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. Author manuscript; available in PMC: 2011 Apr 11.
Published in final edited form as: Cell Cycle. 2008 Feb 19;7(9):1231–1237. doi: 10.4161/cc.7.9.5795

U-box-type ubiquitin E4 ligase, UFD2a attenuates cisplatin mediated degradation of ΔNp63α

Aditi Chatterjee 1, Sunil Upadhyay 1, Xiaofei Chang 1, Jatin K Nagpal 1, Barry Trink 1, David Sidransky 1,*
PMCID: PMC3073353  NIHMSID: NIHMS280793  PMID: 18418053

Abstract

ΔNp63α, the dominant negative isoform of the p63 family is an essential survival factor in head and neck squamous cell carcinoma. This isoform has been shown to be down regulated in response to several DNA damaging agents, including cisplatin. But little is understood about the post-translational protein stability of ΔNp63α. In this present study we demonstrate for the first time that ΔNp63α physically interacts with U-box-type E4 ubiquitin ligase UFD2a. UFD2a stabilizes ΔNp63α, and ubiquitylation of ΔNp63α is attenuated by UFD2a both in the presence and absence of cisplatin. Ectopic expression of UFD2a increased the half-life of ΔNp63α in association with a significant enhancement of the repressive transcriptional activity of ΔNp63α. Downregulation of endogenous UFD2a by RNAi resulted in degradation of ΔNp63α. Taken together, our current study provides an insight onto the regulation of ΔNp63α protein levels in response to cisplatin and also suggests that UFD2a might play an important role in the regulation of cisplatin mediated cell death by p63.

Keywords: ΔNp63α, UFD2a, ubiquitylation, cisplatin, protein stability

Introduction

p63, a p53 homologue exhibits high structural similarity to p53 and yet has significant structural and functional differences.1–5 Both p63 and p73 genes encode proteins that possess transactivation, DNA binding and oligomerization domains similar to p53 domains. However, unlike p53, p73 and p63 genes, each contain 2 independent promoters and make significant use of differential splicing at the gene’s 3′ end, hence yielding an array of at least 6 unique proteins that form 2 distinct classes: those containing an amino acid terminus (called TAp73 and TAp63) and those that are truncated (called ΔNp73 and ΔNp63) with no amino-terminus region.6–10 Additionally, both genes can be alternatively spliced to generate proteins with different carboxyl termini. As a result of this alternative splicing six splice variants can be generated from the two promoters of p63 with different C termini, termed as α, β and γ.5,11 Further both p63α and p73α contain an additional domain, not present in p53 known as the SAM (sterile alpha motif) domain.12,13 Recent studies have shown that ΔNp63α protein levels are decreased after UV and paclitaxel treatment and that the protein levels were regulated in a proteosome-dependent manner in UV treated cells.14 Earlier studies from our lab have demonstrated degradation of ΔNp63α by cisplatin, which was impaired by the a proteosomal inhibitor MG-132.15

Protein ubiquitylation is achieved by a multistep mechanism involving several enzymes: an ubiquitin-activating enzyme (E1), an ubiquitin-conjugating enzyme (E2) and an ubiquitin-protein ligase (E3). A new class of ubiquitylation enzyme, a ubiquitin chain assembly factor (E4), was recently discovered and shown to be required for the degradation of certain types of substrate, (including a fusion protein with an NH2-terminal ubiquitin moiety), by a ubiquitin fusion degradation pathway, designated UFD.16,17 Ufd2a/E4 and its homolog in other eukaryotes share a conserved domain of ~70 amino acids termed the U-box. Apoptosis induced by multiple stimuli cleaves Ufd2a which results in significant loss of its activity, indicating that Ufd2a might play an important role in apoptosis signaling.18

Earlier studies from our lab have reported cisplatin mediated degradation of ΔNp63α.15 Our current studies indicate that the other isoforms of p63 remain unaffected in response to cisplatin treatment. Here we investigate the effect of Ufd2a on ΔNp63α. We show that ΔNp63α physically interacts with UFD2a. Ectopic expression of UFD2a led to an increase in the levels of ΔNp63α. We also demonstrate that UFD2a enhances the transcriptional repressive capacity of ΔNp63α. Furthermore, overexpression of UFD2a inhibited cisplatin mediated degradation of ΔNp63α and decreased the ubiquitination levels of ΔNp63α. Our data suggest that UFD2a participates in the regulation of the steady-state level of ΔNp63α and may be one of the major determinants of cellular response to cisplatin.

Results

Cisplatin mediated degradation of ΔNp63α was attenuated in Del 152

It has been previously reported that ΔNp63α undergoes degradation by cisplatin and that this degradation is specifically prevented in the presence of the proteosomal inhibitor MG132.15 In this study we attempted to map the region of ΔNp63α responsible for the above mentioned effect. We generated a series of deletion constructs, termed hereafter as Del 368 (aa 1–368) and Del 152 (aa1–152), from the COOH-terminal end of ΔNp63α (aa 1–587). Construct 587 depicts the full length construct of ΔNp63α. Equal expression of all constructs were tested by western blotting, confirming that no protein degradation was involved (Fig. 1, lanes 4, 7 and 10). 48 h post transfection 022 cells were treated with or without, MG132 (2 μM) for 2 h followed by 75 μM (IC50 for 022) of cisplatin for 24 h. We observed that in the presence of cisplatin steady state levels ΔNp63α, as well as recombinant ΔNp63α showed degradation (Fig. 1, lanes 2 and 5). A similar level of cisplatin mediated degradation was observed in construct Del 368 (Fig. 1A, lane 8), and construct aa 1–219 (data not shown). However the cisplatin mediated degradation was almost abolished in construct Del 152 (Fig. 1, lane 11). The presence of MG132 was able to protect this degradation in 022 cells, as well as in cells expressing constructs 587, Del 368 and Del 152 (Fig. 1, lanes 3, 6, 9 and 12).

Figure 1.

Figure 1

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Regulation of ΔNp63α in response to cisplatin and MG132. (A) Western blot showing the stability of ΔNp63α and the deletion constructs in the presence or absence of cisplatin and MG132 as indicated. 2 μg of each indicated expression plasmid was transfected into JHU 022 cells.

UFD2a and ΔNp63α are endogenously co-expressed and interact

Earlier reports have demonstrated that UFD2a is degraded by cisplatin19 and further ΔNp63α has been reported as an essential survival factor in head and neck carcinoma cells.20 Since ΔNp63α is degraded by cisplatin, we hypothesized that UFD2a might play an important role in the regulation of ΔNp63α stability. To test this hypothesis, we investigated the expression levels of UFD2a before and after DNA damage. 022 cells treated with cisplatin for 24 h showed decreased cellular survival in a dose dependent manner (Fig. 2A). Our experiments also revealed that overexpression of UFD2a in 022 cells did not affect the cisplatin mediated cell death of the cells (data not shown). Moreover, our results indicated that both ΔNp63α and UFD2a underwent substantial degradation as observed in 022 cells when exposed to higher concentrations of cisplatin (Fig. 2B, lanes 2 and 3). These results supported the notion that cisplatin exposure leads to UFD2a and ΔNp63α degradation, and further suggested that UFD2a might play an important role in the regulation of p63 stability. Western blot analysis of known p53 target genes revealed that treatment of 022 cells with cisplatin resulted in increased expression of p21, Noxa and Puma, with degradation of both ΔNp63α and UFD2a (Fig. 2B). Next we investigated if UFD2a might physically interact with ΔNp63α. To verify that the interaction between UFD2a and ΔNp63α occurs at endogenous levels, we performed co-immunoprecipitation experiments in JHU 022 cells, which express moderate levels of ΔNp63α. JHU 022 cells were transfected with the Flag-UFD2a expression construct with or without ΔNp63α in pRC/cytomegalovirus vector. The immunoprecipitation reaction was done with anti-flag matrix, and western blot was preformed using anti-p63 antibody. Endogenous ΔNp63α was co-immunoprecipitated with UFD2a (Fig. 2C, lane 4). Transfecting the cells with Flag-ΔNp63α. and imunoprecipitation with anti-flag-matrix followed by western blot using anti-UFD2a revealed co-immuprecipitation of endogenous UFD2a (Fig. 2D, lane 3). These results confirm the endogenous association of ΔNp63α and UFD2a in JHU 022 cells. Further transfection of 022 cells with deletion constructs Del 368-Flag and Del 152-Flag and imunoprecipitation with anti-flag matrix followed by western blot using anti-UFD2a revealed that Del 368 co-immuoprecipitated with UFD2a (Fig. 2D, lane 6) but no interaction was observed between Del 152 and UFD2a (Fig. 2D, lane 5). Transfection of TAp63α, TAp63β and ΔNp63β in p53 and p63 deficient H1299 cells and treatment with cisplatin revealed that these isoforms were not affected by cisplatin (Fig. 2E).

Figure 2.

Figure 2

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Regulation of ΔNp63α and UFD2a in response to cisplatin. (A) Cellular survival of JHU 022 cells in response to increasing concentration of cisplatin after 24 h exposure. (B) Immunoblot analysis of JHU 022 cells cultured in absence and presence of cisplatin at the indicated concentrations for 24 h. Immunoblot analysis was performed with the indicated antibodies. (C) Complex formation between ΔNp63α and UFD2a. JHU 022 cells were transfected with Flag-UFD2a; immunoprecipitation was performed using anti- flag matrix and Western blot was performed using anti-p63 antibody (lane 4). (D) JHU 022 cells were transfected with Flag-ΔNp63α, Flag-ΔNp63α-Del 368 and Flag-ΔNp63α-Del 132. Immunoprecipitation was performed using anti-flag matrix and the membrane was blotted with anti-UFD2a antibody (lanes 3, 5 and 6 respectively). (E) p53 and p63 deficient, H1299 cells were transfected with the indicated isoforms of p63 and immunoblot analysis was performed after treatment of the cultured cells in the presence of cisplatin at the indicated concentration for 24 h with anti-p63 antibody.

UFD2a affects both the steady level and half-life of ΔNp63α

To elucidate the possible effect of UFD2a in the stabilization of ΔNp63α, JHU 022 cells were co-transfected with ΔNp63α and with increasing concentrations of UFD2a. DNA concentration in each transfection was kept constant using pCDNA3.1 plasmid. After 24 h of transfection, the cells were treated with or without 75 μM cisplatin. We observed that forced overexpression of UFD2a greatly enhanced the steady state levels of ΔNp63α in JHU 022 cells (Fig. 3A). Further the cisplatin mediated degradation of ΔNp63α was also blocked by UFD2a (Fig. 3A, lane 5 and 6). We also observed that the presence of UFD2a stabilized ΔNp63α levels in transfected JHU022 cells (Fig. 3B and A, lanes 2 and 3). Further, similar stabilization of ΔNp63α protein was observed even in the presence of cisplatin (Fig. 3B and A, lanes 5 and 6). We could corroborate the stabilizing effect of UFD2a on ΔNp63α in the p53 and p63 deficient lung cancer cell line, H1299, where enforced expression of UFD2a did stabilize ΔNp63α, both in the presence and absence of 75 μM cisplatin (Fig. 3C). Treatment of 022 cells with UFD2a siRNA, led to the degradation of the ΔNp63α protein both in the presence and absence of cisplatin (Fig. 3D), further supporting the notion that UFD2a stabilizes ΔNp63α. Analysis of TAp63α, TAp63β and ΔNp63β in H1299 cells with increasing concentration of UFD2a in absence of cisplatin did not show any significant difference in the stability of these isoforms (Fig. 3E).

Figure 3.

Figure 3

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UFD2a inhibits cisplatin mediated degradation of ΔNp63α. (A) JHU 022 cells were transfected with UFD2a expression plasmid, and 24 h post transfection the cells were treated with or without cisplatin (75 μM) as indicated. Western blot was performed using anti- p63 and anti-UFD2a antibodies, to assess the endogenous levels of p63. (B) JHU 022 cells were transfected with a steady concentration of 2 μg of expression plasmid for ΔNp63α (A) or empty vector (B), as indicated with increasing concentration of UFD2a (1 and 1.5 μg). 24 h post transfection, cells were treated with 75 μM cisplatin for 24 h before cellular lysates were subjected to immunoblotting with the indicated antibodies. (C) p53 and p63 deficient H1299 cells were transfected with 2 μg of ΔNp63α expression plasmid with increasing concentrations of UFD2a (0.5, 1 and 1.5 μg). 24 h post transfection the cells were treated with or without cisplatin as indicated and subjected to Western blotting with anti p63 and anti-UFD2a antibodies. (D) JHU 022 cells were transfected with increasing concentrations of UFD2a siRNA, and 24 h after transfection the cells were treated with or without 75 μM cisplatin for 24 h and subjected to Western blot analysis using anti-p63 antibodies to assess the endogenous levels of p63. (E) H1299 cells were transfected with the indicated isoforms of p63 with increasing concentrations of expression plasmid UFD2a. DNA amount in each transfection was kept constant using empty vector pRC. Immunoblot analysis was performed with anti-p63 antibody. (F) UFD2a increases the half life of ΔNp63α. JHU 022 cells were co-transfected with 1 μg of ΔNp63α expression plasmid with or without 1.5 μg of Flag-UFD2a as indicated. 24 h post transfection the cells were treated with 25 μg/ml of cycloheximide for the indicated time periods and cellular lysates were subjected to Western blot using anti-p63 antibody.

To further show the UFD2a-dependent stabilization in ΔNp63α protein levels, we analyzed the half-life of ΔNp63α in the presence or absence of UFD2a. JHU 022 cells were transfected with ΔNp63α plasmid and 24 h after transfection the cells were treated with or without 25 μM of cycloheximide. The presence of UFD2a, increased the half life of ΔNp63α in the presence of cycloheximide (Fig. 3F) at the indicated time points.

ΔNp63α ubiquitylation is decreased by UFD2a

Having demonstrated that ΔNp63α interacts with UFD2a and that UFD2a stabilizes the protein, we next assessed if the ubiquitin-proteasome pathway could be involved in this process. JHU022 cells were co-transfected with Flag-ΔNp63α and Ub-HA expression plasmid with or without increasing concentrations of His-UFD2a. 48 h post transfection, protein was collected, immunoprecipetated with anti-HA-matrix and subjected to western blot analysis with anti-Flag antibody. Our results demonstrate that presence of UFD2a decreased the ubiquitylation level of ΔNp63α protein (Fig. 4, lanes compare lanes 3 and 5). Further treatment of JHU022 cells with cisplatin also did not affect the over all ubiquitylation pattern of ΔNp63α (Fig. 4, compare lanes 3 and 6) but the presence of UFD2a did result in decreased ubiquitylation of ΔNp63α (Fig. 4, compare lanes 6 and 8).

Figure 4.

Figure 4

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Ubiquitylation of ΔNp63α is affected by UFD2a. JHU 022 cells were co-transfected with 1 μg each of expression plasmid Flag-ΔNp63α and HA-Ub with or without increasing concentration of His-UFD2a (1 and 1.5 μg), in the presence and absence of cisplatin as indicated. 24 h post transfection, cellular lysates were immunoprecipitated with anti-HA matrix and immunoblotting was performed using the anti-Flag antibody.

UFD2a enhances the repressive transcriptional activity of ΔNp63α

To further investigate the functional consequences of the interaction between UFD2a and ΔNp63α, we tested the effect of UFD2a on the repressive transcriptional activity of ΔNp63α on 2 well known targets in JHU022 cells. As seen in Figure 5A, forced expression of ΔNp63α resulted in a remarkable reduction of endogenous p21WAF1, which was further decreased in the presence of UFD2a (1.6 fold). Similar transcriptional repression results were also observed with the Bax-luciferace construct, where the presence of UFD2a led to a 1.4 fold downregulation of the Bax promoter activity in combination with ΔNp63α (Fig. 5B).

Figure 5.

Figure 5

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UFD2a enhances transcriptional repression of ΔNp63α. JHU 022 cells were transfected with p21 or Bax luciferace promoter construct with or without His-UFD2a as indicated in combination with Renilla luciferace plasmid. The amount of DNA per transfection was kept constant by using empty pBasic vector. At 24 h post transfection, the firefly luciferase activity was determined. The transfection efficiency was standardized against Renilla luciferase. Results shown are representative of three independent experiments. * indicated p ≤ 0.002.

Discussion

The p63 protein is expressed in the basal cells of stratified epithelium such as breast, prostate, skin, cervix and other tissues.5,21 ΔNp63α has been reported to be the predominant splice variant, if not the only one that is expressed in these basal epithelial cells.5,21,22 Ectopic expression of ΔN splice variants have been reported to decrease p53 target promoter activity (e.g., p21), suggesting a role for ΔNp63α in maintaining cell proliferation.5,23 Consistent with these studies we demonstrate here that degradation of ΔNp63α in response to cisplatin results in stabilization of p53 target genes like p21, Noxa and Puma. Recent studies demonstrate the necessity of ΔNp63α in the cellular survival of head and neck squamous cell carcinoma.20 Recent reports have demonstrated the interaction of p63 and ubiquitin ligase E3. Ours appears to be the first study reporting the endogenous association of U-box-type ubiquitin protein ligase family E4, UFD2a and ΔNp63α.

Consistent with our earlier publication, we observed a decrease in ΔNp63α protein levels on cisplatin treatment and a reversion of the same when treated with MG132.15 We now demonstrate a physical interaction between UFd2a and ΔNp63α. We also demonstrate that deletion of ΔNp63α to aa 132 abolishes this interaction. We further show that cisplatin mediated degradation of ΔNp63α is prevented in the presence of E4 ubiquitination factor, UFD2a and that ΔNp63α protein expression is stabilized by UFD2a. Our results also indicate that ubiquitylation of ΔNp63α is attenuated by UFD2a both in the presence and absence of cisplatin, which may provide an insight into the regulation of ΔNp63α protein. Further experiments are required to study the precise mechanism of UFD2a mediated stabilization of ΔNp63α.

One ubiquitin ligase may possess different functional activity on the proteins belonging to the same family, as demonstrated by MDM2. It has been shown that MDM2 promotes ubiquitination and degradation of p53 and that disruption of the interaction between them is essential for p53 stabilization.24 In a sharp contrast to p53, MDM2 resulted in an increase in the amount of p73 and does not induce the ubiquitination and degradation of p73.25,26 p63 has been shown to interact with MDM2, although the functional effects of this are currently controversial.26 Another ubiquitin ligase, NEDL2, a HECT-type E3 ligase, has also been reported to bind and ubiquitinate p73, resulting in its stabilization rather than degradation.27 Studies by Osada et al., reveal that ΔN isoforms of p63 are much more stable than wild-type p63, suggesting that the NH2-terminal transactivation domain of p63 regulates its stability through a pathway that is sensitive to MG-132.28 These observations imply that p63 stability is regulated through a pathway distinct from that used by p53 or p73. Earlier studies with TAp73α and UFD2a indicates that UFD2a is degraded by DNA damage, stabilizing TAp73α.19 In contrast, we demonstrate here that ΔNP63α is stabilized by UFD2a and that the cisplatin mediated degradation of ΔNP63α is also prevented in the presence of UFD2a. Consistent with our earlier findings, we show here that degradation of ΔNp63α by cisplatin is prevented in the presence of MG132. We further show that the cisplatin mediated degradation of ΔNp63α is attenuated by deletion construct Del 132. Taken together the above results demonstrate that ΔNp63α protein levels are regulated in a proteosome-dependent manner and that the carboxyl terminal is involved in sensitizing/degrading ΔNp63α in response to cisplatin.

In contrast to ΔNP63α which is degraded with cisplatin treatment, TAp73α is stabilized with cisplatin treatment.19 Downregulation of ΔNp63α by other DNA damaging agents like UV and paclitaxel has also been well documented.14 Ubiquitin ligase Itch has been shown to degrade both TAp63α and ΔNp63α,29,30 but both Itch and ΔNp73α were found to be rapidly downregulated in response to DNA damaging agents like doxorubicin and etoposide.31,32 Further downregulation of Itch in response to DNA damage has been shown to increase the levels of TAp73α but rapidly degrade ΔNp73α.31 Similarly, degradation of UFD2a with the DNA damaging agent cisplatin leads to a parallel decrease of ΔNp63α. Our data also show that presence of UFD2a leads to increase in steady state levels of ΔNp63α both in absence and presence of cisplatin and also increased the half life of ΔNp63α.

Our results also demonstrate that TAp63α remained unaffected by cisplatin and UFD2a. We attribute this difference to the presence of the transactivating domain in the TAp63α which is in agreement with earlier studies that have reported differences in the functional profile and regulation of ΔNp63α and TAp63α.33–35 Treatment of other isoforms of p63 (TAp63β and ΔNp63β) with cisplatin or UFD2a did not affect the protein stability, suggesting that a second pathway might specifically target these isoforms, (beyond the scope of this manuscript). One possibility could be due to the lack of a SAM domain in these isoforms. Studies are currently on going to determine the difference of this stability pattern in p63β isoforms.

Consistent with earlier reports by Westfall et al.,14 our results indicate that ΔNp63α represses transactivation of both the p21 and Bax promoter-luciferace vector. We further show that presence of UFD2a (which we demonstrate to stabilize ΔNp63α) further represses the promoter-luciferace activity of both p21 and Bax. We show here that ΔNp63α physically interacts with UFD2a, and that the ubiquitylation pattern of ΔNp63α was decreased by UFD2a in the presence or absence of cisplatin. It is becoming evident that not all cellular proteins are subjected to ubiquitination-dependent protesomal turnover. Ornithine decarboxylase (ODC) is degraded by the proteasome independently of ubiquitin modification.36 Further degradation of p21WAF1 by the proteasome does not require direct ubiquitination of p21WAF1.37 It has also been demonstrated by Jin et al., that MDM2 binds to p21WAF1 and induces proteasomal turnover by affecting the ubiquitination level of the protein.38 It is thus clear that the degradation/stabilization mechanism of ΔNp63α is different from those of p73 and p53, indicating that another as yet unknown pathway may target ΔNp63α. Finally, the ability of UFD2a to promote the stabilization of ΔNp63α in response to a chemotherapeutic drug may prevent cells from apoptotic cell death in response to DNA damage induced by chemotherapeutic drugs. It is thus conceivable that reduction of UFD2a levels might enhance anticancer treatments.

Materials and Methods

Plasmids

ΔNp63α-Flag was obtained PCR amplification of ΔNp63α cDNA and cloned into the BamHI and NotI sites of pCDNA3.1/hygro (Invitrogen, Carlsbad, CA) and was called Flag-587. Deletion constructs of ΔNp63α were generated using PCR amplification and cloned into the BamHI and NotI of the same plasmid. The deletion constructs were named as Del 368 and Del 152 respectively. TAp63α, TAp63β, ΔNp63α and ΔNp63β were cloned into the pRC/cytomegalovirus vector as described in.39

Plasmid HA-Ub construct was kindly provided by Dr. Bohmann (University of Rochester, NY, USA), Flag-UFD2a was provided by Dr. James Mahoney (Johns Hopkins University, Baltimore, USA). His-UFD2a was obtained by PCR amplification of UFd2a c-DNA and cloned into pCDNA3.1/V5-His directional TOPO cloning vector (Invitrogen). Bax-Luciferace expression vector was kindly provided by Dr. John Reed (Burnham Institute for Medical Research, CA, USA) and p21WAF1-Luciferace construct was a kind gift from Prof. Bert Vogelstein (Johns Hopkins University, Baltimore, USA). All clones were sequenced to rule out any mutation.

Cell cultures and transfection

Human head and neck cancer cell line JHU 022 and Human lung carcinoma cell line (H1299) were maintained in RPMI supplemented with 10% heat-inactivated fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA), 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were maintained in a humidified atmosphere containing 5% CO2 at 37°C. H1299 and JHU 022 cells were transiently transfected with the indicated expression plasmids using FuGENE HD transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN) in accordance with the manufacturer’s specifications. UFD2a siRNA was obtained from Dharmacon (Chicago, IL). 022 cells were transfected with 20 and 50 nM “UFD2a on-target plus SMART pool siRNA” or scramble siRNA. 48 h post transfection the cells were treated with or without 75 μM cisplatin before harvesting them for cellular lysates.

Antibodies and immunoblot analysis

Monoclonal anti-p63 (4A4), monoclonal anti-HA and monoclonal anti-p21WAF1 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), monoclonal anti-FLAG (M2) and anti-β-Actin antibodies were obtained from Sigma Chemicals (St. Louis, MO), monoclonal anti-UFD2a antibody were from BD Biosciences (Franklin lakes, NJ). Cells were lysed in RIPA lysis buffer containing 1 mM PMSF and protease inhibitor mixture (Sigma Chemical Co., St Louis, MO). The protein concentration of the lysates was determined by Lowry protein assay (Bio-Rad Laboratories, Hercules, CA). Equal amounts of protein were mixed with Laemmli sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.1 M DTT and 0.01% bromophenol blue), run on 4–12% NuPAGE and electro-blotted onto Nitrocellulose membrane (Bio-Rad Laboratories). The membrane was blocked with PBS supplemented with 0.1% Tween 20 and 5% nonfat milk for 1 h at room temperature, and probed with primary antibody for 1 h at room temperature followed by HRP-conjugated appropriate secondary antibody. Signals from immunoreactive bands were detected by enhanced chemiluminescence (ECL, GE Health Care, Piscataway, NJ) according to the manufacturer’s instructions.

Immunoprecipitation analysis

Cells were transfected with various constructs and after 48 h were washed with PBS and lysed using TRITON-X lysis buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl, 1 mM EDTA; 1% TRITON-X100) containing protease and phosphatase inhibitor cocktails (Sigma Chemical Co.,) Lysates were pre-cleaned with protein A-sepharose beads and then incubated for 2 h at 4°C with primary antibody or affinity matrix. The immune complexes were precipitated with protein A-Sepharose beads for 4 h at 4°C, and the nonspecific bound proteins were removed by washing the beads with the NP-40 lysis buffer three times at 4°C. The beads were loaded in Laemmli sample buffer directly and analyzed by immunoblotting with anti-p63 and anti-HA antibody.

Ubiquitination assay

022 cells were transiently cotransfected with the expression plasmid for HA-tagged ubiquitin, and Flag-ΔNp63α together with or without increasing amounts of the His-UFD2a expression plasmid in the presence and absence of 75 μM of cisplatin as indicated. Total amounts of transfected DNA were kept constant by pcDNA3 (Invitrogen) per transfection. 48 h after transfection, cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl, 1 mM EDTA; 1% TRITON-X100) containing protease and phosphor inhibitors (Sigma Chemical Co). Ubiquitinated proteins were recovered by HA resin (Sigma) and analyzed by immunoblotting with the anti-Flag antibody.

Measurement of p63 half-life

Cycloheximide (25 μg/ml) (Sigma) was added to 022 cells 24 h after transfection with the indicated combination of plasmids. Protein levels were determined by collecting cells at the indicated time points and immunoblotting was performed as described above. The membrane was re-probed with β-Actin to confirm for equal loading.

Promoter luciferase activity assays

022 cells were transiently transfected with the Bax and p21WAF1 reporter plasmids along with 500 ng of ΔNp63α expression plasmid (where indicated) in the presence or absence of 1.5 μg of His-UFD2a and the empty pGL3-Basic vector were using FuGENE HD according to the manufacturer’s protocol (Roche, Indianapolis IN). The transfection efficiency was determined by the Renilla luciferase gene-containing pRL-CMV plasmid (Promega, Madison, WI). 48 h after transfection, cells were washed twice with phosphate-buffered saline (PBS) and lysed in a lysis buffer (5x PLBR, Promega, Madison, WI) with gentle shaking at room temperature for 20 min. Cell lysates were centrifuged at 13,000 rpm on a table top centrifuge for 2 min to pellet the cell debris. The supernatants were transferred to a fresh tube, and the dual luciferase activity in cell extracts was determined according to the manufacturer’s protocol (Promega). Briefly, each assay mixture contained 2 μl (2 μg) of the cell lysates and 10 μl of a firefly luciferase-measuring buffer (LAR ll R, Promega, Madison, WI). The firefly luciferase activity was measured by a luminometer (the luminometer was programmed to perform a 2-s pre-measurement delay, followed by a 10-s measurement period for each reporter assay). After measuring the firefly luciferase activity (Stop & GloR, Promega, Madison, WI), a Renilla luciferase-measuring buffer was added, and the Renilla luciferase activity was then measured. Each transfection was performed in duplicate, and all experiments were repeated at least three times.

Acknowledgments

This research was supported by national Institutes of Health Grant R01 DE 13561-05A1, titled “The Role of P40 Squamous Cell Carcinoma”.

Abbreviations

SAM

sterile alpha motif

His

histidine

JHU

johns hopkins university

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