) was cloned into the GST into pGBT9 or pGBKT7 in frame with the coding region for the expression vector pGEX-5X-1, and the plasmid transformed into

DNA binding domain of the yeast Gal4 transcription factor Rosetta cells. Transformants were grown to OD600 0.5–1.0 in LB

(Gal4DBD). Fragments of eIF3K were cloned into pACT2 (Clontech) medium and were induced with 0.1 mM IPTG for 3 h at 37 1C. in frame with the coding region for the activation domain of the Cells were harvested, aliquoted and stored at 80 1C. Cells

Gal4 transcription factor (Gal4AD). An alternative splice isoform of (80 μL wet pellet volume) were resuspended in 800 μLof eIF3K (eIF3K isoform 2, see Supplementary information Fig. 2) purification buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0.1% was isolated from total HeLa RNA extracts, reverse transcribed NP-40, 1 mM PMSF) containing 0.4 mg lysozyme and incubated on and cloned into the mammalian expression plasmid pcDNA3.1 (-) ice for 20 min. Cell suspensions were sonicated for five rounds of containing an influenza hemagglutinin (HA) epitope tag on the 10 s each at 30% output on a Misonex Sonicator 3000 and C-terminus (eIF3K-Iso2-HA). For expression of GST-eIF3K in E. coli,the centrifuged at 21 000g for 15 min at 4 1C. Protein concentration in coding sequence for eIF3K was cloned into pGEX-5 1vector(GELife the supernatant was quantifiedbyaBradfordassayanddilutedtoa Sciences). Maltose binding protein (MBP)–PML-I recombinant plasmid final volume of 750 μLinpurification buffer. 60 μL of glutathione EXPERIMENTAL CELL RESEARCH 319 (2013) 2554– 2565 2563

Sepharose 4B beads (GE Life Sciences) were added to the soluble lysates were mixed with mouse monoclonal anti-FLAG M2 fraction and the suspensions rocked for 30 min at room tempera- magnetic beads (Sigma) or mouse IgG bound to PureProteome™ ture. Beads were washed three times in purification buffer. Protein G magnetic beads (Millipore) and incubated overnight at For expression of full-length PML-I and PML-IV protein in insect 4 1C. Beads were washed four times with lysis buffer and eluted cells, the Bac-to-Bac baculovirus expression system was used with 2 protein sample buffer and boiling. Recovered proteins according to the manufacturer's instructions (Life Technologies). were analyzed by SDS-PAGE and Western Blotting with rabbit The coding sequence for PML-I and PML-IV were cloned into anti-ECS (DDDDK) antibody (Bethyl Laboratories, A190-102A) and Fastbac expression vectors and transformed into Max efficiency rabbit anti-eIF3K (Novus Biologicals, NB100-93304). DH10Bac competent cells (Life Technologies). Bacmids were transfected into sf-9 cells with Cellfectin (Life Technologies) to Immunofluorescence produce recombinant baculovirus particles. Virus was collected from the culture medium and used to infect sf-9 cells for 48 h. Protein colocalization was visualized by fluorescence microscopy. Infected cell lysates were prepared by resuspending cell pellets in Cells were plated onto coverslips in a 6-well plate and transfected ten volumes of insect cell lysis buffer (50 mM Tris pH 7.4, 250 mM with the relevant plasmids. Forty-eight hours post-transfection, cells sucrose, 150 mM NaCl, 0.1% Triton X-100, and 2 mM PMSF). were washed with PBS and fixed in 3% paraformaldehyde. Cells were Suspensions were sonicated for six rounds of 10 s at 30% output. permeabilized with 0.5% Triton X-100 in PBS, and blocked with 4% Lysates were supplemented with 15% glycerol, aliquoted and BSA in PBS. Cells were then immunolabeled with primary antibodies 1 stored at 80 C. Suspensions were centrifuged at 21,000g for (1 h, diluted in blocking buffer), washed with PBS and incubated fi 30 min. Protein concentration in the supernatant was quanti ed with fluorescently labeled secondary antibodies (45 min, diluted in by a Bradford assay, and volumes of supernatants containing blocking buffer). Following washes in PBS, cells were incubated with fi equal levels of PML-I and PML-IV were diluted to a nal volume of 1 μg/mL of 4',6-diamidino-2-phenylindole (DAPI) stain to visualize μ 400 L in insect cell lysis buffer. DNA. Fluorescent images were captured with Zeiss Axiovert 200M Microscope and Hamamatsu Orca R2 Camera or with Zeiss Cell In vitro binding assays Observer Microscope under a 63 immersion oil objective lens. Images were processed using only linear adjustments (e.g. bright- fi fi In vitro af nity puri cation assays were performed by pre-clearing ness/contrast) with Slidebook (Intelligent Imaging Innovations, μ 2 g of protein lysates with glutathione Sepharose 4B or amylose Boulder, CO) and Adobe Photoshop CS5. Quantification of eIF3K resin for GST and MBP tagged proteins, respectively and incubated colocalization with PML nuclear bodies was determined by count- – – with GST, GST-eIF3K or MBP and MBP PML-I (750 882 aa) bound ing 50–100 eIF3K positive cells per experimental condition and 1 beads for 3 h at 4 C.Thebeadswerewashedthreetimeswithwash calculating the percentage of cells with no, partial or complete buffer (PBS supplemented with 0.1% Triton X-100 and 50 mM NaCl) colocalization between eIF3K and PML nuclear bodies. The means fi and once with PBS. The co-puri ed proteins were eluted by boiling and standard errors from at least three independent experiments in 2 SDS loading buffer and analyzed by Western blotting. were calculated and statistical significance was determined using For testing interaction of GST-eIF3K with His6-PML-I and His6- the student's t-test in Microsoft Excel 2007. PML-IV, 400 μL of diluted insect cell lysate was added to 20 μLof gluthione Sepharose beads containing bound GST or GST-eIF3K and rocked for 1 h at room temperature. Beads were washed four Acknowledgments times for 5 min at room temperature in 1 mL purification buffer fi containing 300 mM NaCl. Co-puri ed proteins were eluted by This research was funded by an operating grant to G.D. from the μ boiling for 5 min in 60 L of protein gel loading buffer. Canadian Institutes of Health Research (CIHR, MOP-84260). J.S., D.C. and J.P. are supported by trainee awards from the Beatrice Co-immunoprecipitation assay and western blotting Hunter Cancer Research Institute with funds provided as part of the Terry Fox Foundation Strategic Health Research Training For co-immunoprecipitation of endogenous proteins, untrans- Program in Cancer Research at CIHR. B.T. received a studentship fected human embryonic kidney (HEK-293T) cells were lysed from the Nova Scotia Health Research Foundation. ice-cold lysis buffer (50 mM Tris–HCl pH 8, 300 mM NaCl, 0.25% Nonidet P-40, 1 protease inhibitor cocktail). Cleared lysates were incubated overnight at 4 1C with mouse anti-eIF3K antibody Appendix A. Supporting information that was pre-bound to PureProteome™ Protein G magnetic beads (Millipore). The beads were washed 3 times with wash buffer Supplementary data associated with this article can be found in – (50 mM Tris HCl pH 8, 150 mM NaCl, 0.1% Nonidet P-40, 5 mM the online version at http://dx.doi.org/10.1016/j.yexcr.2013.09.001. EDTA) followed by one wash with PBS. The bound proteins were eluted by boiling in 2 SDS sample buffer and analyzed by reference Western blotting. For co-immunoprecipitation of tagged PML and eIF3K, HEK-293A cells were transfected with N-terminal FLAG tagged PML-I and C-terminal HA-tagged eIF3K expression [1] M.A. Andrade, C. Petosa, S.I. O'Donoghue, C.W. Muller, P. Bork, Comparison of ARM and HEAT protein repeats, J. Mol. Biol. 309 plasmids using Lipofectamine 2000 (Life Technologies) following (2001) 1–18. the manufacturer's instructions. At 48 h post transfection, cells [2] K. Asano, T.G. Kinzy, W.C. Merrick, J.W. Hershey, Conservation were lysed with ice-cold lysis buffer (50 mM Tris–HCl pH 8, and diversity of eukaryotic translation initiation factor eIF3, J. 150 mM NaCl, 0.5% Nonidet P-40, 1 protease inhibitor). Cleared Biol. Chem. 272 (1997) 1101–1109. 2564 EXPERIMENTAL CELL RESEARCH 319 (2013) 2554– 2565

[3] C.A. Ascoli, G.G. Maul, Identification of a novel nuclear domain, [23] R.D. Gietz, R.H. Schiestl, A.R. Willems, R.A. Woods, Studies on the J. Cell Biol. 112 (1991) 785–795. transformation of intact yeast cells by the LiAc/SS-DNA/PEG [4] R. Bernardi, P.P. Pandolfi, Role of PML and the PML-nuclear body procedure, Yeast 11 (1995) 355–360. in the control of programmed cell death, Oncogene 22 (2003) [24] A. Guo, P. Salomoni, J. Luo, A. Shih, S. Zhong, W. Gu, P.P. Pandolfi, 9048–9057. The function of PML in p53-dependent apoptosis, Nat. Cell Biol. 2 [5] R. Bernardi, P.P. Pandolfi, Structure, dynamics and functions of (2000) 730–736. promyelocytic leukaemia nuclear bodies, Nat. Rev. Mol. Cell Biol. [25] C. Gurrieri, P. Capodieci, R. Bernardi, P.P. Scaglioni, K. Nafa, 8 (2007) 1006–1016. L.J. Rush, D.A. Verbel, C. Cordon-Cardo, P.P. Pandolfi, Loss of the [6] K.L. Borden, RING fingers and B-boxes: zinc-binding protein-protein tumor suppressor PML in human cancers of multiple histologic interaction domains, Biochem. Cell Biol. 76 (1998) 351–358. origins, J. Natl. Cancer Inst. 96 (2004) 269–279. [7] K.S. Browning, D.R. Gallie, J.W. Hershey, A.G. Hinnebusch, [26] B.R. Henderson, A. Eleftheriou, A comparison of the activity, U. Maitra, W.C. Merrick, C. Norbury, Unified nomenclature for the sequence specificity, and CRM1-dependence of different nuclear subunits of eukaryotic initiation factor 3, Trends Biochem. Sci. 26 export signals, Exp. Cell Res. 256 (2000) 213–224. (2001) 284. [27] J.W. Hershey, K. Asano, T. Naranda, H.P. Vornlocher, P. Hanachi, [8] D. Burke, D. Dawson, T. Stearns, Methods in Yeast Genetics: A W.C. Merrick, Conservation and diversity in the structure of Cold Spring Harbor Laboratory Course Manual, Cold Spring translation initiation factor EIF3 from humans and yeast, – Harbor Laboratory Press, Woodbury, New York, 2000. Biochimie 78 (1996) 903 907. [9] W. Condemine, Y. Takahashi, M. Le Bras, H. de Thé, A nucleolar [28] A.M. Ishov, A.G. Sotnikov, D. Negorev, O.V. Vladimirova, N. Neff, targeting signal in PML-I addresses PML to nucleolar caps in T. Kamitani, E.T. Yeh, J.F. Strauss III, G.G. Maul, PML is critical for stressed or senescent cells, J. Cell Sci. 120 (2007) 3219–3227. ND10 formation and recruits the PML-interacting protein daxx to fi [10] W. Condemine, Y. Takahashi, J. Zhu, F. Puvion-Dutilleul, this nuclear structure when modi ed by SUMO-1, J.Cell Biol. 147 – S.Guegan,A.Janin,H.deThé,Characterizationofendogenous (1999) 221 234. human promyelocytic leukemia isoforms, Cancer Res. 66 (2006) [29] K. Jensen, C. Shiels, P.S. Freemont, PML protein isoforms and the – 6192–6198. RBCC/TRIM motif, Oncogene 20 (2001) 7223 7233. [11] K. De Martelaere, B. Lintermans, G. Haegeman, P. Vanhoenacker, [30] T. Kamitani, K. Kito, H.P. Nguyen, H. Wada, T. Fukuda-Kamitani, fi Novel interaction between the human 5-HT7 receptor isoforms E.T. Yeh, Identi cation of three major sentrinization sites in PML, – and PLAC-24/eIF3k, Cell. Signal. 19 (2007) 278–288. J. Biol. Chem. 273 (1998) 26675 26682. [12] H. de Thé, C. Lavau, A. Marchio, C. Chomienne, L. Degos, A. [31] S. Karki, L.A. Ligon, J. DeSantis, M. Tokito, E.L. Holzbaur, PLAC-24 is a cytoplasmic dynein-binding protein that is recruited to sites Dejean, The PML-RAR alpha fusion mRNA generated by the t of cell–cell contact, Mol. Biol. Cell 13 (2002) 1722–1734. (15;17) translocation in acute promyelocytic leukemia encodes a [32] M.H. Koken, F. Puvion-Dutilleul, M.C. Guillemin, A. Viron, functionally altered RAR, Cell 66 (1991) 675–684. G. Linares-Cruz, N. Stuurman, L. de Jong, C. Szostecki, F. Calvo, [13] G. Dellaire, D.P. Bazett-Jones, PML nuclear bodies: dynamic C. Chomienne, The t(15;17) translocation alters a nuclear body in sensors of DNA damage and cellular stress, Bioessays 26 (2004) a retinoic acid-reversible fashion, EMBO J. 13 (1994) 1073–1083. 963–977. [33] H.K. Lai, K.L. Borden, The promyelocytic leukemia (PML) protein [14] G. Dellaire, R.W. Ching, H. Dehghani, Y. Ren, D.P. Bazett-Jones, suppresses cyclin D1 protein production by altering the nuclear The number of PML nuclear bodies increases in early S phase by a cytoplasmic distribution of cyclin D1 mRNA, Oncogene 19 (2000) fission mechanism, J.Cell Sci. 119 (2006) 1026–1033. 1623–1634. [15] G. Dellaire, R. Farrall, W.A. Bickmore, The nuclear protein database [34] H.K. Lin, S. Bergmann, P.P. Pandolfi, Cytoplasmic PML function in (NPD): sub-nuclear localisation and functional annotation of the TGF-beta signaling, Nature 431 (2004) 205–211. nuclear proteome, Nucleic Acids Res. 31 (2003) 328–330. [35] Y.M. Lin, Y.R. Chen, J.R. Lin, W.J. Wang, A. Inoko, M. Inagaki, [16] G. Dellaire, E.M. Makarov, J.J. Cowger, D. Longman, H.G. Suther- Y.C. Wu, R.H. Chen, eIF3k Regulates apoptosis in epithelial cells land, R. Luhrmann, J. Torchia, W.A. Bickmore, Mammalian PRP4 by releasing caspase 3 from keratin-containing inclusions, J.Cell kinase copurifies and interacts with components of both the U5 Sci. 121 (2008) 2382–2393. snRNP and the N-CoR deacetylase complexes, Mol. Cell Biol. 22 [36] M. Masutani, N. Sonenberg, S. Yokoyama, H. Imataka, Reconsti- – (2002) 5141 5156. tution reveals the functional core of mammalian eIF3, EMBO J. 26 [17] C. Desbois, R. Rousset, F. Bantignies, P. Jalinot, Exclusion of Int-6 (2007) 3373–3383. from PML nuclear bodies by binding to the HTLV-I Tax onco- [37] G.G. Maul, Nuclear domain 10, the site of DNA virus transcription – protein, Science 273 (1996) 951 953. and replication, Bioessays 20 (1998) 660–667. [18] C.H. Eskiw, G. Dellaire, D.P. Bazett-Jones, Chromatin contributes [38] G.L. Mayeur, C.S. Fraser, F. Peiretti, K.L. Block, J.W. Hershey, to structural integrity of promyelocytic leukemia bodies through Characterization of eIF3k: a newly discovered subunit of mam- a SUMO-1-independent mechanism, J. Biol. Chem. 279 (2004) malian translation initiation factor elF3, Eur. J. Biochem. 270 – 9577 9585. (2003) 4133–4139. [19] R.D. Everett, P. Lomonte, T. Sternsdorf, R. van Driel, A. Orr, Cell [39] C. Morris-Desbois, V. Bochard, C. Reynaud, P. Jalinot, Interaction cycle regulation of PML modification and ND10 composition, J. between the Ret finger protein and the Int-6 gene product and Cell Sci. 112 (Pt 24) (1999) 4581–4588. co-localisation into nuclear bodies, J. Cell Sci. 112 (Pt 19) (1999) fi [20] M. Fagioli, M. Alcalay, P.P. Pandol , L. Venturini, A. Mencarelli, 3331–3342. A. Simeone, D. Acampora, F. Grignani, P.G. Pelicci, Alternative [40] D. Negorev, G.G. Maul, Cellular proteins localized at and inter- splicing of PML transcripts predicts coexpression of several acting within ND10/PML nuclear bodies/PODs suggest functions carboxy-terminally different protein isoforms, Oncogene 7 of a nuclear depot, Oncogene 20 (2001) 7234–7242. (1992) 1083–1091. [41] L.A. Nguyen, P.P. Pandolfi, Y. Aikawa, Y. Tagata, M. Ohki, [21] V. Fogal, M. Gostissa, P. Sandy, P. Zacchi, T. Sternsdorf, K. Jensen, I. Kitabayashi, Physical and functional link of the leukemia- P.P. Pandolfi, H. Will, C. Schneider, S.G. Del, Regulation of p53 associated factors AML1 and PML, Blood 105 (2005) 292–300. activity in nuclear bodies by a specific PML isoform, EMBO J. 19 [42] L. Phan, X. Zhang, K. Asano, J. Anderson, H.P. Vornlocher, (2000) 6185–6195. J.R. Greenberg, J. Qin, A.G. Hinnebusch, Identification of a [22] P.S. Freemont, The RING finger. A novel protein sequence translation initiation factor 3 (eIF3) core complex, conserved in motif related to the zinc finger, Ann. N. Y. Acad. Sci. 684 (1993) yeast and mammals, that interacts with eIF5, Mol. Cell. Biol. 18 174–192. (1998) 4935–4946. EXPERIMENTAL CELL RESEARCH 319 (2013) 2554– 2565 2565

[43] E. Pick, K. Hofmann, M.H. Glickman, PCI complexes: beyond the L.J. Gay, S.W. Hulyk, D.K. Villalon, D.M. Muzny, E.J. Sodergren, proteasome, CSN, and eIF3 Troika, Mol. Cell 35 (2009) 260–264. X. Lu, R.A. Gibbs, J. Fahey, E. Helton, M. Ketteman, A. Madan, [44] A. Reymond, G. Meroni, A. Fantozzi, G. Merla, S. Cairo, L. Luzi, D. S. Rodrigues, A. Sanchez, M. Whiting, A. Madan, A.C. Young, Riganelli, E. Zanaria, S. Messali, S. Cainarca, A. Guffanti, Y. Shevchenko, G.G. Bouffard, R.W. Blakesley, J.W. Touchman, S. Minucci, P.G. Pelicci, A. Ballabio, The tripartite motif family E.D. Green, M.C. Dickson, A.C. Rodriguez, J. Grimwood, identifies cell compartments, EMBO J. 20 (2001) 2140–2151. J. Schmutz, R.M. Myers, Y.S. Butterfield, M.I. Krzywinski, fi [45] P. Salomoni, P.P. Pandol , The role of PML in tumor suppression, U. Skalska, D.E. Smailus, A. Schnerch, J.E. Schein, S.J. Jones, – Cell 108 (2002) 165 170. M.A. Marra, Generation and initial analysis of more than 15,000 [46] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning: A full-length human and mouse cDNA sequences, Proc. Natl. Acad. Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989. Sci. USA 99 (2002) 16899–16903. fi [47] T.H. Shen, H.K. Lin, P.P. Scaglioni, T.M. Yung, P.P. Pandol , The [53] T. Ulbricht, M. Alzrigat, A. Horch, N. Reuter, A. von Mikecz mechanisms of PML-nuclear body formation, Mol. Cell 24 (2006) V. Steimle, E. Schmitt, O.H. Kramer, T. Stamminger, P. Hemmerich, 331–339. PML promotes MHC class II gene expression by stabilizing the class [48] X. Shen, Y. Yang, W. Liu, M. Sun, J. Jiang, H. Zong, J. Gu, II transactivator, J.Cell Biol. 199 (2012) 49–63. Identification of the p28 subunit of eukaryotic initiation factor 3 [54] E. Van Damme, K. Laukens, T.H. Dang, X. Van Ostade, A manually (eIF3k) as a new interaction partner of cyclin D3, FEBS Lett. 573 curated network of the PML nuclear body interactome reveals an (2004) 139–146. important role for PML-NBs in SUMOylation dynamics, Int. J. Biol. [49] J. Shi, J.W. Hershey, M.A. Nelson, Phosphorylation of the eukar- Sci. 6 (2010) 51–67. yotic initiation factor 3f by cyclin-dependent kinase 11 during [55] M. Vernier, V. Bourdeau, M.F. Gaumont-Leclerc, O. Moiseeva, apoptosis, FEBS Lett. 583 (2009) 971–977. V. Begin, F. Saad, A.M. Mes-Masson, G. Ferbeyre, Regulation of [50] T. Sternsdorf, K. Jensen, B. Reich, H. Will, The nuclear dot protein E2Fs and senescence by PML nuclear bodies, Genes Dev. 25 sp100, characterization of domains necessary for dimerization, – subcellular localization, and modification by small ubiquitin-like (2011) 41 50. modifiers, J. Biol. Chem. 274 (1999) 12555–12566. [56] Z.G. Wang, L. Delva, M. Gaboli, R. Rivi, M. Giorgio, C. Cordon- fi [51] T. Sternsdorf, K. Jensen, H. Will, Evidence for covalent modifica- Cardo, F. Grosveld, P.P. Pandol , Role of PML in cell growth and – tion of the nuclear dot-associated proteins PML and Sp100 by the retinoic acid pathway, Science 279 (1998) 1547 1551. PIC1/SUMO-1, J. Cell Biol. 139 (1997) 1621–1634. [57] Z. Wei, P. Zhang, Z. Zhou, Z. Cheng, M. Wan, W. Gong, Crystal fi [52] R.L. Strausberg, E.A. Feingold, L.H. Grouse, J.G. Derge, structure of human eIF3k, the rst structure of eIF3 subunits, R.D. Klausner, F.S. Collins, L. Wagner, C.M. Shenmen, G.D. Schuler, J. Biol. Chem. 279 (2004) 34983–34990. S.F. Altschul, B. Zeeberg, K.H. Buetow, C.F. Schaefer, N.K. Bhat, [58] J. Yu, J. Lan, C. Wang, Q. Wu, Y. Zhu, X. Lai, J. Sun, C. Jin, H. Huang, R.F. Hopkins, H. Jordan, T. Moore, S.I. Max, J. Wang, F. Hsieh, PML3 interacts with TRF1 and is essential for ALT-associated PML L. Diatchenko, K. Marusina, A.A. Farmer, G.M. Rubin, L. Hong, bodies assembly in U2OS cells, Cancer Lett. 291 (2010) 177–186. M. Stapleton, M.B. Soares, M.F. Bonaldo, T.L. Casavant, [59] S. Zhong, P. Hu, T.Z. Ye, R. Stan, N.A. Ellis, P.P. Pandolfi, A role for T.E. Scheetz, M.J. Brownstein, T.B. Usdin, S. Toshiyuki, P. Carninci, PML and the nuclear body in genomic stability, Oncogene 18 C. Prange, S.S. Raha, N.A. Loquellano, G.J. Peters, R.D. Abramson, (1999) 7941–7947. S.J. Mullahy, S.A. Bosak, P.J. McEwan, K.J. McKernan, J.A. Malek, [60] S. Zhong, P. Salomoni, P.P. Pandolfi, The transcriptional role of P.H. Gunaratne, S. Richards, K.C. Worley, S. Hale, A.M. Garcia, PML and the nuclear body, Nat.Cell Biol. 2 (2000) E85–E90.