HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 283, Issue 1, 66-75, January 4, 2008

This Article Abstract Full Text (PDF) Supplemental Data All Versions of this Article: 283/1/66    most recent M704686200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gallegos, J. R. Articles by Lu, H. Search for Related Content PubMed PubMed Citation Articles by Gallegos, J. R. Articles by Lu, H. Social Bookmarking                      What's this?

SCF^βTrCP1 Activates and Ubiquitylates TAp63^*

Jayme R. Gallegos¹, Joel Litersky, Hunjoo Lee, Yi Sun, Keiichi Nakayama^¶||, Keiko Nakayama^¶**, and Hua Lu²

From the Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239, the Department of Biochemistry and Molecular Biology, University of Indiana, Indianapolis, Indiana 46202, the Department of Radiation Oncology, University of Michigan Comprehensive Cancer Center, Ann Arbor, Michigan 48109, the ^¶Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan, ^||CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan, and the ^**Department of Developmental Biology, Center for Translational and Advanced Animal Research, Graduate School of Medicine, Tohoku University, 2-1 Seiryo, Aoba-ku, Sendai 980-8575, Japan

Received for publication, June 7, 2007 , and in revised form, October 26, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
p63 is a member of the p53 tumor suppressor family that is critical for epithelial differentiation and also has an important role in cancer progression. Currently, the molecular mechanisms governing regulation of p63 function remain largely unclear. This study identifies a unique E3 ubiquitin ligase for p63, SCF^βTrCP1. SCF^βTrCP1 is able to bind p63 isoforms, with a higher affinity for the TAp63 isoform. Strikingly, co-expression of TAp63 and βTrCP1 leads to the stabilization of TAp63. This stabilization of TAp63 leads to up-regulation of p21 at the mRNA and protein level by increased binding of TAp63 at the p21 promoter. The up-regulation of p21 causes a subsequent increase in G₁ phase cell cycle arrest. Last, SCF^βTrCP1 is able to ubiquitylate TAp63, and this ubiquitylation, as well as the increased activity of TAp63, is ablated with the expression of a ubiquitin-deficient mutant of βTrCP1 (FβTrCP1). Therefore, our study reveals that SCF^βTrCP1 is an E3 ligase that activates p63 through ubiquitylation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
p63 is a member of the p53 tumor suppressor family. The six major isoforms of p63 have several conserved regions common to the p53 family members, such as the transactivation domain (TA),³ the DNA binding domain, and the oligomerization domain (1–3). The TA isoforms have a full-length TA, whereas the N isoforms do not, and are therefore often considered dominant-negative regulators of the TA isoforms (1–3). Further, the C-terminal regions of these isoforms are alternatively spliced. These variants are termed , β, and (1, 3). The p63 isoforms are the longest and most commonly expressed in adult tissue (2, 3), whereas the p63 isoforms are the shortest and the most potent transcriptional activators of the six isoforms (3, 4). Because of its strong transactivation and its role in the stress response, TAp63 is considered the p63 isoform "most like" p53.

The biological role of p63 is more complex than that of a classical tumor suppressor. p63 knock-out mice have a severe defect in epithelial stratification and fail to form associated tissues, such as teeth, hair, and mammary glands (2, 5). Also, it is debated whether p63 might act as a tumor suppressor or an oncogene (1). Recent data suggest that the Np63 isoforms, when overexpressed, may be tumorigenic, whereas the TA isoforms may play a more important role in tumor suppression (1). Several papers have suggested a reciprocal relationship between Np63 and TAp63, since, under a variety of cellular stimuli, the levels of one are often high when those of the other are low, and vice versa (1). What is clear is that a tightly controlled balance of p63 isoform levels in the cell is critical to homeostasis.

This tight balance is maintained through both direct binding of p53 family member transcription sites and heterodimerization among the p53 family members. Although commanding a unique set of target genes, p63 also shares several target genes with the other p53 family members, such as p21 (6–8). In a p53-deficient background, TAp63 may rescue the growth-inhibitory function of p53 by activating some of these common target genes and causing growth arrest in response to various forms of stress (6–8). The elucidation of both common and isoform-specific signaling pathways will be critical to our understanding of the role of p63 in development and oncogenesis.

In an effort to understand one of the biochemical mechanisms that regulate p63, we searched for a p63-specific regulator that could modulate p63 ubiquitylation and, therefore, its activity. A search of the literature revealed that the inhibitor of IB kinase knock-out mouse displayed an epidermal phenotype very similar to that of the p63 knock-out mouse (9). The inhibitor of IB kinase kinase complex works in conjunction with the E3 ubiquitin ligase complex SCF^βTrCP1 to degrade the IB and allow subsequent up-regulation of NFB (10). βTrCP1 (β-transducin repeats-containing protein 1, Fwd1, etc.) is one of the many substrate recognition components of the SCF complex (10). Humans have two βTrCP proteins, which act in homo- or heterodimers as part of the SCF complex: βTrCP1 and βTrCP2. When exogenously expressed, βTrCP1 is nuclear, whereas βTrCP2 is cytoplasmic (10). βTrCP has two major protein motifs: an F-box motif that connects βTrCP to the rest of the SCF complex though its interaction with Skp1 and a WD domain, which binds the many substrates of βTrCP (10). βTrCP proteins can recognize a canonical DSX_2–3S motif, where the Ser residues are usually phosphorylated (10). However, several noncanonical sequences and regions have also been found. These regions usually depend on a cluster of highly charged or phosphorylated residues and are found in proteins, such as Emi1 and Cdc25a (10–12).

Therefore, it is very plausible that SCF^βTrCP1 may also regulate the p63 pathway. Our study, as described here, tests this idea. We have chosen to focus on the p63 isoforms, because they are the most potent regulators of transcription and may be important in modulating the activity of the other family members during cell growth. Our data demonstrate that SCF^βTrCP1 binds TAp63 and, to a lesser degree, Np63. This interaction takes place at sites on both the N and the C termini of TAp63. Further, this binding leads to subsequent stabilization of TAp63, leading to increased activation of p21, because of increased binding of TAp63 to the p21 promoter. In turn, the up-regulation of p21 leads to an increase in G₁ phase cells. Further, βTrCP1 is able to ubiquitylate TAp63 in vitro and in cells, demonstrating that although it regulates the activity of TAp63, βTrCP1 also has a dual role as an E3 ligase to TAp63. This E3 ligase activity is critical to the increased activation of TAp63 by βTrCP1, since a βTrCP1 mutant that can bind but not ubiquitylate TAp63 (FβTrCP1) is unable to mediate this activity. Thus, this study provides insight into a novel ubiquitin-dependent activation mechanism for TAp63.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell Culture—Hi-5 insect cells were maintained in HfQ SFX-insect cell medium (Perbio) with 1x penicillin/streptomycin (Cellgro) at 25 °C. Human embryonic kidney epithelial cells (HEK293) and human non-small cell lung carcinoma cells (H1299) were cultured as described previously (7). Embryonic mouse embryonic fibroblasts (MEFs) were maintained according to the previous protocol (13). HaCaT and HEKn-E6/E7 cells were obtained from Dr. Mihail Iordanov (Oregon Health and Science University) and cultured as described previously (14, 15).

Antibodies and Vectors—p63 was detected using Pab4A4 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or p63-NTA antibody (generated by our laboratory). Polyclonal -GFP antibody (Santa Cruz Biotechnology) was used for exogenous GFP-p63 IPs, and -His antibody (Qiagen) was used to detect the His-tagged p63, as indicated. βTrCP1 was detected using -FLAGM2 antibody (Sigma) or endogenous βTrCP1 antibodies, N-15 or H-300 (Santa Cruz Biotechnology). Tubulin was detected using antibody from Sigma. p21 was detected using p21 polyclonal sc-H164 (Santa Cruz Biotechnology). Bax was detected using Bax Ab5 (Neomarkers). pCDNA3.1 empty vector, pEGFP-TAp63 (and related mutants F1–F4), pEGFP-Np63, and pCDNA3His-ubiquitin were generated as described previously (7, 16). pCDNA3FLAG-βTrCP1, pCDNA3FLAG-FβTrCP1, and baculoviral expression constructs of Roc-1, HA-Cul1, His-Skp1, and FLAG-βTrCP1 were kindly provided by Dr. Yi Sun (University of Michigan) (17).

Transient Transfection/siRNA Transfection—Transient transfection used Transfectin (Bio-Rad) reagent according to the manufacturer's directions at 70% confluence for all cell lines. For each experiment, a 1:2 µg ratio of pEGFP-TAp63 to pCDNA3-FLAGβTrCP1 vector or a 1:2.5 µg ratio of pEGFP-TAp63 to pCDNA3-FLAGFβTrCP1 vector was used and scaled to the appropriate size of the plate (for ChIP, 0.2 µg of pEGFP-TAp63:2 µg of pCDNA3-FLAGβTrCP1 or 2.5 µg of pCDNA3-FLAGFβTrCP1 was used per 10-cm plate). siRNA oligonucleotide infection was performed using cells at 50% confluence. For each experiment, a βTrCP1/2 siRNA oligonucleotide pool (Santa Cruz Biotechnology) or scramble siRNA oligonucleotide mix was added to each plate at a final concentration of 100 nM. The cells were harvested 48 h post-transfection.

Preparation of Purified His-TAp63, GST-FLAG-βTrCP1, and SCF^βTRCP1 Complex—All of the His-TAp63, GST-FLAG-βTrCP1, and His-TAp63 fragments (F1–F4) were purified as described previously (7, 16). His-TAp63 was purified using Ni²⁺-nitrilotriacetic acid purification, followed by purification on a Poros HQ affinity column (PerSeptive Biosystems). The SCF^βTrCP1 complex was purified as described previously (17). The fractions were run on SDS-PAGE and checked by Western blot using polyclonal -Roc1 (Neomarkers), monoclonal 12CA5 anti-HA (generated in the Lu laboratory), monoclonal -FLAGM2, and monoclonal -His antibodies to detect separate complex components.

Western Blot Analysis—Clarified whole cell lysates were loaded directly onto an SDS gel and probed with antibodies, as noted in the figure legends, according to the previous protocol (7, 16). Band density was calculated using Adobe Photoshop or Optiquant software and normalized to tubulin. For Western blot analysis (WB) indicating protein levels after co-expression, cells were harvested 48 h post-transfection.

In Vitro Binding—Protein-protein association assays were conducted as reported (7, 16).

Co-immunoprecipitation—GFP-TAp63, GFP-Np63, and FLAG-βTrCP1 vectors were co-expressed in HEK293 or H1299 cells, according to protocols above and the figure legends. Clarified whole cell lysate (concentration indicated per figure) was prebound for 2 h using 2 µg of antibody, monoclonal IgG, or polyclonal IgG at 4 °C, and then 30 µl of protein G-agarose bead slurry (Santa Cruz Biotechnology) was added, and the samples continued to rotate at 4 °C overnight. The samples were then washed once with general lysis buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40), once with SNNTE buffer (50 mM Tris-HCl, pH 7.4, 5 mM EDTA, 500 mM NaCl, 1% Nonidet P-40, 50% sucrose), and a second time with lysis buffer. The samples were then prepared using SDS sample buffer (50 mM Tris-Cl, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromphenol blue, 10% glycerol) and analyzed by WB according to the above protocols. For endogenous assays, the co-IP was performed using the noted endogenous antibodies under the same conditions as above with 800 µg of protein lysate harvested from keratinocytes.

Stability Assay—Cells were plated onto 60-mm culture dishes and transfected, as noted above, for regular transfection or siRNA depletion. 48 h post-transfection, cells were treated with 100 µg/ml cycloheximide and harvested at the indicated time points before preparation for WB.

Semiquantitative and Real Time PCR—For RT-PCR, cells were plated according to the protocols above. If transfection was used, cells were harvested 24 h post-transfection. The RNA was isolated using TRIZOL reagent (Invitrogen), according to the manufacturer's directions. cDNA was produced, and RT-PCR was performed using our previous protocol and the same primers for p63, p21, β-actin, and bax, as noted previously (7, 16). βTrCP1 primers were as follows: 5'-ATTGTGCCCAAGCAGCGGAAAC-3' and 3'-TGTTCTCAGCGATGTGGTCCAG-5'. Endogenous mouse TAp63 primers were kindly provided by Dr. M. Koster in the laboratory of Dr. D. Roop. Image J software was used to estimate band density. qPCR was performed with 2 µl of cDNA as described previously using the listed primers against p21 and glyceraldehyde-3-phosphate dehydrogenase (18). Levels were normalized to input and glyceraldehyde-3-phosphate dehydrogenase and then graphed using Microsoft Excel. Data were analyzed for statistical significance using Student's t test.

Flow Cytometry—H1299 cells were transfected with vectors as indicated. After 1.5 days, the cells were treated with 150 ng/ml nocodazole to arrest them in G₂, fixed, permeabilized, and stained with propidium iodide (PI), as noted previously (7, 16). Cells were analyzed for DNA content with a BD Biosciences FACScan flow cytometer. Data were analyzed using a multicycle software program using a polynomial S-phase algorithm.

Chromatin Immunoprecipitation—H1299 cells were transfected according to the protocol above. 24 h post-transfection, the cells were fixed for ChIP and processed according to the previous protocol (18), except that the IP was carried out with 2 µg of ChIP grade Pab4A4 (Abcam) or 2 µg of monoclonal IgG (Sigma). 2 µl of the purification elutant was loaded onto a 96-well plate, and qPCR was performed as noted above using primers against regions of the p21 promoter or gene taken from a previous publication (19). Each sample was normalized to input using Microsoft Excel.

In Vitro Ubiquitylation Assay—The SCF^βTrCP1-mediated in vitro ubiquitylation assay was performed similarly to previous assays (20), with some modifications. The p63 in vitro ubiquitylation assay was carried out in 40 µl of a reaction mixture containing 50 mM Tris-HCl (pH 8.0), 5 mM MgCl₂, 0.5 mM dithiothreitol, 2 mM NaF, 3 µM okadaic acid, and the noted concentrations of His-TAp63, 10 µl of SCF^βTrCP1, 400 ng of UBA-1 (Boston Biochem), 400 ng of UbcH3 (Boston Biochem), 1000 ng of HA-ubiquitin, 5 mM ATP (Amersham Biosciences), and 1.5 mM ATPS (Fisher ICN). The mixture was incubated at 37 °C for 1.5 h and analyzed afterward by SDS-PAGE followed by WB, as noted above.

In Vivo Ubiquitylation Assay—10-cm plates were transfected as noted above. 24 h post-transfection, the cells were treated with 10 µM MG132 (a protease inhibitor) for 6 h. The cells were harvested, and the pellet was split in half. Half underwent standard WB sample preparation (above), and half was prepared using our previous protocol (21).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
βTrCP1 Expression Stabilizes TAp63—To determine if βTrCP1 could act as a regulator for TAp63, we wished to determine if βTrCP1 would have an effect on the stability of TAp63, since βTrCP1 is a well known regulator of protein stability. To answer this question, we used primary MEFs at passage 2 derived from βTrCP1^+/– and βTrCP1^–/– littermates (13) and examined the endogenous levels of TAp63 mRNA and protein. Strikingly, there was a significantly lower amount of TAp63 protein in the βTrCP1^–/– MEFs compared with the βTrCP1^+/– MEFs (30%), although the TAp63β protein level is not affected (Fig. 1A, top). Since the p63 antibody is pan-specific, O3C keratinocytes were used as positive control for the p63 isoforms (23, 24). Further, when the mRNA levels were examined using semiquantitative RT-PCR with TA-specific p63 primers, little change was observed (less than 0.5-fold) in TAp63 between the βTrCP1^+/– and the βTrCP1^–/– MEFs (Fig. 1A, bottom). Since the protein level of TAp63β is minor compared with the level of TAp63, by WB, this RT-PCR measurement is representative of the TAp63 RNA levels. Next, to determine if this observation was specific to βTrCP1 expression, we repeated this experiment in H1299 cells, a cell line that is p53 null and has undetectable endogenous expression of p63 and p73 by WB. In fact, in the presence of increased βTrCP1, the protein level of TAp63 increased significantly above the level of TAp63 alone (Fig. 1B, lane 4 versus lane 2). Therefore, TAp63 protein levels are also increased by a subsequent increase of βTrCP1. These results suggest that βTrCP1 must mediate the endogenous protein stability of TAp63 through a post-translational mechanism.

In order to verify these intriguing observations, we measured the half-life of exogenous TAp63 in the presence βTrCP1. Again, to our surprise, although βTrCP1 has been shown to be involved in the degradation of its substrates, our data demonstrate that it stabilizes TAp63. As shown in Fig. 1C, although the exogenous GFP-TAp63 alone has a half-life of 2–3 h, comparable with others' published data (25), the GFP-TAp63 co-expressed with the FLAG-βTrCP1 was highly stabilized over 6 h. We also confirmed this result using a labeled pulse-chase (data not shown).

Further, to examine the effect of βTrCP on endogenous p63 stability, we used a human keratinocyte line with several p63 isoforms and βTrCP1 (HaCaT). The cells were treated with siRNA oligonucleotides against βTrCP1/2 (since βTrCP2 often rescues βTrCP1 if knocked down alone). An initial experiment was performed to determine the efficiency of knockdown at both 24 and 48 h post-knockdown to find the optimal time to start the degradation assay. Knockdown was confirmed, at 0.47-fold versus the scramble control at 48 h (Fig. 1D). This degree of reduction is biologically significant, since several groups have shown that βTrCP1 is affected by both haploinsufficiency and dominant-negative mutants in vivo (10), compared with the control, by WB (Fig. 1D). Therefore, for our half-life, time 0 for the cycloheximide exposure in Fig. 1E is 48 h after oligonucleotide transfection. As predicted from the data above, the half-life of p63 in the βTrCP1/2 knockdown cells was shorter (about 1.5 h) versus the control (about 3.5 h) (Fig. 1E). The βTrCP1/2 siRNA treatment also reduced the level of endogenous steady-state p63 (data not shown), supporting the result in Fig. 1A. Therefore, βTrCP1 co-expression stabilizes TAp63 exogenously and endogenously.

View larger version (25K): [in this window] [in a new window]   FIGURE 1. βTrCP1 expression stabilizes TAp63. A, βTrCP1 knock-out mice have lower endogenous protein levels of TAp63. βTrCP1+/– or βTrCP1–/– primary MEFs were harvested for either WB (top) or RT-PCR (bottom). In the WB, 150 µg of whole cell lysate for MEFs was loaded beside 30 µg of whole cell lysate from O3C mouse keratinocytes. B, βTrCP1 expression stabilizes raises steady-state protein levels of exogenous TAp63. H1299 cells were transfected for 48 h and harvested for WB. 50 µg of whole cell lysis was used. Loading control denotes nonspecific band. C, βTrCP1 expression stabilizes TAp63 post-cycloheximide treatment. HEK293 cells were transfected with GFP-TAp63 and FLAG-βTrCP1. 48 h later, they were treated with cycloheximide (CHX) and harvested over the indicated time course. Shown is representative WB with quantification of p63 signal intensity/tubulin signal intensity of three experiments graphed below. *, p 0.03. D, βTrCP1 levels are reduced in βTrCP1/2-depleted cells. HaCaT cells were treated with control scramble siRNA oligonucleotides or βTrCP1/2 siRNA oligonucleotides and harvested after 24 or 48 h. 80 µg of whole cell lysate was used for WB analysis. The protein levels were quantified and normalized to tubulin. The numbers above are calculated -fold reduction shown. The lane numbers are denoted below. E, βTrCP1/2 knockdown destabilizes endogenous p63 post-cycloheximide treatment. HaCaT cells were transfected with TrCP1/2 siRNA oligonucleotides or scramble control oligonucleotides. 48 h later, they were treated with cycloheximide and harvested over the indicated time course. Quantification of p63 signal intensity/tubulin signal intensity of representative blot graphed below.

View larger version (18K): [in this window] [in a new window]   FIGURE 2. p63 binds βTrCP1. A, TAp63 and Np63 bind βTrCP1 in cells. GFP-TAp63, GFP-Np63, and FLAG-βTrCP1 were expressed in HEK293 cells. 448 µg of clarified whole cell lysate co-immunoprecipitated per sample, with 15.6% of the prebound lysate loaded as input. WB was performed using the indicated antibodies. B, TAp63 binds βTrCP1 in vitro. GST-βTrCP1 was bound to GSH-agarose bead. 60 ng of purified His-TAp63 was bound to bead, 10% input, and binding samples were analyzed by WB. C, TAp63 and TAp63 bind βTrCP1 endogenously. HEKn-E6/E7 cells were harvested, and 850 µg of total lysate was immunoprecipitated using the noted antibodies (left). The panel on the right shows the IgG control taken from the same gel, same exposure.

View larger version (23K): [in this window] [in a new window]   FIGURE 3. βTrCP1 binds TAp63 N and C termini. A, a schematic of wild-type TAp63 and the TAp63 fragments. The transactivation (TA; striped), DNA binding domain (DBD; black pattern), and oligomerization domain (OD; white pattern) are indicated. The fragments (F1–F4) and the encompassing amino acid residues are noted. B, in vitro mapping of βTrCP1 binding to TAp63. GST0 or GST-βTrCP1 was incubated with 40 ng of His-TAp63 F1–F4, as indicated. 100% input was loaded as a control. The upper band in lanes 3 and 10 is nonspecific. Below is a summary of the binding of βTrCP1 to TAp63. TAp63 is shown as in A, with the stronger and weaker interaction with βTrCP1 shown as the solid and dashed lines, respectively.

View larger version (9K): [in this window] [in a new window]   FIGURE 4. TAp63 activation of p21, but not Bax, is increased by βTrCP1 expression. A, TAp63 activation of p21, but not Bax, is increased by βTrCP1 expression at the mRNA level. RT-PCR for p21, bax, βTrCP1, p63, and β-actin was performed in H1299 cells as shown (right). qPCR was also performed, and p21 was normalized to glyceraldehyde-3-phosphate dehydrogenase (right) Representative data are shown. *, p < 0.0002. B, TAp63 activation of p21, but not Bax, is increased by βTrCP1 expression at the protein level. 70 µg of whole cell lysate was analyzed by WB.

View larger version (10K): [in this window] [in a new window]   FIGURE 5. TAp63 binding at the p21 promoter increases in the presence of βTrCP1 in cells. A, schematic of the p21 promoter. The primer regions used for ChIP at the p21 promoter are shown by gray boxes with labels according to primer set. +, a CATG core sequence; *, a CGTG core sequence. B, TAp63 binding to the p21 promoter increases in the presence ofβTrCP1 in cells. H1299 cells were transfected and subjected to ChIP using the indicated ChIP antibody/qPCR primer combinations. Representative data are shown. *, p 0.02.

TAp63 Binding at the p21 Promoter Increases in the Presence of βTrCP1 in Cells—Since βTrCP1 stabilized TAp63, and βTrCP1 also augmented TAp63 activation of p21 levels, we next asked if βTrCP1 could enhance the presence of TAp63 at the p21 promoter. The regions on the promoter that were used for analysis began at amino acids –2283, –20, and +7878 and spanned 50 bp downstream in each case. These regions are called P1, P2, and P3, respectively (Fig. 5A). The P1 primers include a high affinity site for the p53 family members, whereas the P3 primers are a negative control. Last, sequence analysis of the p21 promoter revealed both CATG sequences and CGTG sequences that may be favorable for p63 binding near the P2 primer set (4). Thus, we also tested this region for TAp63 binding. Consistent with our earlier results, TAp63 alone resulted in a 20-fold increase of promoter binding over the vector alone control, whereas co-expression of TAp63 with βTrCP1 resulted in an increase of over 50-fold of TAp63 bound to the p21 promoter (Fig. 5B). The P3 primers in the gene were negative. Interestingly, P2 primers also could detect an active site of TAp63 recruitment on the p21 promoter (Fig. 5B), demonstrating the existence of a novel site for p63 binding at the p21 promoter. Therefore, the increased stability of TAp63 in the presence of βTrCP1 leads to increased TAp63 bound to the promoter, using both a canonical p53 family binding region and a novel p63 binding region, accounting for the activation of p21 noted above.

View larger version (12K): [in this window] [in a new window]   FIGURE 6. TAp63 activation of growth arrest is increased by βTrCP1 expression. Representative curves of H1299 cells treated with nocodazole, subjected to fluorescence-activated cell sorting, and gated for GFP-positive signal are shown below as generated by FloJo software and plotted to scale on the same axis, as indicated. G1, 2n peak by PI staining; G2, 4n by PI staining. The percentage of GFP-positive cells in each phase of the cell cycle was determined, and data for three experiments were plotted (top). *, p = 0.02.

p63 Is Ubiquitylated by SCF^βTrCP1—Our data above identify βTrCP1 as a regulator of TAp63, which acts by increasing TAp63 through a post-translational mechanism. Since SCF^βTrCP1 is an active member of the SCF complex, we next examined whether SCF^βTrCP1 could ubiquitylate TAp63. First, we wished to examine the general ubiquitylation of p63 in H1299 cells. Using a Ni²⁺-nitrilotriacetic acid binding assay, GFP-TAp63, Myc-TAp63, and Myc-Np63 showed a shift to higher weight moieties in the presence of ubiquitin (Fig. 7A, left, lanes 4, 6, and 8), but not when transfected alone, demonstrating that all of the isoforms in this case are ubiquitylated. Interestingly, the N isoform, although expressed at equivalent levels, has less ubiquitylation than the TA isoforms, as shown by the much lighter laddering. Therefore, several isoforms of p63 are ubiquitylated, with the TA isoforms ubiquitylated to a greater degree, suggesting that ubiquitin may be one way to account for the differences in p63 isoform activity in the cell. Next, to ask if the ubiquitylation we observed was βTrCP1-dependent, we performed an in vitro ubiquitylation assay using a purified SCF complex, p63, E1, E2, and ubiquitin. In fact, in the presence of all of the ubiquitylation components, TAp63 shifted to several ubiquitylated forms of the protein, which were absent in the control lanes (Fig. 7B, lane 7). We then tested whether FLAG-βTrCP1 expression would increase the ubiquitylation of TAp63 in H1299 cells. In this assay, we used FLAG-βTrCP1 or the F-box mutant of βTrCP1, FLAG-FβTrCP1. FβTrCP1 is a dominant negative truncation mutant of βTrCP1 that lacks the F-box. Therefore, the FβTrCP1 protein can bind the substrates of βTrCP1, but those substrates are not presented to the SCF complex for ubiquitylation (data not shown) (10, 26). Consequently, the level of generally ubiquitylated TAp63 precipitated in cells (Fig. 7C, lane 3) increased specifically in the presence of FLAG-βTrCP1 (lane 5) and decreased in the presence of the FLAG-FβTrCP1 (lane 7). Further, as an additional control to ensure that our result was not due to the function of another SCF complex, we also performed experiments co-expressing TAp63 with another SCF substrate recognition component, such as Skp2, and these experiments were negative (data not shown). Thus, this finding demonstrates that SCF^βTrCP1 could act as a specific E3 ubiquitin ligase for TAp63 and that the ubiquitylation of TAp63 requires the presence of the rest of the SCF complex. Therefore, while also acting functionally as a regulator of TAp63, βTrCP1 acts as an E3 ubiquitin ligase for TAp63.

View larger version (21K): [in this window] [in a new window]   FIGURE 7. SCFβTrCP1 ubiquitylates TAp63. A, TAp63 isoforms are more ubiquitylated in cells. H1299 cells were analyzed by WB with 50 µg of whole cell lysis (bottom) or by Ni2+-nitrilotriacetic acid precipitation (top). B, SCFβTrCP1 ubiquitylates TAp63 in vitro. Shown is 100 ng of purified His-TAp63 and ATP reaction buffer with noted substrates, analyzed with -p63 antibody. *, degradation of purified TAp63. C, SCFβTrCP1 ubiquitylates TAp63 in cells. Ubiquitylation was analyzed as in A.

View larger version (13K): [in this window] [in a new window]   FIGURE 8. p63 activation by SCFβTrCP1 is mediated by its ubiquitylation. A, FβTrCP1 co-expression does not stabilize TAp63. H1299 cells were transfected with GFP-TAp63, FLAG-βTrCP1, and FLAG-FβTrCP1. 48 h later, they were treated with cycloheximide (CHX) and harvested over the indicated time course. B, FβTrCP1 co-expression does not activate p21. qPCR was performed on p21, as in Fig. 4. Representative data are shown. *, p = 0.022 increase from TAp63 alone control; **, p < 0.003 decrease from TAp63 + βTrCP1 sample. C, FβTrCP1 expression decreases the amount of TAp63 bound to the p21 promoter. H1299 cells were transfected and subjected to ChIP using the indicated ChIP antibody/qPCR primer combinations. Representative data are shown; the top panel shows data from the P1 primer set, and the bottom panel shows data from the P2 primer set on the same experiment. For the P1 graph,*, p = 0.006 increase from TAp63 alone control; **, p < 0.002 decrease from TAp63 + βTrCP1 sample. For the P2 graph,*, p = 0.005 increase from TAp63 alone control; **, p = 0.013 decrease from TAp63 + βTrCP1 sample.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The literature on various E3 ligases in signaling, in terms not only of degradation but also of activation, is increasing at an incredible rate. Although our report is the first to demonstrate a direct role for SCF^βTrCP1 in stabilization and activation of its substrate, βTrCP1 is involved in activation pathways, such as its well established role in aiding in the activation of the NF-B pathway by degrading IB (27). Additionally, the association between transcriptional activators and E3 ligases is also well trodden experimental ground. In fact, another E3 ligase that forms a complex with the SCF core is Skp2, which co-activates the oncogene c-myc (28, 29). Our data clearly demonstrate that TAp63 is stabilized exogenously at subsaturating levels of both TAp63 and βTrCP1 and destabilized endogenously by knockdown of βTrCP1 and βTrCP2 (Fig. 1, C–E). Our endogenous data in primary MEFs shows that the TAp63 protein level is markedly reduced with the loss of βTrCP1 (Fig. 1A). Consistent with this result, increased levels of βTrCP1 raise the steady-state levels of TAp63 (Figs. 1B and 4B), again while the mRNA levels remain largely even (Figs. 1A and 4A). Taken together, these data demonstrate that the stabilization and activation of TAp63 by SCF^βTrCP1 are present endogenously.

An intriguing observation is the clear effect of the ubiquitin modification itself on TAp63 stability. Our data show that the p63 isoforms have a distinctly differing degree of ubiquitylation, demonstrating that ubiquitylation could be used as one mechanism to distinguish specific activity of each p63 isoform (Fig. 7A). Further, TAp63 is ubiquitylated in the presence of βTrCP1, but not FβTrCP1 (Fig. 7, B and C), suggesting that this binding includes the entire SCF complex. Our data clearly show that significantly more TAp63 is recruited to the p21 promoter in the presence of βTrCP1 (Figs. 5B and 8C) after its stabilization, accounting for the significant activation of p21 at both the mRNA and protein level (Figs. 4, A and B, and 8B). However, strong evidence in Fig. 8 shows that in each case when the ubiquitylation activity is lost through expression of the FβTrCP1 mutant, which can bind the substrates of βTrCP1 but not carry them to the SCF complex for ubiquitylation, TAp63 stability (Fig. 8A), its up-regulation of p21 (Fig. 8B), and its binding to the p21 promoter (Fig. 8C) are lost as well. Adding further detail to our model are the facts that the stability of TAp63 is initiated off chromatin, since our IP data are taken from a whole cell lysate that would not contain chromatin-associated proteins, and that this signaling event most likely occurs in the nucleus, since βTrCP1 and TAp63 both are nuclear proteins and are therefore aptly placed for activation (7, 10). Although ubiquitin is traditionally thought of as a degradation signal, the list of proteins that use ubiquitin as an activating stimulus is growing. Some ubiquitin linkages have been linked to activation, and a recent study shows that forked polyubiquitin chains created by some E2s, including UbcH5c, may also be a signal used for a function other than proteasomal degradation (27, 30). Further, p53 is targeted for ubiquitylation by E4F1, and this modification stabilizes it for a p21-specific activation cascade very similar to what we observe with p63 (31). It is also unsurprising that p63 is targeted by an SCF complex, since p53 is also recognized by a cullin-containing complex during viral infection (22). The characterization of βTrCP1 ubiquitylation of TAp63 would be an interesting future study, especially with the conservation of this stabilizing role of ubiquitin among two of the three p53 family members (1). For now, what is clear in light of our data and earlier studies is that the relationship among p63 DNA binding, subsequent transactivation activity, and stability is most likely the result of subtle modifications and interactions tailored to each isoform.

Specific isoform interaction would be provided by binding affinity and presence of βTrCP1 in the cell. βTrCP1 binds TAp63 and Np63 isoforms in vitro and in cells at both the N- and C-terminal segments of the protein (Figs. 2 and 3), regions known to be important for moderating stability among the p53 family members. It is quite intriguing that βTrCP1 makes both N- and C-terminal contacts with varying interaction at those regions, because the difference in binding strength could easily provide a mechanism for the modulation of isoform-specific activity. Other than conferring a selective TA isoform-specific advantage in binding to and subsequent activation by βTrCP1, this dual binding site may also serve three distinct purposes. First, the placement of the N-terminal site may occlude the binding of other substrates, allowing for selective activation of specific downstream targets involved in growth arrest, but not apoptosis, as our later mRNA/protein activation and cell cycle profile suggests (Figs. 4 and 6). In this respect, the lack of binding in the case of endogenous TAp63β may be explained in that other interacting proteins may have a higher affinity for TAp63β at those sites (Fig. 2C). Second, it could also prevent export to the cytoplasm, in the case of MDM2 occlusion, or prevent immediate degradation by ligases, such as Itch. Last, it is also possible that βTrCP1 binding facilitates the placement of other post-translational modifications on p63. Further, in each of these cases, not only the βTrCP1 affinity for the individual p63 isoforms, but also βTrCP1 affinity for its other substrates would play a critical role in its regulation of p63 under our model, since SCF^βTrCP1 protein levels have been demonstrated as a limiting factor in endogenous signaling (10, 27). Our data show that βTrCP1 also binds TAp63 and NP63; this binding could provide another level of regulation through isoform competition. Likewise, future studies will probably elucidate the effect of the specific balance of p63 isoforms, as well as the other p53 family members, on this pathway in vivo, since these studies focus largely on TAp63 and our binding and ubiquitylation data suggest that differences in binding and ubiquitylation would be important for the regulation of p63 signaling through this mechanism. Therefore, most likely a fine balance exists between the availability of both βTrCP1 and p63 impacting the ability of βTrCP1 to act as a regulator of p63.

View larger version (14K): [in this window] [in a new window]   FIGURE 9. Model of TAp63 ubiquitylation and activation by SCFβTrCP1. TAp63 binds SCFβTrCP1 (top). SCFβTrCP1 then ubiquitylates TAp63 on unknown sites, stabilizing it; thus, more TAp63 binds the endogenous p21 promoter (bottom). p21 transcription is increased due to the increased binding of TAp63 to the promoter.

	FOOTNOTES

 
^* This work is supported in part by NCI, National Institutes of Health Grants CA 93614, CA 095441, and CA 079721 (to H. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ Recipient of funding from an Oregon Health and Science University Department of Dermatology predoctoral fellowship (National Institutes of Health) and an Oregon Health and Science University Ophthalmology and Immunology predoctoral research fellowship (National Institutes of Health).

² To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Indiana, 635 Barnhill Dr., MS 4053, Indianapolis, IN 46202. Tel.: 317-278-0920; E-mail: hualu{at}iupui.edu.

³ The abbreviations used are: TA, transactivation domain; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; GFP, green fluorescent protein; EGFP, enhanced GFP; HA, hemagglutinin; siRNA, small interfering RNA; IP, immunoprecipitation; ChIP, chromatin immunoprecipitation; WB, Western blot; RT, reverse transcription; qPCR, quantitative PCR; MEF, mouse embryo fibroblast.

	ACKNOWLEDGMENTS

 
We thank Yetao Jin, Mushui Dai, and Joylene Gaska for help in editing and final preparation of the manuscript. The Myc-Np63 construct and O3C cells were kindly provided by the laboratory of Dr. Molly Kulesz-Martin. The human keratinocyte lines were kindly provided by the laboratory of Dr. Mihail Iordanov.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Moll, U. M., and Slade, N. (2004) Mol Cancer Res. 2, 371–386[Abstract/Free Full Text]
2. Yang, A., Schweitzer, R., Sun, D., Kaghad, M., Walker, N., Bronson, R. T., Tabin, C., Sharpe, A., Caput, D., Crum, C., and McKeon, F. (1999) Nature 398, 714–718[CrossRef][Medline] [Order article via Infotrieve]
3. Yang, A., Kaghad, M., Wang, Y., Gillett, E., Fleming, M. D., Dotsch, V., Andrews, N. C., Caput, D., and McKeon, F. (1998) Mol Cell 2, 305–316[CrossRef][Medline] [Order article via Infotrieve]
4. Osada, M., Park, H. L., Nagakawa, Y., Yamashita, K., Fomenkov, A., Kim, M. S., Wu, G., Nomoto, S., Trink, B., and Sidransky, D. (2005) Mol. Cell. Biol. 25, 6077–6089[Abstract/Free Full Text]
5. Mills, A. A., Zheng, B., Wang, X. J., Vogel, H., Roop, D. R., and Bradley, A. (1999) Nature 398, 708–713[CrossRef][Medline] [Order article via Infotrieve]
6. Osada, M., Ohba, M., Kawahara, C., Ishioka, C., Kanamaru, R., Katoh, I., Ikawa, Y., Nimura, Y., Nakagawara, A., Obinata, M., and Ikawa, S. (1998) Nat Med 4, 839–843[CrossRef][Medline] [Order article via Infotrieve]
7. Zeng, S. X., Dai, M. S., Keller, D. M., and Lu, H. (2002) EMBO J. 21, 5487–5497[CrossRef][Medline] [Order article via Infotrieve]
8. Bourdon, J. C., Fernandes, K., Murray-Zmijewski, F., Liu, G., Diot, A., Xirodimas, D. P., Saville, M. K., and Lane, D. P. (2005) Genes Dev. 19, 2122–2137[Abstract/Free Full Text]
9. Sil, A. K., Maeda, S., Sano, Y., Roop, D. R., and Karin, M. (2004) Nature 428, 660–664[CrossRef][Medline] [Order article via Infotrieve]
10. Fuchs, S. Y., Spiegelman, V. S., and Kumar, K. G. (2004) Oncogene 23, 2028–2036[CrossRef][Medline] [Order article via Infotrieve]
11. Peters, J. M. (2003) Mol Cell 11, 1420–1421[CrossRef][Medline] [Order article via Infotrieve]
12. Jin, J., Shirogane, T., Xu, L., Nalepa, G., Qin, J., Elledge, S. J., and Wade Harper, J. (2003) Genes Dev. 17, 3062–3074[Abstract/Free Full Text]
13. Nakayama, K., Hatakeyama, S., Maruyama, S., Kikuchi, A., Onoe, K., Good, R. A., and Nakayama, K. I. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 8752–8757[Abstract/Free Full Text]
14. Boukamp, P. P. R., Breitkreutz, D., Hornung, J., Markham, A., and Fusenig, N. E. (1988) J. Cell Biol. 106, 761–771[Abstract/Free Full Text]
15. Iordanov, M. S., Choi, R. I., Ryabinina, O. P., Dinh, T. H., Bright, R. K., and Magun, B. E. (2002) Mol. Cell Biol. 15, 5380–5394
16. MacPartlin, M., Zeng, S., Lee, H., Stauffer, D., Jin, Y., Thayer, M., and Lu, H. (2005) J. Biol. Chem. 280, 30604–30610[Abstract/Free Full Text]
17. Tan, M., Gallegos, J. R., Gu, Q., Huang, Y., Li, J., Jin, Y., Lu, H., and Sun, Y. (2006) Neoplasia 8, 1042–1054[CrossRef][Medline] [Order article via Infotrieve]
18. Li, Y., Zeng, S. X., Landais, I., and Lu, H. (2007) J. Biol. Chem. 282, 6936–6945[Abstract/Free Full Text]
19. Gomes, N. P., Bjerke, G., Llorente, B., Szostek, S. A., Emerson, B. M., and Espinoza, J. M. (2006) Genes Dev. 20, 601–612[Abstract/Free Full Text]
20. Honda, R., Tanaka, H., and Yasuda, H. (1997) FEBS Lett. 420, 25–27[CrossRef][Medline] [Order article via Infotrieve]
21. Jin, Y., Zeng, S. X., Lee, H., and Lu, H. (2004) J. Biol. Chem. 279, 20035–20043[Abstract/Free Full Text]
22. Querido, E., Blanchette, P., Yan, Q., Kamura, T., Morrison, M., Boivin, D., Kaelin, W. G., Conaway, R. C., Conaway, J. W., and Branton, P. E. (2001) Genes Dev. 15, 3104–3117[Abstract/Free Full Text]
23. King, K. E., Ponnamperuma, R. M., Gerdes, M. J., Tokino, T., Yamashita, T., Baker, C. C., and Weinberg, W. C. (2006) Carcinogenesis 27, 53–63[Abstract/Free Full Text]
24. Kulesz-Martin, M., Lagowski, J., Fei, S., Pelz, C., Sears, R., Powell, M. B., Halaban, R., and Johnson, J. (2005) J. Investig. Dermatol. Symp. Proc. 10, 142–152[CrossRef][Medline] [Order article via Infotrieve]
25. Rossi, M., Aqeilan, R. I., Neale, M., Candi, E., Salomoni, P., Knight, R. A., Croce, C. M., and Melino, G. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 12753–12758[Abstract/Free Full Text]
26. Belaidouni, N., Peuchmaur, M., Perret, C., Florentin, A., Benarous, R., and Besnard-Guerin, C. (2005) Oncogene 24, 2271–2276[CrossRef][Medline] [Order article via Infotrieve]
27. Krappmann, D., and Scheidereit, C. (2005) EMBO Rep. 6, 321–326[CrossRef][Medline] [Order article via Infotrieve]
28. Kim, S. J., Herbst, A., Tworkowski, K. A., Salghetti, S. E., and Tansey, W. P. (2003) Mol. Cell 11, 1177–1188[CrossRef][Medline] [Order article via Infotrieve]
29. Von der Lehr, N., Johnsson, S., and Larsson, L. (2003) Cell Cycle 5, 403–407
30. Kim, H. T., Kim, K. P., Lledias, F., Kisselev, A. F., Scaglione, K. M., Skowyra, D., Gygi, S. P., and Goldberg, A. L. (2007) J. Biol. Chem. 282, 17375-17386[Abstract/Free Full Text]
31. Le Cam, L., Linares, L. K., Paul, C., Julien, E., Lacroix, M., Hatchi, E., Triboulet, R., Bossis, G., Shmueli, A., Rodriguez, M. S., Coux, O., and Sardet, C. (2006) Cell 127, 775–788[CrossRef][Medline] [Order article via Infotrieve]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				M. MacPartlin, S. X. Zeng, and H. Lu Phosphorylation and Stabilization of TAp63{gamma} by I{kappa}B Kinase-{beta} J. Biol. Chem., June 6, 2008; 283(23): 15754 - 15761. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Supplemental Data All Versions of this Article: 283/1/66    most recent M704686200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gallegos, J. R. Articles by Lu, H. Search for Related Content PubMed PubMed Citation Articles by Gallegos, J. R. Articles by Lu, H. Social Bookmarking                      What's this?