The ubiquitin–protein ligase Itch regulates p73 stability
2005; Springer Nature; Volume: 24; Issue: 4 Linguagem: Inglês
10.1038/sj.emboj.7600444
ISSN1460-2075
AutoresMario Rossi, Vincenzo De Laurenzi, Eliana Munarriz, Douglas R. Green, Yun‐Cai Liu, Karen H. Vousden, Gianni Cesareni, Gerry Melino,
Tópico(s)Ubiquitin and proteasome pathways
ResumoArticle27 January 2005free access The ubiquitin–protein ligase Itch regulates p73 stability Mario Rossi Mario Rossi Department of Biology, University of Rome Tor Vergata, Rome, Italy Medical Research Council, Toxicology Unit, Leicester University, Leicester, UK Search for more papers by this author Vincenzo De Laurenzi Vincenzo De Laurenzi Biochemistry Laboratory, IDI-IRCCS, C/O Department of Experimental Medicine and Biochemical Sciences, University of Rome 'Tor Vergata', Rome, Italy Medical Research Council, Toxicology Unit, Leicester University, Leicester, UK Search for more papers by this author Eliana Munarriz Eliana Munarriz Medical Research Council, Toxicology Unit, Leicester University, Leicester, UK Search for more papers by this author Douglas R Green Douglas R Green La Jolla Institute for Allergy and Immunology, San Diego, CA, USA Search for more papers by this author Yun-Cai Liu Yun-Cai Liu La Jolla Institute for Allergy and Immunology, San Diego, CA, USA Search for more papers by this author Karen H Vousden Karen H Vousden Beatson Institute for Cancer Research, Garscube Estate, Bearsden, Glasgow, UK Search for more papers by this author Gianni Cesareni Corresponding Author Gianni Cesareni Department of Biology, University of Rome Tor Vergata, Rome, Italy Search for more papers by this author Gerry Melino Corresponding Author Gerry Melino Biochemistry Laboratory, IDI-IRCCS, C/O Department of Experimental Medicine and Biochemical Sciences, University of Rome 'Tor Vergata', Rome, Italy Medical Research Council, Toxicology Unit, Leicester University, Leicester, UK Search for more papers by this author Mario Rossi Mario Rossi Department of Biology, University of Rome Tor Vergata, Rome, Italy Medical Research Council, Toxicology Unit, Leicester University, Leicester, UK Search for more papers by this author Vincenzo De Laurenzi Vincenzo De Laurenzi Biochemistry Laboratory, IDI-IRCCS, C/O Department of Experimental Medicine and Biochemical Sciences, University of Rome 'Tor Vergata', Rome, Italy Medical Research Council, Toxicology Unit, Leicester University, Leicester, UK Search for more papers by this author Eliana Munarriz Eliana Munarriz Medical Research Council, Toxicology Unit, Leicester University, Leicester, UK Search for more papers by this author Douglas R Green Douglas R Green La Jolla Institute for Allergy and Immunology, San Diego, CA, USA Search for more papers by this author Yun-Cai Liu Yun-Cai Liu La Jolla Institute for Allergy and Immunology, San Diego, CA, USA Search for more papers by this author Karen H Vousden Karen H Vousden Beatson Institute for Cancer Research, Garscube Estate, Bearsden, Glasgow, UK Search for more papers by this author Gianni Cesareni Corresponding Author Gianni Cesareni Department of Biology, University of Rome Tor Vergata, Rome, Italy Search for more papers by this author Gerry Melino Corresponding Author Gerry Melino Biochemistry Laboratory, IDI-IRCCS, C/O Department of Experimental Medicine and Biochemical Sciences, University of Rome 'Tor Vergata', Rome, Italy Medical Research Council, Toxicology Unit, Leicester University, Leicester, UK Search for more papers by this author Author Information Mario Rossi1,3, Vincenzo De Laurenzi2,3, Eliana Munarriz3, Douglas R Green4, Yun-Cai Liu4, Karen H Vousden5, Gianni Cesareni 1 and Gerry Melino 2,3 1Department of Biology, University of Rome Tor Vergata, Rome, Italy 2Biochemistry Laboratory, IDI-IRCCS, C/O Department of Experimental Medicine and Biochemical Sciences, University of Rome 'Tor Vergata', Rome, Italy 3Medical Research Council, Toxicology Unit, Leicester University, Leicester, UK 4La Jolla Institute for Allergy and Immunology, San Diego, CA, USA 5Beatson Institute for Cancer Research, Garscube Estate, Bearsden, Glasgow, UK *Corresponding authors: Medical Research Council, Toxicology Unit, Hodgkin Building, Leicester University, Lancaster Road, PO Box 138, Leicester LE1 9HN, UK. Tel.: +44 116 252 5551; Fax: +44 116 252 5616; E-mail: [email protected] of Biology, University of Rome 'Tor Vergata', Via della Ricerca Scientifica, Rome 00133, Italy. Tel.: +39 06 72594315; Fax: +39 06 2023500; E-mail: [email protected] The EMBO Journal (2005)24:836-848https://doi.org/10.1038/sj.emboj.7600444 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info p73, a member of the p53 family of transcription factors, is upregulated in response to DNA damage, inducing cell cycle arrest and apoptosis. Besides indications that this p73 response is post-transcriptional, little is known about the underlying molecular mechanisms of p73 protein degradation. Ubiquitination and proteasomal-dependent degradation of p53 are regulated by its transcriptional target MDM2. However, unlike p53, p73 binds to, but is not degraded by, MDM2. Here we describe the binding of p73 to Itch, a Hect ubiquitin–protein ligase. Itch selectively binds and ubiquitinates p73 but not p53; this results in the rapid proteasome-dependent degradation of p73. Upon DNA damage Itch itself is downregulated, allowing p73 protein levels to rise and thus interfere with p73 function. In conclusion, we have identified a key mechanism in the control of p73 protein levels both in normal as well as in stress conditions. Introduction p73 is a member of the p53 family of transcription factors, and, like p53, has a modular structure (Figure 1A) (Kaghad et al, 1997). p73 shares a high degree of sequence homology with p53 and can bind to p53-responsive elements, activating the transcription of p53 target genes, such as those inducing cell cycle arrest and promoting apoptosis (De Laurenzi et al, 1998, 2000; Catani et al, 2002). Figure 1.p73 binds to Itch. (A) Schematic representation of the modular structure of the p73α, p73δ, p53, and Itch proteins. The main structural domains are indicated: transactivation domain (TA), DNA-binding domain (DBD), oligomerization domain (OD), sterile alpha motif (SAM), amino-terminal C2 domain (C2), WW domains (WW), and carboxyl-terminal Hect domain (HECTc). The p73 region from Met452 to Ala489, containing the PPxY motif, used as bait in the phage library panning experiment, is also shown. Bars indicate the regions of Itch expressed by the clones selected in the screening. (B) T7 plaques from the unselected library and from the enriched phage pool, transferred to a cellulose membrane and probed with GST-PY are shown. PCR demonstrating the enrichment of clones containing the Itch WW domains is shown in the lower panel. (C) GST pull-down. Hek293 cells were transfected with HA-TAp73α (TAp73α) or empty vector (pCDNA), and lysates were incubated with GST alone, or a GST fusion protein containing the four WW domains of Itch (GST-WW). The retained proteins were detected with anti-p73 antibody (upper panel). The same blot was reprobed with anti-GST polyclonal serum (lower panel). Co-IP of overexpressed proteins: HA-TAp73α or HA-TAp53 (D) or HA-ΔNp73α (E) or TAp73αY487F, TAp73αY407F, and TAp73αY407F/Y487F (F) were transiently transfected in Hek293 cells with either Myc-Itch or Myc-Itch MUT expression vectors. Cells were treated with or without MG132 before lysis. Cell extracts were IP with anti-Myc antibody. The immune complexes were subjected to western blot analysis with anti-HA antibody (upper panels). The same blots were re-probed with anti-Myc antibody (middle panels). Aliquots of total cell extracts from unprocessed cells (25 μg/lane) were directly subjected to immunoblot analysis with anti-HA antibody (lower panels). (G) Co-IP of endogenous p73 and Itch proteins. Cells were IP with antibodies against either p73 (mix of clones C17 and C20, Santa Cruz) or p53 (mix of clones D01 and 1801, Santa Cruz) and western blot was performed with antibody against: Itch, p73, p53, and Actin. As a control, IP was performed also with an anti-Actin antibody. Download figure Download PowerPoint Unlike p53, however, p73 is expressed as different isoforms (Kaghad et al, 1997; Ueda et al, 1999). Most of the variation generated by alternative splicing occurs at the 3′ end, in a part of the sequence that does not have a counterpart in p53. At least six different p73 proteins (α–η) are generated (De Laurenzi et al, 1998, 1999; Ueda et al, 1999). In addition, the p73 gene exploits an alternative promoter and an extra exon (exon 3′) to generate N-terminally truncated isoforms (ΔNp73). These variants lack the transactivation domain and act as 'dominant negatives', blocking the function of either p53 or p73 full-length proteins (Yang et al, 2000; Grob et al, 2001; Sayan et al, 2004). The relative levels of TA and ΔN isoforms determine cell fate, resulting in either growth arrest and death or uncontrolled proliferation. TAp73 steady-state protein levels are upregulated in response to DNA damage in a fashion distinct from p53 (Agami et al, 1999; Gong et al, 1999; Yuan et al, 1999), while ΔNp73 is rapidly degraded (Maisse et al, 2004). These observations suggest an important differential role for these isomers in carcinogenesis (Melino et al, 2002, 2003; Stiewe and Putzer, 2002; Zaika et al, 2002). Despite its importance, however, very little is known of the molecular mechanisms underlying the regulation of p73 protein steady-state levels. MDM2, the E3 ubiquitin (Ub) ligase that regulates the degradation of the cognate protein p53 via a proteasomal-dependent pathway, binds to p73 but does not promote its degradation (Balint et al, 1999; Dobbelstein et al, 1999; Lohrum and Vousden, 1999; Ongkeko et al, 1999; Zeng et al, 1999). In order to define the degradation pathway of p73, we searched a human cDNA library displayed on bacteriophage capsids for p73-specific binding partners. In order to identify mechanisms distinct from those of p53, we used a p73 C-terminal fragment as a bait, since this region is not present in p53 and contains a PPxY (PY) sequence that has been characterized as a binding motif for a class of WW domains (Figure 1A) (Sudol, 1996). We found that p73 binds Itch, a human Ub–protein ligase (E3). The Itch mouse homologue gene is absent in the non-agouti-lethal 18H (Itchy) mice, which display profound immune defects (Perry et al, 1998; Fang et al, 2002). Itch belongs to the Nedd4-like E3 family, and is characterized by a modular organization that includes: an N-terminal protein kinase C-related C2 domain, multiple WW domains, and a C-terminal HECT (homologous to the E6-associated protein carboxyl-terminus) Ub–protein ligase domain (Harvey and Kumar, 1999) (Figure 1A). Ub–protein ligases are involved in the multistep process that leads to ubiquitination of protein substrates. In this pathway, E3 catalyzes the final transfer of Ub to a specific substrate, thus governing the specificity of substrate recognition (Hicke, 2001; Kloetzel, 2001; Weissman, 2001). Here we show that Itch binds and ubiquitinates TAp73 and ΔNp73, but not p53, and determines p73 proteasome-dependent degradation. We also show that Itch expression is downregulated upon DNA damage, thereby allowing stabilization of the TA and ΔN p73 proteins. Our data also show that a second as yet unidentified mechanism is responsible for selective ΔNp73 degradation in response to DNA damage. Therefore, we have identified a relevant mechanism in the control of p73 levels both in normal as well as in stress conditions. Results Identification of Itch as a novel p73 interacting partner To identify new p73-binding proteins, not shared by p53, we fused a fragment of TAp73α (Met 452–Ala 489) to GST protein (Figure 1A) and used it as bait in a 'phage display' screening (Cesareni et al, 1999; Castagnoli et al, 2001). This fragment contains a region that is not homologous to p53 and contains a protein-protein binding motif, known as a PY motif (Strano et al, 2001) (Figure 1A) characterized by the consensus sequence PPxY. This motif binds to a 40 amino-acid long structural domain known as WW domain, organized to form a three-stranded, antiparallel β sheet, containing two tryptophan (W) residues, spaced 20–22 amino acids apart (Sudol, 1996). After three rounds of affinity selection, performed on a human brain cDNA library displayed by a T7 phage vector, we analyzed the resulting phage population by a plaque assay. The affinity-selected phage pool contained a high percentage (25%) of positive clones that bound the bait (Figure 1B). By comparing the frequency of positive plaques before and after selection, we estimated an enrichment of at least 60-fold (Figure 1B). Several clones contained overlapping protein fragments (all containing the WW domains) encoded by the Itch gene (Figure 1A). The enrichment of clones displaying Itch WW domains was further confirmed by performing PCR reactions with specific oligonucleotides flanking the WW domains (from Pro 317 to Pro 520) (Figure 1B). In contrast, clones containing the β-actin gene were rapidly lost during the selection process (Figure 1B). p73, but not p53, associates with Itch In order to verify that Itch associates with p73, we performed an in vitro pull-down assay. Human embryonal kidney (Hek)293 cells were transfected with either an empty vector or with a vector encoding HA-tagged TAp73α. Cell lysates were mixed separately with a sepharose resin containing either GST or the WW region of Itch fused to GST (GST-WW). TAp73α was efficiently retained by GST-WW, while no significant binding to GST alone was detected (Figure 1C). The interaction was also confirmed by co-immunoprecipitation (co-IP) of overexpressed TAp73α and Itch. As shown in Figure 1D, immunoprecipitation (IP) of Myc-tagged Itch resulted in co-IP of TAp73α. Addition of proteasome inhibitor MG132 resulted in stronger interaction. As expected, since p53 does not contain the PY motif (Figure 1A), it did not bind to Itch and could not be precipitated with Itch, regardless of the presence of the proteasome inhibitor (Figure 1D). Similarly, TAp73δ, that also lacks the PY motif (Figure 1A), could not be co-IP with Itch (data not shown). The N-terminally truncated form ΔNp73α also bound Itch (Figure 1E). To confirm that the interaction requires the PY motif of p73, we generated mutants of both the PY motifs found in p73 (Figure 1A). Figure 1F shows that mutants containing the Y487F substitution lost the ability to bind Itch, while the single mutant TAp73αY407F did not. In order to confirm that this interaction also occurs in cells at physiological concentrations, we performed co-IP of endogenous proteins (Figure 1G). Again IP of endogenous p73 co-precipitated Itch, while IP of p53 did not. These data clearly demonstrate that endogenous p73α isoforms can bind to the WW domains of Itch through their PY motif and that the interaction is selective for p73 and not shared by p53. p73 is ubiquitinated by Itch We next investigated whether p73 can serve as a substrate for the Ub–protein ligase activity of Itch. We used a recombinant Itch (GST-Itch) (Qiu et al, 2000), in a reconstituted in vitro ubiquitination system containing Ub, wheat E1, human E2 (UbcH7), ATP, and in vitro synthesized radiolabelled [35S]TAp73α protein as substrate. In the presence of purified GST-Itch, the TAp73α protein was ubiquitinated, as shown by the appearance of discrete higher molecular weight TAp73α species (Figure 2A, lane 1). To demonstrate that the appearance of ubiquitinated forms of TAp73α requires an intact Itch Hect domain, we used a previously described inactive mutant of Itch (GST-Itch MUT) (C830A) (Winberg et al, 2000). As shown in Figure 2A, this mutant, that retains the ability to bind TAp73α (Figure 1D), lost the ability to promote TAp73α ubiquitination. The inability of Itch to ubiquitinate p73δ and p53 suggests that the PY motifs are required, since these two proteins lack these motifs (Figures 1A and 2A, lanes 4 and 7). These in vitro data also show that no other factor was required for this reaction to occur. Figure 2.Itch ubiquitinates p73. (A) TAp73α and TAp73δ and p53 proteins were in vitro translated in the presence of [35S]Met and incubated with purified Itch (GST-Itch) or its catalytically inactive mutant (GST-Itch MUT) in the presence of ATP, Ub, and bacterially expressed E1 and E2 (UbcH7). Lanes 10–12 show in vitro translation with the empty vector (pCDNA). Lanes 13–16 show an aliquot (1/10) of the in vitro translated proteins used in the ubiquitination reaction. To demonstrate that p73 ubiquitination could also occurs in a more physiological system, Hek293 cells were transiently co-transfected with expression plasmids for Ub-HA, Flag-TAp73α or Flag-p53 (B), Flag-ΔNp73α (C), or Flag-TAp73αY487F, Flag-TAp73αY407F, and Flag-TAp73αY407F/Y487F (D) and Myc-Itch or Myc-Itch MUT. At 48 h after transfection, cells were treated with MG132 and then collected. Lysates were subjected to IP using an anti-Flag antibody. Immune complexes were revealed with anti-HA antibody (upper panels). No Ub-HA conjugates are present when Ub-HA is omitted from the reaction (B—lanes 10–18). The p73 and p53 protein expression levels are demonstrated by probing the same membranes with anti-Flag antibodies (middle panels), and those of Itch by probing the same membrane with anti-Myc antibodies (lower panels). Download figure Download PowerPoint We next examined if Itch can catalyze p73 ubiquitination in cells. Extracts of Hek293 cells transfected with plasmids expressing, HA-tagged Ub (Ub-HA), Myc-tagged Itch (Myc-Itch), and Flag-tagged TAp73α or p53 (Flag-TAp73α and Flag-p53), were subjected to IP with anti-Flag antibodies and detected with anti-HA and anti-Flag western blots. As shown in Figure 2B (lane 5), Ub-HA TAp73α conjugates were detected upon co-transfection only with wild-type (WT) Itch (Myc-Itch WT), and not with the catalytically inactive mutant of Itch (Myc-Itch MUT) (Figure 2B, lane 6). Similarly, ΔNp73α was ubiquitinated by Itch (Figure 2C), showing that the N-terminal part of the protein is not required for the ubiquitination. In contrast, p53 was not ubiquitinated by Itch (Figure 2B, lanes 7–9). Although ubiquitination of TAp73αY487F mutant was reduced, a greater reduction was seen with the double mutant TAp73αY407F/Y487F (Figure 2D). Ubiquitination of the TAp73αY407F mutant was similar to that of WT p73. The right panel (lanes 10–18) in Figure 2B demonstrates the specificity of the reaction since no higher molecular weight bands were observed in the absence of Ub-HA. These data clearly show that p73 but not p53 is ubiquitinated by Itch, suggesting that this protein plays an important role in the regulation of p73 steady-state protein levels. Itch regulates the stability of p73 in cells Since ubiquitination of proteins is usually associated with their turnover (Weissman, 2001), we investigated if Itch can regulate p73 protein abundance. We measured TAp73α levels in whole-cell extracts in the presence or absence of Itch. Representative data from several independent experiments demonstrate that co-expression of Myc-Itch and TAp73α in cells results in a striking decrease of TAp73α levels (Figure 3A), indicating that the Itch-dependent ubiquitination targets p73 for degradation. Consistently, the catalytic mutant of Itch was not able to alter the concentration of p73 (Figure 3A, lane 3) and the TAp73αY487F (Figure 3B) mutant levels were not affected by Itch overexpression. Figure 3.Effect of Itch expression on the steady-state levels of p73. (A) Hek293 cells were transfected with either HA-TAp73α and p53 or HA-TAp73δ (A) or Flag-TAp73αY487F, Flag-TAp73αY407F, and Flag-TAp73αY407F/Y487F (B), together with either Myc-Itch or Myc-Itch MUT. At 48 h after transfection, cells were treated or not with MG132. Equal amounts of total protein cell lysates were subjected to western blotting analysis using anti-HA antibody or anti-Flag antibody (upper rows) to detect the steady-state levels of p73s and p53 proteins. The same blots were re-probed with anti-Myc antibody in order to detect the expression levels of Itch (middle rows), and with anti-Actin antibody to show equal loading (lower rows). 35S pulse chase: H1299 cells were transfected with HA-TAp73α (C) or HA-ΔNp73α (D), together with Myc-Itch or pCDNA-Myc. At 48 h post-transfection, cells were labelled with 250 μCi/ml of Redivue PRO-MIX (L-[35S] in vitro cell-labelling mix). Unlabelled Met and Cys (1 mg/ml) were added and cells were collected at the indicated time points. IP was performed with anti-HA (Y-11) polyclonal antibody. Immunoprecipitates were washed and run on SDS–PAGE and detected by autoradiography. For cycloheximide-blocking experiments, Hek293 cells were transfected with either HA-TAp73α (E), HA-ΔNp73α (F), or HA-p53 (G), together with either Myc-Itch or pCDNA-Myc. At 24 h after transfection, cells were treated with cycloheximide and collected at different time points. Equal amounts of total protein cell lysates were subjected to western blotting analysis using anti-HA antibody. To demonstrate equal loading, the same blots were stripped and re-probed with anti-β-Tubulin or anti-Hsp-70 antibodies for p73s and p53 blots, respectively. (C) Western blots were subjected to densitometric analysis and results were normalized based on β-Tubulin or Hsp-70 expression levels, respectively, and reported in graphical form (lower panels). Download figure Download PowerPoint As polyubiquitination generally targets proteins for proteasomal degradation (Weissman, 2001), we determined the effect of MG132 on the steady-state levels of TAp73α. As shown in Figure 3A (lanes 4–6), addition of MG132 to cells blocked the Itch-mediated TAp73α degradation and resulted in the accumulation of the ubiquitinated forms of TAp73α (data not shown). This is consistent with previous reports showing that proteasome inhibition leads to the stabilization of endogenous p73 protein (Balint et al, 1999). Again, TAp73δ and p53 levels were not affected by Itch (Figure 3A). We further confirmed these results by measuring p73 half-life in the presence or absence of Itch using two different methods. Both pulse chase using [35S]Met and Cys (Figure 3C and D), and cycloheximide blockade (Figure 3E and F), showed a marked decrease of TAp73 and ΔNp73 half-lives in the presence of Itch. Under similar experimental conditions, no change in p53 half-life was observed (Figure 3G). To further confirm the importance of Itch in controlling p73 steady-state levels, we reduced endogenous Itch levels by siRNA. To this end, we used Tet-On-inducible Saos-2 cells expressing p73 (Melino et al, 2004). Figure 4A shows that, in this inducible cell line, reduction in Itch expression by siRNA resulted in more rapid induction and higher levels of p73 protein. Moreover, after withdrawal of induction, p73 levels decline more slowly when Itch expression is reduced (Figure 4B). Similarly, the steady-state levels of endogenous TAp73 and ΔNp73 isoforms increased in Saos-2 cells when Itch was downregulated (Figure 4C). This confirms that basal Itch levels are important in controlling basal p73 levels. In agreement, endogenous levels of ΔNp73 (which is the only isoform detectable in these cells) were increased in mouse embryo fibroblasts (MEFs) derived from non-agouti-lethal 18H Itch-deficient mice (MEF Itch−/−) (Figure 4D). Re-introduction of WT Itch into MEFs Itch−/− resulted in reduction of endogenous ΔNp73 levels in these cells (Figure 4E). Figure 4.Effects of Itch downregulation on p73 protein levels. (A) Saos-2-TAp73α inducible cells were transfected with siRNA oligonucleotides targetting the Itch sequence or with a scrambled oligonucleotide. After 48 h, cells were induced to express TAp73α for the indicated time points with doxycycline (inducer). p73 levels increase more rapidly and reach higher levels when Itch is downregulated. The lower panel shows endogenous Itch levels. Graphs show densitometric analysis of the p73 western blots normalized for β-Tubulin. (B) Saos-2-TAp73α-inducible cells were transfected with oligos targetting the Itch sequence, or with a scrambled oligo. Cells were induced to express TAp73α for 14 h with doxycycline, the inducer was removed and cells collected at the indicated time points. p73 levels decay more rapidly in cells transfected with the scrambled oligo compared to those in which Itch is downregulated. The lower panel shows endogenous Itch levels. Graphs show densitometric analysis of the p73 western blots normalized for β-Tubulin. (C) Saos-2 cells were transfected with oligos targeting the Itch sequence or with a scrambled oligo and collected 48 h later. Itch downregulation (lower panel) results in an increase of TA and ΔN p73a levels (upper panels). (D) Western blot for endogenous p73 of WT MEFs (MEF+/+), non-agouti-lethal 18H Itch-deficient MEFs (MEF Itch−/−) and a spontaneously immortalized clone of these MEFs (MEF Itch−/− Immortalized). ΔNp73 levels (the only form detectable in these cells) are higher in MEFs Itch−/−. (E) Immortalized MEFs Itch−/− were transfected with Myc-Itch WT and collected 48 h later. Re-introduction of Itch results in ΔNp73 downregulation. Download figure Download PowerPoint Not all Itch family members had the same effect on p73. Nedd4 bound (Figure 5A) both TAp73 and ΔNp73, but was not capable of catalyzing the ubiquitination of these proteins (Figure 5C), and therefore did not affect their degradation (Figure 5B). Miyazaki et al (2003) demonstrated that NEDL2, another Nedd4 family member, binds, ubiquitinates and stabilizes p73. Thus, different Nedd4-like E3 ligases, although all capable of binding the p73 PY motifs, exert different effects on these proteins. Figure 5.Nedd4 binds p73 but fails to ubiquitinate it. (A) Hek293 cells were transfected with HA-TAp73α, HA-ΔNp73α, HA-p53 or an empty vector, together with Myc-Itch or Myc-Nedd4 in the presence of MG132. Cells were subjected to IP using anti-HA antibody and analyzed by western blot, using an antibody against Myc. Itch and Nedd4 co-IP with p73. (B) Hek293 cells were transfected with HA-TAp73α, HA-ΔNp73α or HA-p53, together with Myc-Itch or Myc-Nedd4. Cell extracts were analyzed by western blot, using an antibody against HA. Expression of Itch but not of Nedd4 results in TA and ΔNp73 downregulation. (C) Cells transfected with the indicated plasmids together with a plasmid expressing Ub-HA were analyzed by western blot using an antibody against HA. Higher molecular bands characteristic of ubiquitination appear when Itch but not Nedd4 is overexpressed. Download figure Download PowerPoint Itch decreases p73-dependent transcriptional activity To evaluate if the interaction between p73 and Itch influences its transcriptional activity, we co-transfected H1299 cells with TAp73α and Myc-Itch or Myc-Itch MUT and assessed TAp73α transcription activity by luciferase reporter assay using different p53/p73-responsive promoters (Bax, p21, and MDM2). As shown in Figure 6A, C and E, consistent with a reduction in TAp73 protein levels, co-transfection of Myc-Itch reduced the transcriptional activity of TAp73α on all the promoters tested in a dose-dependent manner. As expected, the mutated form of Itch had no effect on the transcriptional activity of p73 (Figure 6B, D and F). The reduction of the promoter activity was paralleled by a reduction in endogenous levels of p73 target proteins such as p21 (Figure 6G). Figure 6.Itch expression reduces the transcriptional activity of p73. H1299 cells were transfected with the indicated combinations of plasmids (at the different indicated ratios) encoding TAp73α, together with Myc-Itch WT (Itch wt) or Myc-Itch MUT (Itch Mut), or empty control vector (pCDNA), together with a Bax- (A, B) or MDM2- (C, D) or p21- (E, F) luciferase reporter plasmid and Renilla luciferase reporter plasmid. Cell extracts were prepared 36 h later and luciferase activity was determined. Results are represented as fold induction of luciferase activity as compared with the control cells. Histograms show the mean of three independent experiments; bars indicate standard deviation. (G) H1299 cells were transfected with HA-TAp73α, together with Myc-Itch at the indicated ratios. Equal amounts of cell extracts were subjected to western blot with anti p21 antibody (upper panel), anti-HA (middle panel), and anti-Myc (lower panel). Download figure Download PowerPoint Itch is downregulated in response to DNA damage Since TAp73 protein levels increase in response to DNA damage (Agami et al, 1999; Gong et al, 1999; Yuan et al, 1999), we investigated whether Itch expression is also modulated after DNA damage. In Saos-2 cells, following treatment with doxorubicin, to induce DNA damage, endogenous Itch protein levels were downregulated in a time- (Figure 7A) and dose- (not shown) dependent manner. The reduction of Itch levels was paralleled by an increase in endogenous TAp73 levels (Figure 7A). Reduction of Itch also paralleled an increase in apoptosis (Figure 7B). This response of Itch to doxorubicin treatment is not cell type specific: Figure 7C shows a similar experiment using HeLa, H1299, and Hek293 cell lines treated with doxorubicin. These results suggest that Itch could be an important regulator of TAp73 levels and that upon DNA damage Itch is downregulated, allowing TAp73 levels to rise. The relative contribution of this mechanism in determining apoptosis of damaged cells remains to be quantified. This pathway is p53 independent since the effect on cell cycle and apoptosis was also observed in cell lines lacking p53, such as Saos-2. Figure 7.Itch is downregulated by DNA-damaging agents. Saos-2 cells were treated with 2 μM doxorubicin (Doxo) for 24 or 48 h, then collect
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