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SIAH2 and TRIAD1
The International Journal of Biochemistry & Cell Biology 44 (2012) 132–138
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Differential regulation of PML–RAR␣ stability by the ubiquitin ligases SIAH1/SIAH2 and TRIAD1 Kristin Pietschmann a , Marc Buchwald a , Sylvia Müller a , Shirley K. Knauer b , Manfred Kögl c , Thorsten Heinzel a , Oliver H. Krämer a,∗ a
Institute of Biochemistry and Biophysics, Center for Molecular Biomedicine (CMB), Department of Biochemistry, University of Jena, Hans-Knöll-Str. 2, 07745 Jena, Germany Center for Medical Biotechnology (ZMB), Department of Molecular Biology II, University of Duisburg-Essen, Campus Essen, Universitätsstraße 5, 45141 Essen, Germany c German Cancer Research Center, Preclinical Target Development, and Genomics and Proteomics Core Facilities, 69120 Heidelberg, Germany b
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Article history: Received 29 June 2011 Received in revised form 5 October 2011 Accepted 16 October 2011 Available online 22 October 2011 Keywords: Ubiquitinylating enzymes TRIAD1 SIAH UBCH8 Proteasomal degradation PML–RAR␣
a b s t r a c t The ubiquitin proteasome system plays an important role in normal and malignant hematopoiesis and relies on the concerted action of three enzyme families. The E2 ubiquitin conjugase UBCH8 (ubiquitin conjugating enzyme [human] 8) cooperates with the E3 ubiquitin ligases SIAH1 and SIAH2 (seven in absentia homolog 1/2) to mediate the proteasomal degradation of oncoproteins. One such protein is the leukemia fusion protein PML–RAR␣ (promyelocytic leukemia–retinoic acid receptor␣) that is associated with acute promyelocytic leukemia. A limited number of UBCH8 interaction partners that participate in the UBCH8-dependent depletion of cancer-relevant proteins are known. We report here that TRIAD1 (two RING fingers and DRIL [double RING finger linked] 1), an E3 ubiquitin ligase relevant for the clonogenic growth of myloid progenitors, binds UBCH8 as well as PML–RAR␣. Moreover, there is concurrent induction of TRIAD1 and UBCH8 upon combinatorial treatment of acute promyelocytic leukemia cells with the pro-apoptotic epigenetic modulator valproic acid and the differentiation inducing agent all-trans retinoic acid. However, in sharp contrast to SIAH1/SIAH2 and UBCH8, TRIAD1 binding to PML–RAR␣ has no effect on its turnover. In summary, our data exclude TRIAD1 as crucial regulator of the leukemic determinant PML–RAR␣, but highlight the prominence of the UBCH8/SIAH axis in PML–RAR␣ degradation. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction In light of the crucial role of the ubiquitin proteasome system (UPS) for health and disease, detailed characterization of this system and its regulators is highly warranted (Kirkin and Dikic, 2011). Tagging of proteins with lysine K48-linked polyubiquitin chains allows their rapid elimination via the proteasome, an intracellular multi-protease complex (Hochstrasser, 2009). Ubiquitinylation is catalyzed by an E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzymes, and E3 ubiquitin ligases. The E1 activates ubiquitin
Abbreviations: APL, acute promyelocytic leukemia; ATRA, all-trans retinoic acid; HDACi, histone deacetylase inhibitor; PML–RAR␣, promyelocytic leukemia–retinoic acid receptor ␣; SIAH, seven in absentia homolog; TRIAD1, two RING fingers and DRIL (double RING finger linked) 1; UBCH, ubiquitin conjugating enzyme (human); VPA, valproic acid. ∗ Corresponding author. Tel.: +49 3641 949362; fax: +49 3641 949352. E-mail addresses: [email protected] (K. Pietschmann), [email protected] (M. Buchwald), [email protected] (S. Müller), [email protected] (S.K. Knauer), [email protected] (M. Kögl), [email protected] (T. Heinzel), [email protected] (O.H. Krämer). 1357-2725/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2011.10.008
ATP-dependently and transfers it to an E2 enzyme. RING domain E3s mediate substrate binding and recruit E2s, thereby allowing the transfer of the E2 ubiquitin cargo to a substrate. Dedicated substrate/E3 pairing permits precise ubiquitinylation. Specificity of ubiquitin ligases is additionally guaranteed by their ability to associate with different ubiquitin conjugases. Furthermore, ubiquitin ligases themselves can be targeted for proteasomal degradation by their cognate ubiquitin conjugase(s). In line with these findings we could show in a previous study that the E3 ubiquitin ligase SIAH1 (seven in absentia homolog 1) and the E2 ubiquitin conjugase UBCH8 (ubiquitin conjugating enzyme [human] 8) cooperatively promote the turnover of the UBCH8 interacting E3 ligase RLIM (RING finger LIM domain-binding protein) (Krämer et al., 2008). Various studies indicate that the UPS regulates proliferation, differentiation, and apoptosis during hematopoiesis (Crawford et al., 2008; Heuzé et al., 2008; Marteijn et al., 2006). An example for successful therapeutic activation of the UPS is acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemias characterized by blasts blocked at the promyelocytic stage (Okuno et al., 2004). In 95% of all cases, APL cells express PML–RAR␣ (promyelocytic leukemia–retinoic acid receptor ␣) encoded by
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the translocation t(15;17) (Perissi et al., 2010). In contrast to the RAR␣ transcription factor, PML–RAR␣ is not activated upon physiological concentrations of retinoic acid. Subsequently, differentiation genes positively controlled by RAR␣ become repressed by PML–RAR␣ under physiological conditions (Boukarabila et al., 2009; Epping et al., 2007; Mercurio et al., 2010; Perissi et al., 2010). Pharmacological doses of all-trans retinoic acid (ATRA) evoke proteasomal degradation of PML–RAR␣ and a co-repressor/coactivator exchange turning PML–RAR␣ into a potent transcriptional activator. Consequently, promyelocytic leukemia cells become mature white blood cells (Brown et al., 2009; Duprez et al., 2003; Mercurio et al., 2010; Perissi et al., 2010). HDACI (histone deacetylase inhibitors) alter gene expression and cellular signaling by inducing histone and non-histone protein acetylation (Buchwald et al., 2009; Spange et al., 2009). Such agents are able to correct dysregulated repressive transcription patterns. Moreover, by augmenting the expression of the ubiquitin conjugase UBCH8, these drugs trigger the proteasomal degradation of proteins associated with tumorigenesis. One example is PML–RAR␣, as it is targeted for degradation by the HDACimodulated UBCH8–SIAH1–proteasome axis (Buchwald et al., 2009; Krämer et al., 2008). Contrary to retinoids, HDACi mainly evoke caspase-dependent apoptosis of PML–RAR␣-positive NB4 cells. A deeper insight of regulatory mechanisms controlling the abundance of this oncoprotein may provide new avenues for therapy of patients suffering from APL (Buchwald et al., 2010; Krämer et al., 2008; Mercurio et al., 2010; Müller and Krämer, 2010; Perissi et al., 2010). In this study we aimed for the identification of so far unknown UBCH8 interacting proteins that might participate in the UBCH8-dependent degradation of cancer-relevant oncoproteins. By systematic yeast two-hybrid (Y2H) screens we identified the ubiquitin ligase TRIAD1 (Two RING fingers and DRIL [double RING finger linked]-1) as a novel UBCH8 interaction partner. In vitro binding and cellular co-localization studies confirmed the association of TRIAD1 with UBCH8. Co-expression studies revealed that TRIAD1 itself is not targeted for degradation by UBCH8 and its cooperating SIAH ubiquitin ligases. We furthermore disclose the interaction of TRIAD1 with the UBCH8 substrate PML–RAR␣. Moreover, we show that ATRA and the HDACi valproic acid (VPA) concurrently induce UBCH8 and TRIAD1 expression in cells derived from the bone marrow of a patient with APL in relapse. Our work though also demonstrates that the interaction of TRIAD1 with PML–RAR␣ and UBCH8 neither leads to PML–RAR␣ protein destabilization nor does it interfere with the very potent UBCH8/SIAH1- or UBCH8/SIAH2mediated degradation of PML–RAR␣. 2. Materials and methods 2.1. Drugs and chemicals The proteasome inhibitor Z-Leu-Leu-Leu-al (MG132) was purchased from Axxora; ATRA and VPA were from Sigma–Aldrich.
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1999); TRIAD1-Myc (Marteijn et al., 2005). The plasmid encoding GST-UBCH7 was created using the primers 5 -CGGGATCCCGATGGCGGCCAGCAGGAGGC-3 and 5 GGAATTCCTTAGTCCACAGGTCGCTTTTCCCCTA-3 . The PCR product was ligated into BamHI/EcoRI sites of pGEX-5X-1 (GE Healthcare). The UBCH7-V5 encoding plasmid was constructed by PCR amplification using 5 -GCCACCATGGCGGCCA-GCAGGAGGC-3 and 5 -GTCCACAGGTCGCTTTTCCCCATATT-3 and ligated into the pcDNA3.1/V5-His-TOPO vector as stated by the manufacturer (Invitrogen). All constructs were verified by sequencing. 2.4. Yeast two-hybrid screen Automated Y2H screens were performed as described in references (Albers et al., 2005; Albert et al., 2003; Lamesch et al., 2007 human genes). Human cDNA libraries from testis and brain (Clonetech) as well as a library of individually cloned full-length open reading frames (from cDNAs of 10.070 different genes) were screened for full-length UBCH8 as the bait. 2.5. Quantitative real-time PCR Cellular mRNA was isolated and cDNA was synthesized as explained in (Krämer et al., 2008). Data obtained were analyzed with the delta-Cq quantification model (Hellemans et al., 2007) using three reference genes (HMBS, GAPDH, and RPL13A). These were verified with the geNorm program (Vandesompele et al., 2002). Primer sequences for quantitative real-time PCR (qPCR) were: TRIAD1 fwd 5 -CGGGTACAGGAGCCTAGAGCTCGCCG-3 and rev 5 GGATTGTGGCACAGTCTGTGGGTGCG-3 ; UBCH8 fwd 5 -TGGCGAGCATGCGAGTGGTGAAGG-3 and rev 5 -CTGGACAGGTTCCGCAGGTATGGG-3 ; GAPDH fwd 5 -TGCACCACCAACTGCTTAGC-3 and rev 5 -GGCATGGACTGTGGTCATGAG-3 ; RPL13A fwd 5 -CCTGGAGGAGAAGAGGAAAGAGA-3 and rev 5 -TTGAGGACCTCTGTGTATTTGTCAA-3 ; HMBS fwd 5 -GGCAATGCGGCTGCAA-3 and rev 5 -GGGTACCCACGCGAATCAC-3 . 2.6. Transfection assays Transient protein expression in HEK293T cells was achieved with PEI (Sigma–Aldrich) or Lipofectamine2000 (Invitrogen) (Krämer et al., 2009). Cells were harvested after 24 h. Empty vectors pcDNA3.1 or pSG5, as applicable regarding the coding plasmids, were used to obtain equal amounts of total DNA transfected (2 g/106 cells). We noted that it is important to use the same plasmid backbone for this as each appeared to have a different impact on general transfection efficiency. A plasmid encoding green fluorescent protein (pEGFP, 0.05 g) was co-transfected to monitor transfection efficiency where applicable. 2.7. Cell lysis, immunoblot, immunofluorescence, and microscopy
2.2. Cell lines Cell lines were cultured as stated (Buchwald et al., 2010; Krämer et al., 2008). 2.3. Plasmids The following plasmids have been described previously: UBCH8-V5, GST-UBCH8, FLAG-PML–RAR␣, Myc-PML–RAR␣GFP, pEGFP, SIAH1C72S ; SIAH1, SIAH2 (Krämer et al., 2008); HA-SIAH1 (Crone et al., 2011); Myc-SIAH2 (Germani et al.,
Lysate preparation and immunoblot techniques are summarized in (Buchwald et al., 2010; Krämer et al., 2008). Microscopy analyses of HeLa cells were performed as described (Knauer et al., 2007). NB4 suspension cells were fixed on frosted glass slides by cytospin centrifugation for 10 min at 100 g before staining. 2.8. GST pull-down This method was carried out as described in references (Buchwald et al., 2010; Krämer et al., 2008).
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2.9. Antibodies Antibodies used for immunoblot were purchased from Santa Cruz Biotechnology: SIAH-1, #sc-5506; SIAH-2, #sc-5507; TRIAD1, #sc-134113; C/EBP, #sc-150; GFP, #sc-9996; HSP90, #sc-13119; Caspase 3, #sc-7272; Sigma–Aldrich: Actin, A2066; Tubulin, #T5168; FLAG, #F-3165; Invitrogen: V5-Tag, #46-0705; New England Biolabs: Caspase 3 cleaved, #9664; Myc-Tag, #2276; Abgent: UBCH8, #AP21185b; Aviva: TRIAD1, ARP34418; Upstate: H3-Ac, 06-599. The antibody against RAR␣/PML–RAR␣ was a gift from Dr. Gronemeier, Straßbourg. 2.10. Flow cytometry analyses and apoptosis assays R-phycoerythrin (RPE)-conjugated anti-CD11b (Dako, #R0841) was used to stain CD11b-positive NB4 cells. For staining, 5 × 105 cells were washed twice with PBS (137 mM NaCl, 8 mM Na2 HPO4 , 2.7 mM KCl, 1.4 mM KH2 PO4 , pH 7.25) and re-suspended in PBA buffer (PBS, 2 mg/ml BSA, 0.05% NaN3 ) with 5 l of RPE-anti-CD11b. Fluorescence was analyzed using the FACS-Canto device (BD Biosciences) (Krämer et al., 2008; Schneider et al., 2010). 3. Results 3.1. Interaction of UBCH8 with TRIAD1 By conducting Y2H screens to identify novel interacting partners of UBCH8 we isolated 145 clones coding for TRIAD1 (Fig. 1a). TRIAD1 (alternatively HARI2 or ARIH2) is a RING-type ubiquitin ligase characterized by a tripartite cysteine-rich domain (Marteijn et al., 2005). The related protein ARIH1 (HHARI), which is a known UBCH8 interacting ubiquitin ligase (Moynihan et al., 1999), and the mitochondrial protein TIMM8A (Tranebjaerg et al., 2001) were also identified to interact with UBCH8 as bait. To validate the UBCH8/TRIAD1 interaction found in the Y2H screens we performed GST pull-down experiments. This approach approved the previously described interaction of TRIAD1 with UBCH7, an E2 related to UBCH8 (Marteijn et al., 2005), and confirmed the binding of UBCH8 and TRIAD1 from whole cell lysates (Fig. 1b). Additionally, TRIAD1 could be co-immunoprecipitated with UBCH7 and UBCH8 (Fig. 1c). In summary, we verified the interaction of TRIAD1 and UBCH8 with three different binding assays. 3.2. Stability of TRIAD1 and interplay between TRIAD1 and SIAH1/SIAH2 Ubiquitin ligases can be very unstable and their amounts are often modulated by associated ubiquitin conjugases and ligases (Depaux et al., 2006; Hu and Fearon, 1999; Krämer et al., 2008; Nagel et al., 2011). Based on these data we investigated whether overexpression of UBCH7 or UBCH8 evokes degradation of TRIAD1. However, co-expression of these proteins had no significant impact on TRIAD1 protein levels (Fig. 2a). SIAH proteins are very potent ubiquitin ligases that interact with UBCH8 and can hierarchically promote proteasomal degradation of other E3 ubiquitin ligases (Calzado et al., 2009; House et al., 2009; Krämer et al., 2008; Nagel et al., 2011). Of note, TRIAD1 also contains structures resembling the PML–RAR␣ coiled coil domain which allows PML–RAR␣ to be recognized as a target by SIAHs (Fanelli et al., 2004; Marteijn et al., 2005). Since we could co-precipitate overexpressed SIAH1 with TRIAD1 (data not shown), we considered to test if SIAH1/SIAH2 targeted TRIAD1 for degradation. Since levels of either E2 or E3 enzymes could be limiting for substrate degradation, we also co-expressed UBCH7 or UBCH8 together with SIAH1 or SIAH2 and assessed TRIAD1 protein stability by immunoblotting.
Fig. 1. Physical interaction of UBCH8 with TRIAD1. (a) Full-length UBCH8 was used as bait protein for yeast two-hybrid screening of three prey libraries. The table displays prey proteins, which have been identified to interact with UBCH8 in these screens. Numbers of times each clone has been isolated and number of different cDNA libraries (o, open-reading-frame; t, testis; b, brain), in which this interaction has been found are also listed. (b) Heterologously expressed GST-UBCH8 was precipitated after incubation with cell lysates from HEK293T cells expressing TRIAD1-Myc. As negative control, pull-down was performed with GST alone. GST-UBCH7 served as positive control (input represents 10% of cell lysates used for pull-down). The precipitates were probed with antibodies against the Myc-Tag. Levels of GST fusion proteins were detected by Ponceau-staining of the membranes (data not shown). The lower panel displays densitometric analyses for TRIAD1 protein bands (means ± SE, n = 3). (c) UBCH7-V5 or UBCH8-V5 were co-expressed with TRIAD1-Myc in HEK293T cells. UBCH7/8 was immunoprecipitated from cell lysates. The presence of TRIAD1, UBCH7 and UBCH8 in the precipitates was analyzed by immunoblotting (pre, pre-immune serum; IP, immunoprecipitation; n = 2).
However, TRIAD1 remained stable upon expression of these ubiquitin conjugases and ligases (Fig. 2b). We conclude that SIAH1/2 and UBCH7/8 unlikely regulate the protein turnover of TRIAD1. SIAH proteins are known to undergo rapid proteasomal degradation. Furthermore, they act as their own E3 ubiquitin ligases to facilitate autoubiquitinylation (Depaux et al., 2006; Hu and Fearon, 1999). This finding prompted us to compare the general turnover rates of SIAH and TRIAD1 proteins. We treated HEK293T cells ectopically expressing these ubiquitin ligases with the proteasome inhibitor MG132 and analyzed their protein stability. Whereas the levels of TRIAD1 remained unchanged upon proteasomal inhibition, detectable amounts of SIAH1 and SIAH2 increased strongly under such conditions (Fig. 2c). These data reveal dissimilar stability profiles and separate degradation pathways for TRIAD1, SIAH1, and SIAH2.
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Fig. 2. Overexpressed TRIAD1 is not degraded by UBCH7, UBCH8, SIAH1, and SIAH2, and is not stabilized by proteasomal inhibition. (a) TRIAD1-Myc was expressed with UBCH7-V5 or UBCH8-V5. Protein levels were analyzed by immunoblot. Tubulin served as loading control. (b) HEK293T cells were transfected with vectors for TRIAD1-Myc, HA-SIAH1, Myc-SIAH2, UBCH7-V5, or UBCH8-V5. Protein levels were detected by immunoblot analyses as indicated. (c) HEK293T cells were transfected with vectors for TRIAD1-Myc, SIAH1, or SIAH2. Cells were treated with MG132 (20 M, 8 h) followed by immunoblot (S1, SIAH1; S2, SIAH2).
3.3. Induced expression of UBCH8 and TRIAD1 by single or combinatorial treatment of APL cells with VPA and ATRA HDACi as well as ATRA can induce PML–RAR␣ degradation in APL cells (Brown et al., 2009; Krämer et al., 2008). We previously showed that treatment of APL cells with the HDACi VPA upregulates the expression of the TRIAD1 binding partner UBCH8, which promotes proteasomal cleavage of PML–RAR␣ (Krämer et al., 2008). Recently, TRIAD1 mRNA was shown to be induced by ATRA in NB4 cells (Marteijn et al., 2005). These data suggest a link between the expression of TRIAD1, UBCH8, PML–RAR␣, and ATRA or HDACi. Therefore, we analyzed the effect of both substances on the regulation of TRIAD1 and UBCH8 by single or co-treatment of NB4 APL cells with VPA and ATRA. Several attempts failed to detect endogenous TRIAD1 at the protein level with antibodies commercially available. Of note, these did also not detect overexpressed Myc-TRIAD1 in HEK293T cells, although its expression was confirmed with antibodies against the Myc epitope. Due to the lack of working antibodies for the detection of TRIAD1 at protein level, we performed quantitative real-time PCR (qPCR) to analyze the regulation of TRIAD1 mRNA expression. As HDACi relax chromatin structures and affect the expression of a wide variety of genes, expression stability of reference genes used for normalization has to be validated. We established a qPCR system with three different reference genes. We noticed a significant induction of TRIAD1 mRNA in NB4 cells by combinatorial treatment with VPA plus ATRA. In contrast, VPA single treatment did not alter TRIAD1 mRNA levels and ATRA only marginally affected expression of this mRNA at 24 h (Fig. 3a). This finding contrasts a previous work that reported ATRA-induced upregulation of TRIAD1 following 24 h of stimulation. This discrepancy might be explained by differences in the applied mRNA detection methods (Northern blot versus qPCR) and the elaborated normalization method we used in our system. Nevertheless, we detected an approximately
Fig. 3. VPA and ATRA alter expression of TRIAD1 and UBCH8. (a) NB4 cells were treated with 1.5 mM VPA (V), 1 M ATRA (A), or left untreated (C) for 24 h. Expression of TRIAD1 mRNA was analyzed by quantitative real-time PCR. Relative expression relates to corresponding values for samples from untreated cells, set as 1 (means ± SE; n = 3; **p < 0.01). GAPDH, HMBS, and RPL13A served as reference genes. (b) NB4 cells were treated with 1.5 mM VPA (V), 1 M ATRA (A), or left untreated (C) for 24 h. Expression of UBCH8 protein was analyzed by immunoblot. Tubulin served as loading control. (c) Activity of ATRA was tested by flow cytometry with an antibody directed against the cell surface protein CD11b (means ± SE, n = 3). (d) Lysates of untreated (C) and ATRA-treated (A) NB4 cells were analyzed for PML–RAR␣ and C/EBP by immunoblot. C/EBP antibodies generated a specific band (>) and a nonspecific band (*) useful as a loading control. (e) NB4 cells were treated with 1.5 mM VPA for 2–24 h (−, untreated). Levels of endogenous PML–RAR␣, UBCH8, Caspase 3 (Casp. 3; fl, full length; cl, cleaved), acetyl-histone H3 (H3-Ac), and Tubulin were determined by immunoblot.
twofold increase of TRIAD1 after 40 h of ATRA treatment (data not shown). To this end our data reveal differential regulation of TRIAD1 in PML–RAR␣-positive leukemia cells upon single or combinatorial treatment with ATRA and VPA. We already described that UBCH8 is induced by HDACi in APL and other leukemic cells (Buchwald et al., 2010; Krämer et al., 2008). Interestingly, we detected enhanced expression of UBCH8
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by VPA and also by ATRA treatment as well as by the combination of both drugs in NB4 cells (Fig. 3b). To ensure activity of ATRA in our experimental setting we tested its potency via detection of typical molecular signs of granulocytic differentiation. We assessed the accumulation of the transcription factor C/EBP (CCAAT/enhancer-binding protein ), which regulates expression of various genes involved in granulocytic differentiation, and the induction of the cell surface marker CD11b (Brown et al., 2009; Duprez et al., 2003; Mercurio et al., 2010; Perissi et al., 2010). ATRA potently induced up to 90% of CD11b positive cells (Fig. 3c) as well as accumulation of C/EBP protein (Fig. 3d). These molecular alterations indicating promyelocyte differentiation correlated with the loss of PML–RAR␣ antagonizing cellular maturation. We equally assured the potency of the HDACi VPA with protein expression analysis showing a time-dependent decrease of PML–RAR␣ and induction of UBCH8, histone hyperacetylation, and caspase activity relevant in apoptosis (Fig. 3e and data not shown). Attenuation of PML–RAR␣ correlated with the induction of UBCH8 before executioner Caspase 3 processing and activation, and thus is congruent with our published data revealing early proteasomal proteolysis of this oncoprotein in NB4 cells treated with VPA (Krämer et al., 2008). 3.4. Effects of TRIAD1, SIAH1, and SIAH2 on PML–RAR˛ stability From these data we conclude that TRIAD1 levels upon VPA or ATRA single treatment do not correlate with PML–RAR␣ degradation induced by these substances (Brown et al., 2009; Krämer et al., 2008). However, the basally expressed TRIAD1 may affect the UBCH8-dependent protein degradation, since proteasomal degradation has been shown to be regulated by limiting amounts of UBCH8 (Krämer et al., 2003). In addition, it remains conceivable that VPA + ATRA-induced TRIAD1 contributes to the degradation of PML–RAR␣. Previously we reported, that UBCH8 in cooperation with the E3 ubiquitin ligase SIAH1 promotes the proteasomal degradation of PML–RAR␣ (Krämer et al., 2008), thereby serving as a reference. Moreover, the fact that TRIAD1 interacts with UBCH8, which can increase PML–RAR␣ ubiquitinylation, raised the question whether TRIAD1 functioned as an additional ubiquitin ligase on PML–RAR␣. To address this issue, we first tested whether there was co-localization of these E2/E3 enzymes and PML–RAR␣. Therefore, we detected ectopically expressed PML–RAR␣ and SIAH1 in HeLa cells by immunofluorescence microscopy. Overexpressed SIAH1 potently promotes proteasomal degradation of PML–RAR␣ and is hardly detectable by immunofluorescence microscopy due to rapid autoubiquitinylation and degradation (Hu and Fearon, 1999; Krämer et al., 2008; Nagel et al., 2011). Thus, we used an established RING-mutant of SIAH1 (SIAH1C72S ), which is deficient in binding E2 ubiquitin conjugases and hence cannot mediate substrate- and autoubiquitinylation (Depaux et al., 2006; Hu and Fearon, 1999; Krämer et al., 2008; Nagel et al., 2011; Winter et al., 2008; Zhao et al., 2011). We could visualize SIAH1C72S and PML–RAR␣ co-localization in a cellular context (Fig. 4a). Microscopical inspection of NB4 APL cells to detect endogenous SIAH1 and PML–RAR␣ supported this observation (Suppl. Fig. S1). In addition to the SIAH1/UBCH8 interaction, we found that the E3 ligase TRIAD1 interacted with UBCH8. Hence, we asked whether TRIAD1 could also recruit PML–RAR␣ to UBCH8. Fluorescence microscopy revealed a partial co-localization of PML–RAR␣ with nuclear UBCH8 and TRIAD1 (Fig. 4b and c). Moreover, a co-immunoprecipitation binding assay biochemically confirmed physical interaction of TRIAD1 and PML–RAR␣ (Fig. 4d). Based on our findings that TRIAD1 interacts with both, UBCH8 and PML–RAR␣, we tested whether this association – equally to the SIAH1/2-mediated degradation of PML–RAR␣ – evoked a loss of this fusion protein. Immunoblot analyses confirmed functionality of SIAH1 as well as SIAH2 against PML–RAR␣. However,
Fig. 4. PML–RAR␣ is degraded by SIAH proteins independently of TRIAD1. (a) HeLa cells were transfected with GFP-PML–RAR␣ and RING mutant SIAH1 (SIAH1C72S ) which was detected by ␣-SIAH1 antibodies (phase, phase contrast microscopy; scale bar = 10 m). (b) GFP-PML–RAR␣ and UBCH8-V5 were expressed in HeLa cells. Localization and overlay of these proteins was revealed with GFP emission signals and probing for V5 (scale bar = 10 m). (c) GFP-PML–RAR␣ and TRIAD1-Myc were expressed in HeLa cells. Localization and overlay of these proteins was revealed with GFP emission signals and probing for Myc (scale bar = 10 m). (d) TRIAD1-Myc was co-expressed with Flag-PML–RAR␣ in HEK293T cells. TRIAD1 was immunoprecipitated with ␣-Myc antibodies and the presence of TRIAD1 and PML–RAR␣ in the precipitates was analyzed by immunoblotting (pre, pre-immune serum; IP, immunoprecipitation; n = 2). (e) FLAG-PML–RAR␣ was coexpressed with SIAH1, SIAH2, and TRIAD1-Myc in HEK293T cells. Expression levels were analyzed by immunoblotting. Tubulin protein levels show equal loading.
co-expressed TRIAD1 had no destabilizing effect (Fig. 4e, lanes 2–4). Recently, overexpressed TRIAD1 was found to interact with the transcriptional repressor GFI1 (growth factor independence 1), thereby blocking other ubiquitin ligases catalyzing its ubiquitinylation and proteasomal degradation (Marteijn et al., 2007). Therefore, we tested whether TRIAD1 also interfered with the SIAH1/2-mediated degradation of PML–RAR␣. However, the specific interaction of TRIAD1 with PML–RAR␣ did not affect SIAH1dependent PML–RAR␣ degradation (Fig. 4e, lanes 5 + 6). Thus, inhibition of proteasomal degradation by TRIAD1 depends on the substrate and most likely also on E2/E3 enzyme pairs cooperating. Fig. 5 summarizes our data shown here as a table and in a model. 4. Discussion In this study, we identify the E3 ubiquitin ligase TRIAD1 as a novel interacting partner of the E2 ubiquitin conjugase UBCH8. Initial data gained in Y2H screens could be verified by pulldown experiments. We also report that both proteins are upregulated by co-treatment of NB4 APL cells with VPA and ATRA. By different
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Fig. 5. Interaction of UBCH8 with SIAH1/2, but not with TRIAD1 targets PML–RAR␣ to proteasomal degradation. Model and table summarize data presented here combined with previous results (Fanelli et al., 2004; Krämer et al., 2008) (+, yes/found/detected; −, no/not detected/unlikely; *, linked to E3 SIAH1 or SIAH2; n.t., not tested).
experimental approaches we reveal that TRIAD1 also interacts and co-localizes with PML–RAR␣. The fact that this association has no effect on its stability argues for a more prominent role for SIAH1/SIAH2 in the control of PML–RAR␣ levels. We present reliable data that demonstrate TRIAD1–UBCH8 interaction. Nevertheless, a previous report, describing a Y2H screen for TRIAD1 binding partners, excluded binding of UBCH8 to this ubiquitin ligase (Marteijn et al., 2005). This discrepancy can be explained by the use of truncated (amino acids 1–119 or 109–493) instead of full-length TRIAD1 in our screen. Apparently, this protein forms interaction surfaces with UBCH8 that are disrupted when expressed discontinuously. Moreover, several E3 ubiquitin ligases have been reported to interact with UBCH8 including TRIAD family members (Beasley et al., 2007; Chuang and Ulevitch, 2004; Fearns et al., 2006; Huang et al., 2006; Moynihan et al., 1999). Our data are further supported by the finding that the UBCH8 related UBCH7 was also shown to bind TRIAD1 (Marteijn et al., 2005). The role of TRIAD1 in proliferation and differentiation of normal and malignant hematopoietic cells has been investigated (Marteijn et al., 2007, 2005, 2009; Wang et al., 2011). These studies demonstrate that TRIAD1 blocks clonogenic growth of primary myeloid progenitor cells but may not promote differentiation. PML–RAR␣ however potently halts myeloid differentiation (Perissi et al., 2010). The fact that TRIAD1 showed no attenuating effect on PML–RAR␣ perfectly corresponds to the notion that TRIAD1 did not enhance differentiation. Furthermore, degradation of PML–RAR␣ is independent of TRIAD1 upregulation in NB4 cells solely treated with VPA or ATRA, standing in perfect agreement with the finding that overexpression of TRIAD1 did not reduce PML–RAR␣ levels. In contrast, both agents induce UBCH8, which in turn cooperates with SIAH1 and SIAH2 in the proteasomal degradation of PML–RAR␣
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(Buchwald et al., 2009; Krämer et al., 2008). Therefore, SIAH1 or SIAH2 together with UBCH8 appear to restrain the development of APL.Until now, no poly-ubiquitinylation substrate of TRIAD1 has been identified, and the role of TRIAD1 for cellular processes therefore remains largely unknown. In addition, previous data suggest that TRIAD1 may mark proteins with ubiquitin in a non-proteolytic manner (Marteijn et al., 2009). Since we found upregulation of TRIAD1 in APL cells treated with the combination of VPA and ATRA, we cannot exclude that TRIAD1 cooperates with UBCH8 in other processes. In order to better understand TRIAD1 functions, future studies have to clarify how association of TRIAD1 and UBCH8 affects targets and cellular fate.In our hands, TRIAD1 is stable upon proteasome inhibition and co-expression with ubiquitin ligases and conjugases. This is congruent with data from Marteijn et al. (2005) demonstrating that inhibition of the proteasome merely stabilizes a minor fraction of poly-ubiquitinylated TRIAD1 without significantly affecting the total TRIAD1 protein level. Furthermore, agents targeting proteasomes specifically affected TRIAD1 functions less efficiently than substances also inhibiting non-proteasomal proteases (Marteijn et al., 2005). These findings and our data suggest a proteasome-independent proteolytic degradation pathway for TRIAD1. Recently, the degradation of the transcriptional repressor GFI1 (Möröy, 2005) was found to be blocked by heterologously overexpressed TRIAD1 (Marteijn et al., 2007). Nonetheless, in our experiments TRIAD1 could not block SIAH1/SIAH2-mediated degradation of PML–RAR␣. Although we cannot exclude that TRIAD1 competes with endogenous expressed E2 and E3 enzymes, our data regarding PML–RAR␣ stability also indicate for dominant functions of SIAH1 and SIAH2 over TRIAD1 in our model system. In sum, we have identified a new UBCH8 interacting ubiquitin ligase, TRIAD1, which equally to UBCH8 is upregulated in APL cells upon combinatorial exposure to VPA plus ATRA. However, our data exclude TRIAD1 as crucial regulator of the leukemic fusion protein PML–RAR␣, and they argue that the specific role of TRIAD1 in APL has still to be defined. It appears that SIAH proteins and TRIAD1 share binding partners though having very different biochemical properties within the cell. Funding This work was supported by grants from the Landesprogramm “ProExzellenz” des Freistaates Thüringen (PE 123-2-1, to OHK), from the Deutsche Krebshilfe (grant number 109265, to OHK), and from the Deutsche Forschungsgemeinschaft (SFB 604, to TH). Acknowledgements We thank S. Reichardt and Dr. G. Greiner for excellent technical assistance, Dr. C. Bier and Dr. S. Drube for supportive discussions and technical help. Dr. R. Marschalek, Dr. N. Varin-Blank, Dr. T.G. Hofmann, Dr. B.A. van der Reijden, and Dr. S. Minucci kindly provided expression constructs for SIAH1, SIAH2, Myc-SIAH2, HASIAH1, TRIAD1-Myc, and tagged PML–RAR␣. Dr. H. Gronemeier kindly provided the antibodies against RAR␣. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biocel.2011.10.008. References Albers M, Kranz H, Kober I, Kaiser C, Klink M, Suckow J, et al. Automated yeast twohybrid screening for nuclear receptor-interacting proteins. Mol Cell Proteomics 2005;4:205–13.
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Differential regulation of PML–RAR␣ stability by the ubiquitin ligases SIAH1/SIAH2 and TRIAD1 Kristin Pietschmann a , Marc Buchwald a , Sylvia Müller a , Shirley K. Knauer b , Manfred Kögl c , Thorsten Heinzel a , Oliver H. Krämer a,∗ a
Institute of Biochemistry and Biophysics, Center for Molecular Biomedicine (CMB), Department of Biochemistry, University of Jena, Hans-Knöll-Str. 2, 07745 Jena, Germany Center for Medical Biotechnology (ZMB), Department of Molecular Biology II, University of Duisburg-Essen, Campus Essen, Universitätsstraße 5, 45141 Essen, Germany c German Cancer Research Center, Preclinical Target Development, and Genomics and Proteomics Core Facilities, 69120 Heidelberg, Germany b
a r t i c l e
i n f o
Article history: Received 29 June 2011 Received in revised form 5 October 2011 Accepted 16 October 2011 Available online 22 October 2011 Keywords: Ubiquitinylating enzymes TRIAD1 SIAH UBCH8 Proteasomal degradation PML–RAR␣
a b s t r a c t The ubiquitin proteasome system plays an important role in normal and malignant hematopoiesis and relies on the concerted action of three enzyme families. The E2 ubiquitin conjugase UBCH8 (ubiquitin conjugating enzyme [human] 8) cooperates with the E3 ubiquitin ligases SIAH1 and SIAH2 (seven in absentia homolog 1/2) to mediate the proteasomal degradation of oncoproteins. One such protein is the leukemia fusion protein PML–RAR␣ (promyelocytic leukemia–retinoic acid receptor␣) that is associated with acute promyelocytic leukemia. A limited number of UBCH8 interaction partners that participate in the UBCH8-dependent depletion of cancer-relevant proteins are known. We report here that TRIAD1 (two RING fingers and DRIL [double RING finger linked] 1), an E3 ubiquitin ligase relevant for the clonogenic growth of myloid progenitors, binds UBCH8 as well as PML–RAR␣. Moreover, there is concurrent induction of TRIAD1 and UBCH8 upon combinatorial treatment of acute promyelocytic leukemia cells with the pro-apoptotic epigenetic modulator valproic acid and the differentiation inducing agent all-trans retinoic acid. However, in sharp contrast to SIAH1/SIAH2 and UBCH8, TRIAD1 binding to PML–RAR␣ has no effect on its turnover. In summary, our data exclude TRIAD1 as crucial regulator of the leukemic determinant PML–RAR␣, but highlight the prominence of the UBCH8/SIAH axis in PML–RAR␣ degradation. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction In light of the crucial role of the ubiquitin proteasome system (UPS) for health and disease, detailed characterization of this system and its regulators is highly warranted (Kirkin and Dikic, 2011). Tagging of proteins with lysine K48-linked polyubiquitin chains allows their rapid elimination via the proteasome, an intracellular multi-protease complex (Hochstrasser, 2009). Ubiquitinylation is catalyzed by an E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzymes, and E3 ubiquitin ligases. The E1 activates ubiquitin
Abbreviations: APL, acute promyelocytic leukemia; ATRA, all-trans retinoic acid; HDACi, histone deacetylase inhibitor; PML–RAR␣, promyelocytic leukemia–retinoic acid receptor ␣; SIAH, seven in absentia homolog; TRIAD1, two RING fingers and DRIL (double RING finger linked) 1; UBCH, ubiquitin conjugating enzyme (human); VPA, valproic acid. ∗ Corresponding author. Tel.: +49 3641 949362; fax: +49 3641 949352. E-mail addresses: [email protected] (K. Pietschmann), [email protected] (M. Buchwald), [email protected] (S. Müller), [email protected] (S.K. Knauer), [email protected] (M. Kögl), [email protected] (T. Heinzel), [email protected] (O.H. Krämer). 1357-2725/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2011.10.008
ATP-dependently and transfers it to an E2 enzyme. RING domain E3s mediate substrate binding and recruit E2s, thereby allowing the transfer of the E2 ubiquitin cargo to a substrate. Dedicated substrate/E3 pairing permits precise ubiquitinylation. Specificity of ubiquitin ligases is additionally guaranteed by their ability to associate with different ubiquitin conjugases. Furthermore, ubiquitin ligases themselves can be targeted for proteasomal degradation by their cognate ubiquitin conjugase(s). In line with these findings we could show in a previous study that the E3 ubiquitin ligase SIAH1 (seven in absentia homolog 1) and the E2 ubiquitin conjugase UBCH8 (ubiquitin conjugating enzyme [human] 8) cooperatively promote the turnover of the UBCH8 interacting E3 ligase RLIM (RING finger LIM domain-binding protein) (Krämer et al., 2008). Various studies indicate that the UPS regulates proliferation, differentiation, and apoptosis during hematopoiesis (Crawford et al., 2008; Heuzé et al., 2008; Marteijn et al., 2006). An example for successful therapeutic activation of the UPS is acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemias characterized by blasts blocked at the promyelocytic stage (Okuno et al., 2004). In 95% of all cases, APL cells express PML–RAR␣ (promyelocytic leukemia–retinoic acid receptor ␣) encoded by
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the translocation t(15;17) (Perissi et al., 2010). In contrast to the RAR␣ transcription factor, PML–RAR␣ is not activated upon physiological concentrations of retinoic acid. Subsequently, differentiation genes positively controlled by RAR␣ become repressed by PML–RAR␣ under physiological conditions (Boukarabila et al., 2009; Epping et al., 2007; Mercurio et al., 2010; Perissi et al., 2010). Pharmacological doses of all-trans retinoic acid (ATRA) evoke proteasomal degradation of PML–RAR␣ and a co-repressor/coactivator exchange turning PML–RAR␣ into a potent transcriptional activator. Consequently, promyelocytic leukemia cells become mature white blood cells (Brown et al., 2009; Duprez et al., 2003; Mercurio et al., 2010; Perissi et al., 2010). HDACI (histone deacetylase inhibitors) alter gene expression and cellular signaling by inducing histone and non-histone protein acetylation (Buchwald et al., 2009; Spange et al., 2009). Such agents are able to correct dysregulated repressive transcription patterns. Moreover, by augmenting the expression of the ubiquitin conjugase UBCH8, these drugs trigger the proteasomal degradation of proteins associated with tumorigenesis. One example is PML–RAR␣, as it is targeted for degradation by the HDACimodulated UBCH8–SIAH1–proteasome axis (Buchwald et al., 2009; Krämer et al., 2008). Contrary to retinoids, HDACi mainly evoke caspase-dependent apoptosis of PML–RAR␣-positive NB4 cells. A deeper insight of regulatory mechanisms controlling the abundance of this oncoprotein may provide new avenues for therapy of patients suffering from APL (Buchwald et al., 2010; Krämer et al., 2008; Mercurio et al., 2010; Müller and Krämer, 2010; Perissi et al., 2010). In this study we aimed for the identification of so far unknown UBCH8 interacting proteins that might participate in the UBCH8-dependent degradation of cancer-relevant oncoproteins. By systematic yeast two-hybrid (Y2H) screens we identified the ubiquitin ligase TRIAD1 (Two RING fingers and DRIL [double RING finger linked]-1) as a novel UBCH8 interaction partner. In vitro binding and cellular co-localization studies confirmed the association of TRIAD1 with UBCH8. Co-expression studies revealed that TRIAD1 itself is not targeted for degradation by UBCH8 and its cooperating SIAH ubiquitin ligases. We furthermore disclose the interaction of TRIAD1 with the UBCH8 substrate PML–RAR␣. Moreover, we show that ATRA and the HDACi valproic acid (VPA) concurrently induce UBCH8 and TRIAD1 expression in cells derived from the bone marrow of a patient with APL in relapse. Our work though also demonstrates that the interaction of TRIAD1 with PML–RAR␣ and UBCH8 neither leads to PML–RAR␣ protein destabilization nor does it interfere with the very potent UBCH8/SIAH1- or UBCH8/SIAH2mediated degradation of PML–RAR␣. 2. Materials and methods 2.1. Drugs and chemicals The proteasome inhibitor Z-Leu-Leu-Leu-al (MG132) was purchased from Axxora; ATRA and VPA were from Sigma–Aldrich.
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1999); TRIAD1-Myc (Marteijn et al., 2005). The plasmid encoding GST-UBCH7 was created using the primers 5 -CGGGATCCCGATGGCGGCCAGCAGGAGGC-3 and 5 GGAATTCCTTAGTCCACAGGTCGCTTTTCCCCTA-3 . The PCR product was ligated into BamHI/EcoRI sites of pGEX-5X-1 (GE Healthcare). The UBCH7-V5 encoding plasmid was constructed by PCR amplification using 5 -GCCACCATGGCGGCCA-GCAGGAGGC-3 and 5 -GTCCACAGGTCGCTTTTCCCCATATT-3 and ligated into the pcDNA3.1/V5-His-TOPO vector as stated by the manufacturer (Invitrogen). All constructs were verified by sequencing. 2.4. Yeast two-hybrid screen Automated Y2H screens were performed as described in references (Albers et al., 2005; Albert et al., 2003; Lamesch et al., 2007 human genes). Human cDNA libraries from testis and brain (Clonetech) as well as a library of individually cloned full-length open reading frames (from cDNAs of 10.070 different genes) were screened for full-length UBCH8 as the bait. 2.5. Quantitative real-time PCR Cellular mRNA was isolated and cDNA was synthesized as explained in (Krämer et al., 2008). Data obtained were analyzed with the delta-Cq quantification model (Hellemans et al., 2007) using three reference genes (HMBS, GAPDH, and RPL13A). These were verified with the geNorm program (Vandesompele et al., 2002). Primer sequences for quantitative real-time PCR (qPCR) were: TRIAD1 fwd 5 -CGGGTACAGGAGCCTAGAGCTCGCCG-3 and rev 5 GGATTGTGGCACAGTCTGTGGGTGCG-3 ; UBCH8 fwd 5 -TGGCGAGCATGCGAGTGGTGAAGG-3 and rev 5 -CTGGACAGGTTCCGCAGGTATGGG-3 ; GAPDH fwd 5 -TGCACCACCAACTGCTTAGC-3 and rev 5 -GGCATGGACTGTGGTCATGAG-3 ; RPL13A fwd 5 -CCTGGAGGAGAAGAGGAAAGAGA-3 and rev 5 -TTGAGGACCTCTGTGTATTTGTCAA-3 ; HMBS fwd 5 -GGCAATGCGGCTGCAA-3 and rev 5 -GGGTACCCACGCGAATCAC-3 . 2.6. Transfection assays Transient protein expression in HEK293T cells was achieved with PEI (Sigma–Aldrich) or Lipofectamine2000 (Invitrogen) (Krämer et al., 2009). Cells were harvested after 24 h. Empty vectors pcDNA3.1 or pSG5, as applicable regarding the coding plasmids, were used to obtain equal amounts of total DNA transfected (2 g/106 cells). We noted that it is important to use the same plasmid backbone for this as each appeared to have a different impact on general transfection efficiency. A plasmid encoding green fluorescent protein (pEGFP, 0.05 g) was co-transfected to monitor transfection efficiency where applicable. 2.7. Cell lysis, immunoblot, immunofluorescence, and microscopy
2.2. Cell lines Cell lines were cultured as stated (Buchwald et al., 2010; Krämer et al., 2008). 2.3. Plasmids The following plasmids have been described previously: UBCH8-V5, GST-UBCH8, FLAG-PML–RAR␣, Myc-PML–RAR␣GFP, pEGFP, SIAH1C72S ; SIAH1, SIAH2 (Krämer et al., 2008); HA-SIAH1 (Crone et al., 2011); Myc-SIAH2 (Germani et al.,
Lysate preparation and immunoblot techniques are summarized in (Buchwald et al., 2010; Krämer et al., 2008). Microscopy analyses of HeLa cells were performed as described (Knauer et al., 2007). NB4 suspension cells were fixed on frosted glass slides by cytospin centrifugation for 10 min at 100 g before staining. 2.8. GST pull-down This method was carried out as described in references (Buchwald et al., 2010; Krämer et al., 2008).
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2.9. Antibodies Antibodies used for immunoblot were purchased from Santa Cruz Biotechnology: SIAH-1, #sc-5506; SIAH-2, #sc-5507; TRIAD1, #sc-134113; C/EBP, #sc-150; GFP, #sc-9996; HSP90, #sc-13119; Caspase 3, #sc-7272; Sigma–Aldrich: Actin, A2066; Tubulin, #T5168; FLAG, #F-3165; Invitrogen: V5-Tag, #46-0705; New England Biolabs: Caspase 3 cleaved, #9664; Myc-Tag, #2276; Abgent: UBCH8, #AP21185b; Aviva: TRIAD1, ARP34418; Upstate: H3-Ac, 06-599. The antibody against RAR␣/PML–RAR␣ was a gift from Dr. Gronemeier, Straßbourg. 2.10. Flow cytometry analyses and apoptosis assays R-phycoerythrin (RPE)-conjugated anti-CD11b (Dako, #R0841) was used to stain CD11b-positive NB4 cells. For staining, 5 × 105 cells were washed twice with PBS (137 mM NaCl, 8 mM Na2 HPO4 , 2.7 mM KCl, 1.4 mM KH2 PO4 , pH 7.25) and re-suspended in PBA buffer (PBS, 2 mg/ml BSA, 0.05% NaN3 ) with 5 l of RPE-anti-CD11b. Fluorescence was analyzed using the FACS-Canto device (BD Biosciences) (Krämer et al., 2008; Schneider et al., 2010). 3. Results 3.1. Interaction of UBCH8 with TRIAD1 By conducting Y2H screens to identify novel interacting partners of UBCH8 we isolated 145 clones coding for TRIAD1 (Fig. 1a). TRIAD1 (alternatively HARI2 or ARIH2) is a RING-type ubiquitin ligase characterized by a tripartite cysteine-rich domain (Marteijn et al., 2005). The related protein ARIH1 (HHARI), which is a known UBCH8 interacting ubiquitin ligase (Moynihan et al., 1999), and the mitochondrial protein TIMM8A (Tranebjaerg et al., 2001) were also identified to interact with UBCH8 as bait. To validate the UBCH8/TRIAD1 interaction found in the Y2H screens we performed GST pull-down experiments. This approach approved the previously described interaction of TRIAD1 with UBCH7, an E2 related to UBCH8 (Marteijn et al., 2005), and confirmed the binding of UBCH8 and TRIAD1 from whole cell lysates (Fig. 1b). Additionally, TRIAD1 could be co-immunoprecipitated with UBCH7 and UBCH8 (Fig. 1c). In summary, we verified the interaction of TRIAD1 and UBCH8 with three different binding assays. 3.2. Stability of TRIAD1 and interplay between TRIAD1 and SIAH1/SIAH2 Ubiquitin ligases can be very unstable and their amounts are often modulated by associated ubiquitin conjugases and ligases (Depaux et al., 2006; Hu and Fearon, 1999; Krämer et al., 2008; Nagel et al., 2011). Based on these data we investigated whether overexpression of UBCH7 or UBCH8 evokes degradation of TRIAD1. However, co-expression of these proteins had no significant impact on TRIAD1 protein levels (Fig. 2a). SIAH proteins are very potent ubiquitin ligases that interact with UBCH8 and can hierarchically promote proteasomal degradation of other E3 ubiquitin ligases (Calzado et al., 2009; House et al., 2009; Krämer et al., 2008; Nagel et al., 2011). Of note, TRIAD1 also contains structures resembling the PML–RAR␣ coiled coil domain which allows PML–RAR␣ to be recognized as a target by SIAHs (Fanelli et al., 2004; Marteijn et al., 2005). Since we could co-precipitate overexpressed SIAH1 with TRIAD1 (data not shown), we considered to test if SIAH1/SIAH2 targeted TRIAD1 for degradation. Since levels of either E2 or E3 enzymes could be limiting for substrate degradation, we also co-expressed UBCH7 or UBCH8 together with SIAH1 or SIAH2 and assessed TRIAD1 protein stability by immunoblotting.
Fig. 1. Physical interaction of UBCH8 with TRIAD1. (a) Full-length UBCH8 was used as bait protein for yeast two-hybrid screening of three prey libraries. The table displays prey proteins, which have been identified to interact with UBCH8 in these screens. Numbers of times each clone has been isolated and number of different cDNA libraries (o, open-reading-frame; t, testis; b, brain), in which this interaction has been found are also listed. (b) Heterologously expressed GST-UBCH8 was precipitated after incubation with cell lysates from HEK293T cells expressing TRIAD1-Myc. As negative control, pull-down was performed with GST alone. GST-UBCH7 served as positive control (input represents 10% of cell lysates used for pull-down). The precipitates were probed with antibodies against the Myc-Tag. Levels of GST fusion proteins were detected by Ponceau-staining of the membranes (data not shown). The lower panel displays densitometric analyses for TRIAD1 protein bands (means ± SE, n = 3). (c) UBCH7-V5 or UBCH8-V5 were co-expressed with TRIAD1-Myc in HEK293T cells. UBCH7/8 was immunoprecipitated from cell lysates. The presence of TRIAD1, UBCH7 and UBCH8 in the precipitates was analyzed by immunoblotting (pre, pre-immune serum; IP, immunoprecipitation; n = 2).
However, TRIAD1 remained stable upon expression of these ubiquitin conjugases and ligases (Fig. 2b). We conclude that SIAH1/2 and UBCH7/8 unlikely regulate the protein turnover of TRIAD1. SIAH proteins are known to undergo rapid proteasomal degradation. Furthermore, they act as their own E3 ubiquitin ligases to facilitate autoubiquitinylation (Depaux et al., 2006; Hu and Fearon, 1999). This finding prompted us to compare the general turnover rates of SIAH and TRIAD1 proteins. We treated HEK293T cells ectopically expressing these ubiquitin ligases with the proteasome inhibitor MG132 and analyzed their protein stability. Whereas the levels of TRIAD1 remained unchanged upon proteasomal inhibition, detectable amounts of SIAH1 and SIAH2 increased strongly under such conditions (Fig. 2c). These data reveal dissimilar stability profiles and separate degradation pathways for TRIAD1, SIAH1, and SIAH2.
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Fig. 2. Overexpressed TRIAD1 is not degraded by UBCH7, UBCH8, SIAH1, and SIAH2, and is not stabilized by proteasomal inhibition. (a) TRIAD1-Myc was expressed with UBCH7-V5 or UBCH8-V5. Protein levels were analyzed by immunoblot. Tubulin served as loading control. (b) HEK293T cells were transfected with vectors for TRIAD1-Myc, HA-SIAH1, Myc-SIAH2, UBCH7-V5, or UBCH8-V5. Protein levels were detected by immunoblot analyses as indicated. (c) HEK293T cells were transfected with vectors for TRIAD1-Myc, SIAH1, or SIAH2. Cells were treated with MG132 (20 M, 8 h) followed by immunoblot (S1, SIAH1; S2, SIAH2).
3.3. Induced expression of UBCH8 and TRIAD1 by single or combinatorial treatment of APL cells with VPA and ATRA HDACi as well as ATRA can induce PML–RAR␣ degradation in APL cells (Brown et al., 2009; Krämer et al., 2008). We previously showed that treatment of APL cells with the HDACi VPA upregulates the expression of the TRIAD1 binding partner UBCH8, which promotes proteasomal cleavage of PML–RAR␣ (Krämer et al., 2008). Recently, TRIAD1 mRNA was shown to be induced by ATRA in NB4 cells (Marteijn et al., 2005). These data suggest a link between the expression of TRIAD1, UBCH8, PML–RAR␣, and ATRA or HDACi. Therefore, we analyzed the effect of both substances on the regulation of TRIAD1 and UBCH8 by single or co-treatment of NB4 APL cells with VPA and ATRA. Several attempts failed to detect endogenous TRIAD1 at the protein level with antibodies commercially available. Of note, these did also not detect overexpressed Myc-TRIAD1 in HEK293T cells, although its expression was confirmed with antibodies against the Myc epitope. Due to the lack of working antibodies for the detection of TRIAD1 at protein level, we performed quantitative real-time PCR (qPCR) to analyze the regulation of TRIAD1 mRNA expression. As HDACi relax chromatin structures and affect the expression of a wide variety of genes, expression stability of reference genes used for normalization has to be validated. We established a qPCR system with three different reference genes. We noticed a significant induction of TRIAD1 mRNA in NB4 cells by combinatorial treatment with VPA plus ATRA. In contrast, VPA single treatment did not alter TRIAD1 mRNA levels and ATRA only marginally affected expression of this mRNA at 24 h (Fig. 3a). This finding contrasts a previous work that reported ATRA-induced upregulation of TRIAD1 following 24 h of stimulation. This discrepancy might be explained by differences in the applied mRNA detection methods (Northern blot versus qPCR) and the elaborated normalization method we used in our system. Nevertheless, we detected an approximately
Fig. 3. VPA and ATRA alter expression of TRIAD1 and UBCH8. (a) NB4 cells were treated with 1.5 mM VPA (V), 1 M ATRA (A), or left untreated (C) for 24 h. Expression of TRIAD1 mRNA was analyzed by quantitative real-time PCR. Relative expression relates to corresponding values for samples from untreated cells, set as 1 (means ± SE; n = 3; **p < 0.01). GAPDH, HMBS, and RPL13A served as reference genes. (b) NB4 cells were treated with 1.5 mM VPA (V), 1 M ATRA (A), or left untreated (C) for 24 h. Expression of UBCH8 protein was analyzed by immunoblot. Tubulin served as loading control. (c) Activity of ATRA was tested by flow cytometry with an antibody directed against the cell surface protein CD11b (means ± SE, n = 3). (d) Lysates of untreated (C) and ATRA-treated (A) NB4 cells were analyzed for PML–RAR␣ and C/EBP by immunoblot. C/EBP antibodies generated a specific band (>) and a nonspecific band (*) useful as a loading control. (e) NB4 cells were treated with 1.5 mM VPA for 2–24 h (−, untreated). Levels of endogenous PML–RAR␣, UBCH8, Caspase 3 (Casp. 3; fl, full length; cl, cleaved), acetyl-histone H3 (H3-Ac), and Tubulin were determined by immunoblot.
twofold increase of TRIAD1 after 40 h of ATRA treatment (data not shown). To this end our data reveal differential regulation of TRIAD1 in PML–RAR␣-positive leukemia cells upon single or combinatorial treatment with ATRA and VPA. We already described that UBCH8 is induced by HDACi in APL and other leukemic cells (Buchwald et al., 2010; Krämer et al., 2008). Interestingly, we detected enhanced expression of UBCH8
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by VPA and also by ATRA treatment as well as by the combination of both drugs in NB4 cells (Fig. 3b). To ensure activity of ATRA in our experimental setting we tested its potency via detection of typical molecular signs of granulocytic differentiation. We assessed the accumulation of the transcription factor C/EBP (CCAAT/enhancer-binding protein ), which regulates expression of various genes involved in granulocytic differentiation, and the induction of the cell surface marker CD11b (Brown et al., 2009; Duprez et al., 2003; Mercurio et al., 2010; Perissi et al., 2010). ATRA potently induced up to 90% of CD11b positive cells (Fig. 3c) as well as accumulation of C/EBP protein (Fig. 3d). These molecular alterations indicating promyelocyte differentiation correlated with the loss of PML–RAR␣ antagonizing cellular maturation. We equally assured the potency of the HDACi VPA with protein expression analysis showing a time-dependent decrease of PML–RAR␣ and induction of UBCH8, histone hyperacetylation, and caspase activity relevant in apoptosis (Fig. 3e and data not shown). Attenuation of PML–RAR␣ correlated with the induction of UBCH8 before executioner Caspase 3 processing and activation, and thus is congruent with our published data revealing early proteasomal proteolysis of this oncoprotein in NB4 cells treated with VPA (Krämer et al., 2008). 3.4. Effects of TRIAD1, SIAH1, and SIAH2 on PML–RAR˛ stability From these data we conclude that TRIAD1 levels upon VPA or ATRA single treatment do not correlate with PML–RAR␣ degradation induced by these substances (Brown et al., 2009; Krämer et al., 2008). However, the basally expressed TRIAD1 may affect the UBCH8-dependent protein degradation, since proteasomal degradation has been shown to be regulated by limiting amounts of UBCH8 (Krämer et al., 2003). In addition, it remains conceivable that VPA + ATRA-induced TRIAD1 contributes to the degradation of PML–RAR␣. Previously we reported, that UBCH8 in cooperation with the E3 ubiquitin ligase SIAH1 promotes the proteasomal degradation of PML–RAR␣ (Krämer et al., 2008), thereby serving as a reference. Moreover, the fact that TRIAD1 interacts with UBCH8, which can increase PML–RAR␣ ubiquitinylation, raised the question whether TRIAD1 functioned as an additional ubiquitin ligase on PML–RAR␣. To address this issue, we first tested whether there was co-localization of these E2/E3 enzymes and PML–RAR␣. Therefore, we detected ectopically expressed PML–RAR␣ and SIAH1 in HeLa cells by immunofluorescence microscopy. Overexpressed SIAH1 potently promotes proteasomal degradation of PML–RAR␣ and is hardly detectable by immunofluorescence microscopy due to rapid autoubiquitinylation and degradation (Hu and Fearon, 1999; Krämer et al., 2008; Nagel et al., 2011). Thus, we used an established RING-mutant of SIAH1 (SIAH1C72S ), which is deficient in binding E2 ubiquitin conjugases and hence cannot mediate substrate- and autoubiquitinylation (Depaux et al., 2006; Hu and Fearon, 1999; Krämer et al., 2008; Nagel et al., 2011; Winter et al., 2008; Zhao et al., 2011). We could visualize SIAH1C72S and PML–RAR␣ co-localization in a cellular context (Fig. 4a). Microscopical inspection of NB4 APL cells to detect endogenous SIAH1 and PML–RAR␣ supported this observation (Suppl. Fig. S1). In addition to the SIAH1/UBCH8 interaction, we found that the E3 ligase TRIAD1 interacted with UBCH8. Hence, we asked whether TRIAD1 could also recruit PML–RAR␣ to UBCH8. Fluorescence microscopy revealed a partial co-localization of PML–RAR␣ with nuclear UBCH8 and TRIAD1 (Fig. 4b and c). Moreover, a co-immunoprecipitation binding assay biochemically confirmed physical interaction of TRIAD1 and PML–RAR␣ (Fig. 4d). Based on our findings that TRIAD1 interacts with both, UBCH8 and PML–RAR␣, we tested whether this association – equally to the SIAH1/2-mediated degradation of PML–RAR␣ – evoked a loss of this fusion protein. Immunoblot analyses confirmed functionality of SIAH1 as well as SIAH2 against PML–RAR␣. However,
Fig. 4. PML–RAR␣ is degraded by SIAH proteins independently of TRIAD1. (a) HeLa cells were transfected with GFP-PML–RAR␣ and RING mutant SIAH1 (SIAH1C72S ) which was detected by ␣-SIAH1 antibodies (phase, phase contrast microscopy; scale bar = 10 m). (b) GFP-PML–RAR␣ and UBCH8-V5 were expressed in HeLa cells. Localization and overlay of these proteins was revealed with GFP emission signals and probing for V5 (scale bar = 10 m). (c) GFP-PML–RAR␣ and TRIAD1-Myc were expressed in HeLa cells. Localization and overlay of these proteins was revealed with GFP emission signals and probing for Myc (scale bar = 10 m). (d) TRIAD1-Myc was co-expressed with Flag-PML–RAR␣ in HEK293T cells. TRIAD1 was immunoprecipitated with ␣-Myc antibodies and the presence of TRIAD1 and PML–RAR␣ in the precipitates was analyzed by immunoblotting (pre, pre-immune serum; IP, immunoprecipitation; n = 2). (e) FLAG-PML–RAR␣ was coexpressed with SIAH1, SIAH2, and TRIAD1-Myc in HEK293T cells. Expression levels were analyzed by immunoblotting. Tubulin protein levels show equal loading.
co-expressed TRIAD1 had no destabilizing effect (Fig. 4e, lanes 2–4). Recently, overexpressed TRIAD1 was found to interact with the transcriptional repressor GFI1 (growth factor independence 1), thereby blocking other ubiquitin ligases catalyzing its ubiquitinylation and proteasomal degradation (Marteijn et al., 2007). Therefore, we tested whether TRIAD1 also interfered with the SIAH1/2-mediated degradation of PML–RAR␣. However, the specific interaction of TRIAD1 with PML–RAR␣ did not affect SIAH1dependent PML–RAR␣ degradation (Fig. 4e, lanes 5 + 6). Thus, inhibition of proteasomal degradation by TRIAD1 depends on the substrate and most likely also on E2/E3 enzyme pairs cooperating. Fig. 5 summarizes our data shown here as a table and in a model. 4. Discussion In this study, we identify the E3 ubiquitin ligase TRIAD1 as a novel interacting partner of the E2 ubiquitin conjugase UBCH8. Initial data gained in Y2H screens could be verified by pulldown experiments. We also report that both proteins are upregulated by co-treatment of NB4 APL cells with VPA and ATRA. By different
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Fig. 5. Interaction of UBCH8 with SIAH1/2, but not with TRIAD1 targets PML–RAR␣ to proteasomal degradation. Model and table summarize data presented here combined with previous results (Fanelli et al., 2004; Krämer et al., 2008) (+, yes/found/detected; −, no/not detected/unlikely; *, linked to E3 SIAH1 or SIAH2; n.t., not tested).
experimental approaches we reveal that TRIAD1 also interacts and co-localizes with PML–RAR␣. The fact that this association has no effect on its stability argues for a more prominent role for SIAH1/SIAH2 in the control of PML–RAR␣ levels. We present reliable data that demonstrate TRIAD1–UBCH8 interaction. Nevertheless, a previous report, describing a Y2H screen for TRIAD1 binding partners, excluded binding of UBCH8 to this ubiquitin ligase (Marteijn et al., 2005). This discrepancy can be explained by the use of truncated (amino acids 1–119 or 109–493) instead of full-length TRIAD1 in our screen. Apparently, this protein forms interaction surfaces with UBCH8 that are disrupted when expressed discontinuously. Moreover, several E3 ubiquitin ligases have been reported to interact with UBCH8 including TRIAD family members (Beasley et al., 2007; Chuang and Ulevitch, 2004; Fearns et al., 2006; Huang et al., 2006; Moynihan et al., 1999). Our data are further supported by the finding that the UBCH8 related UBCH7 was also shown to bind TRIAD1 (Marteijn et al., 2005). The role of TRIAD1 in proliferation and differentiation of normal and malignant hematopoietic cells has been investigated (Marteijn et al., 2007, 2005, 2009; Wang et al., 2011). These studies demonstrate that TRIAD1 blocks clonogenic growth of primary myeloid progenitor cells but may not promote differentiation. PML–RAR␣ however potently halts myeloid differentiation (Perissi et al., 2010). The fact that TRIAD1 showed no attenuating effect on PML–RAR␣ perfectly corresponds to the notion that TRIAD1 did not enhance differentiation. Furthermore, degradation of PML–RAR␣ is independent of TRIAD1 upregulation in NB4 cells solely treated with VPA or ATRA, standing in perfect agreement with the finding that overexpression of TRIAD1 did not reduce PML–RAR␣ levels. In contrast, both agents induce UBCH8, which in turn cooperates with SIAH1 and SIAH2 in the proteasomal degradation of PML–RAR␣
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(Buchwald et al., 2009; Krämer et al., 2008). Therefore, SIAH1 or SIAH2 together with UBCH8 appear to restrain the development of APL.Until now, no poly-ubiquitinylation substrate of TRIAD1 has been identified, and the role of TRIAD1 for cellular processes therefore remains largely unknown. In addition, previous data suggest that TRIAD1 may mark proteins with ubiquitin in a non-proteolytic manner (Marteijn et al., 2009). Since we found upregulation of TRIAD1 in APL cells treated with the combination of VPA and ATRA, we cannot exclude that TRIAD1 cooperates with UBCH8 in other processes. In order to better understand TRIAD1 functions, future studies have to clarify how association of TRIAD1 and UBCH8 affects targets and cellular fate.In our hands, TRIAD1 is stable upon proteasome inhibition and co-expression with ubiquitin ligases and conjugases. This is congruent with data from Marteijn et al. (2005) demonstrating that inhibition of the proteasome merely stabilizes a minor fraction of poly-ubiquitinylated TRIAD1 without significantly affecting the total TRIAD1 protein level. Furthermore, agents targeting proteasomes specifically affected TRIAD1 functions less efficiently than substances also inhibiting non-proteasomal proteases (Marteijn et al., 2005). These findings and our data suggest a proteasome-independent proteolytic degradation pathway for TRIAD1. Recently, the degradation of the transcriptional repressor GFI1 (Möröy, 2005) was found to be blocked by heterologously overexpressed TRIAD1 (Marteijn et al., 2007). Nonetheless, in our experiments TRIAD1 could not block SIAH1/SIAH2-mediated degradation of PML–RAR␣. Although we cannot exclude that TRIAD1 competes with endogenous expressed E2 and E3 enzymes, our data regarding PML–RAR␣ stability also indicate for dominant functions of SIAH1 and SIAH2 over TRIAD1 in our model system. In sum, we have identified a new UBCH8 interacting ubiquitin ligase, TRIAD1, which equally to UBCH8 is upregulated in APL cells upon combinatorial exposure to VPA plus ATRA. However, our data exclude TRIAD1 as crucial regulator of the leukemic fusion protein PML–RAR␣, and they argue that the specific role of TRIAD1 in APL has still to be defined. It appears that SIAH proteins and TRIAD1 share binding partners though having very different biochemical properties within the cell. Funding This work was supported by grants from the Landesprogramm “ProExzellenz” des Freistaates Thüringen (PE 123-2-1, to OHK), from the Deutsche Krebshilfe (grant number 109265, to OHK), and from the Deutsche Forschungsgemeinschaft (SFB 604, to TH). Acknowledgements We thank S. Reichardt and Dr. G. Greiner for excellent technical assistance, Dr. C. Bier and Dr. S. Drube for supportive discussions and technical help. Dr. R. Marschalek, Dr. N. Varin-Blank, Dr. T.G. Hofmann, Dr. B.A. van der Reijden, and Dr. S. Minucci kindly provided expression constructs for SIAH1, SIAH2, Myc-SIAH2, HASIAH1, TRIAD1-Myc, and tagged PML–RAR␣. Dr. H. Gronemeier kindly provided the antibodies against RAR␣. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biocel.2011.10.008. References Albers M, Kranz H, Kober I, Kaiser C, Klink M, Suckow J, et al. Automated yeast twohybrid screening for nuclear receptor-interacting proteins. Mol Cell Proteomics 2005;4:205–13.
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