ICANCER RESEARCH56, 2649-2654. June I. 1996]

ABSTRACT

The levels of the tumor suppressor protein p53 are generally quite lowin normal cells, due in part to its rapid turnover. Previous studies haveimplicated ubiqultin-dependent proteolysis in the turnover of wild-typep53 but have not established whether or not p53 is itself a substrate of theubiquitin system. In this study, Inhibitors ofthe 26S proteasome have beenused to further explore the role of ubiquitin proteolysis in regulating p53turnover. Increased levels of the tumor suppressor protein p53 wereobserved in normal cells, as well as in cells expressing the human papillomavirus 16 E6 oncoprotein, on exposure of the cells to proteasomeinhibitors. Pulse-chase experiments Indicated that the increased p53 levelsresulted from stabilization of the protein. Furthermore, ubiquitin-p53conjugates were detected in untreated as well as i-irradiated cells, mdieating that ubiqulfin-dependent proteolysis plays a role in the normalturnover of p53. Increased levels of the cydlin:cydlin-dependent kinaseinhibitor p21, a downstream effector of p53 functIon, were also observed

in proteasome inhibitor-treated cells, and this increase was due in part toan increase in p21 mRNA.

INTRODUCTION

The tumor suppressor protein p53 is inactivated in the majority ofhuman cancers either by mutation, the action of viral oncoprotemns, orinactivation by the cellular factor mdm-2 (1, 2). Steady-state levels ofwild-type p53 are normally very low in most cells as a consequenceof a short protein half-life. In contrast, p53 levels increase in responseto DNA-damaging agents, and this can lead to a G1 cell cycle arrest(3—5).This G@arrest is dependent on wild-type p53 and is believed toresult from p53-activating transcription of the gene encoding p21, acdk4 inhibitor. The increase in p53 levels following DNA damageresults in large part from stabilization of the p53 protein (5). How thestability of p53 is regulated is currently unknown.

The half-life of p53 in HPV-positive cancer cells and in HPVimmortalized cell lines is significantly shorter than that seen in primary cells (6), and lowered steady-state levels of the protein areobserved (7). A molecular explanation for these phenomena is provided by in vitro studies, which have shown that the E6 proteins ofcancer-associated HPVs (i.e., HPV-16 and -18), in cooperation with acellular factor termed E6AP, can complex p53 in vitro (8, 9) andpromote its ubiquitination and subsequent degradation by the proteasome (10—12).It is unknown whether p53 is normally degraded by theubiquitin-proteasome system in the absence of E6, although recentstudies have suggested that this may be the case. For example,Chowdary et a!. (13) demonstrated increased levels of p53 protein incells in which the ubiquitin system was conditionally inactivated. Inaddition, Ciechanover et a!. (14) have shown limited ubiquitindependent degradation of p53 in vitro in rabbit reticulocyte lysate inthe absence of E6.

Ubiquitin-dependent proteolysis plays an important role in thedegradation of abnormal and short-lived regulatory proteins (15—26).Three enzymatic activities, designated El—E3, are involved in theubiquitination of a protein. Ubiquitin is first activated through itscovalent thioester linkage to the El ubiquitin-activating enzyme.Activated ubiquitin is then transferred to the E2 enzyme, also knownas a ubiquitin-conjugating enzyme, again in the form of a high-energythioester bond. In many cases, ubiquitination requires an E3 protein,also known as a ubiquitin protein ligase, which is involved in therecognition of the target. Some E3s can apparently transfer the activated ubiquitin directly to a substrate (12), whereas in other cases, theE2 enzyme may carry out this function. Additional ubiquitin moietiesare linked sequentially to each other, leading to the formation ofmultiubiquitin chains, and the multiubiquitinated substrate is thendegraded by the 26S proteasome. Ubiquitin hydrolases (isopeptidases)function to cleave ubiquitin from proteins prior or subsequent todegradation, and the free ubiquitin can then be reused in the ubiquiti

nation of other proteins. Enzymes that can participate in the HPVE6-dependent ubiquitination ofp53 have been identified (10—12).TheE3 activity consists of a complex between the E6 protein of eitherHPV-16 or -18 and the cellular E6-associated protein E6AP (8,10—12).Recently, it has been demonstrated that E6AP, in the presenceof E6, can transfer activated ubiquitin directly to p53 (12). It iscurrently unknown whether E6AP is involved in the normal turnoverof p53 in the absence of E6.

Rock et a!. (27) have described a series of peptide aldehydes thatinhibit the chymotryptic and peptidylglutamyl peptidase activities ofthe 26S proteasome in cultured cells. Transient exposure of cells tothese inhibitors has no apparent cytotoxic effects and does not affectoverall rates of protein synthesis (27). Using the inhibitors, Rock et a!.(27) demonstrated a role for the ubiquitin-proteasome system in theprocessing and subsequent presentation of MHC class I-restrictedantigens. Similarly, Palombella et a!. (28) used these same inhibitorsto demonstrate that processing of the p105 NF-KB precursor into theactive p50 subunit of the transcriptional activator occurs in ubiquitinproteasome-dependent manner. In this study, we report an increasedsteady-state level and stability of p53 in cells exposed to the proteasome inhibitors. Ubiquitin-p53 conjugates were detected in untreatedand ‘y-irradiatedcells, providing direct evidence that ubiquitin proteolysis is involved in the degradation of p53. Levels of the cdkinhibitor p2l were also increased in response to the proteasomeinhibitors, and this increase was due in part to an increase in p21mRNA. Our results indicate that the normal turnover of p53 occurs viaubiquitin-dependent proteolysis.

MATERIALS AND METHODS

Cell Strains and Tissue Culture. Human diploid fibroblast cell linesGM6419, GM6419(E6), AGl52l, and AG1521(E6) were generously providedby Hatsumi Nagasawa and John Little (Harvard School of Public Health).AG1S21 cells were derived from the foreskin of an apparently normal infant,whereas GM6419 cells are from an individual who developed retinoblastomatumors and are heterozygous for the wild-type retinoblastoma gene RB. E6-expressing cells were derived by transfection of GM6419 and AGl52l cells

(29) with the HPV-l6 E6 expression plasmid p1436 (30). C33 cells have beendescribed (7). RKO cells were from Michael Brattain (Department of Biochemistry, Medical College of Ohio). Saos-2 cells were a gift of Phil Hinds

2649

Received 1/23/96; accepted 4/1/96.Thecostsof publicationof thisarticleweredefrayedin partby the paymentof page

charges. This article must therefore be hereby marked advertisement in accordance with18U.S.C.Section1734solelyto indicatethis fact.

I This work was supported by NIH Grants POl-CA-50661-0i and ROl-CA64888-l

(P. M. H.).2 Present address: Department of Biochemistry, Rutgers University, Piscataway, NJ.

3 To whom requests for reprints should be addressed, at Department of Pathology,Harvard Medical School, 200 Longwood Avenue, Boston, MA 021 15. Phone: (617)432-2884; Fax: (617) 432-2882.

4 The abbreviations used are: cdk, cyclin-dependent kinase; HPV, human papilloma

virus; Ab, antibody; mAb, monoclonal Ab.

In Vivo Ubiquitination and Proteasome-mediated Degradation of p531

Carl G. Maki, Jon M. Huibregtse,2 and Peter M. Howley3

Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115

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PROTEASOME INHIBITION AFFECTS p53

(Harvard Medical School). U2OS cells were from Ed Harlow (MassachusettsGeneral Hospital). RKO cells were grown in McCoy's 5A media containing10% fetal bovine serum. All other cell strains were maintained in DMEMcontaining 10% fetal bovine serum. y irradiation of cells was carried at theLaboratory of Radiobiology (Harvard School of Public Health).

Proteasome Inhibitors. All proteasome inhibitors used in this study werekindly provided by Myogenics Corp. (Boston, MA). The inhibitor lactacystin(31) as well as the peptide aldehydes MGIOI (Ac-Leu-Leu-NIe-H), MGIO2(N-Ac-Leu-Leu-Nme-H), MGI 15 (Z-Leu-Leu-Nva-H), and MG132 (Z-LeuLeu-Leu-H) were maintained in DMSO at a final concentration of 40 mo.i.

SDS-PAGE, Western Blots, and Immunoprecipitations. Exponentiallygrowing cells were seeded at 50% confluence in 60-mm dishes. Twenty-fourto 36 h later, the proteasome inhibitors in DMSOwere added directly to theculture media at the indicated concentrations. Cultures of the mock-treatedcells were exposed to an equal volume of DMSO (minus inhibitor). Atindicated times thereafter, protein extracts were prepared. For Western blotanalysis without prior immunoprecipitation, cells were washed twice withPBS, scrapedinto0.5 ml lysis buffer[50 nmiTris (pH 8.0), 5 mMEDTA, 150mM NaCI, 0.5% NP4O, and 1 nmi phenylmethylsulfonyl fluoride[, and incubated on ice for 15 mm with occasional light vortexing. Cells were thensonicated for 10 pulses at setting 5, 50% output, using a Branson 450 sonifier.Lysates were spun at 15,000 X g for 15 mm to remove cellular debris. Onehundred @.tgprotein extract from the resulting supernatants were resolved by

SDS-PAGEandtransferredto Immobilon-Pmembranes(Millipore)for detection with p53 or p21 Abs. For radiolabeling proteins, cells were grown inmedia lacking methionine and cysteine and then labeled with 35S-methionineand 35S-cysteine(Express labeling mix; DuPont NEN Research Products) inthe presence or absence of 0.04 mr@iMG132. Labeled cells were chased innormal media containing methionine and cysteine in the presence or absenceof MG] 32 for various times, and protein extracts were prepared as describedabove. Labeled proteins were immunoprecipitated from extracts using anti-p53Ab-421 or Ab-6 (Oncogene Science). Immunoprecipitates were resolved on12% SDS-PAGE gels, and the gels were dried and exposed to film. For

detection of p53-ubiquitin conjugates, cell lysates were prepared in radioimmunoprecipitation assay buffer [2 mt@iTris (pH 7.5), 5 mM EDTA, 150 mt@iNaCI, 1.0% NP4O, 1.0% deoxycholate, 0.025% SDS, and I mMphenylmethylsulfonyl fluoride] and immunoprecipitated with p53 Ab-6. The immunoprecipitates were resolved by SDS-PAGE and transferred to an Immobilon-P

membrane, and the membrane was then autoclaved in water for 15 mm. Themembrane was cut at approximately Mr 60'000' and the upper portion of themembrane was examined by Western blotting with the anti-p53 monoclonalAb 1801 (Oncogene Science) or with the antiubiquitin monoclonal Ab M43(generously provided by Linda Guarino, Texas A&M University; Ref. 32).

RNA Isolation and Analysis. Total RNA was prepared using an RNAisolation kit (RNeasy; Qiagen Corp.). RNA samples (10 pg) were separated on1.2% agarose formaldehyde gels and transferred to Hybond N+ membranes

(Amersham) for Northern blot analysis. A full-length human p21 cDNA (33)was used as a probe to detect the p21 transcripts. Probes were labeled byrandom priming using a Prime-It labeling kit (Stratagene).

RESULTS

Increased Levels of p53 in Response to Proteasome Inhibitors.Previous studies have implicated the proteasome- and ubiquitin-dependent proteolysis in the turnover of wild-type p53 (13, 14) but havenot established whether or not p53 is itself a substrate of the ubiquitinsystem. To gain evidence for the involvement of the proteasome inp53 degradation, p53 levels were examined in cells exposed to specific proteasome inhibitors. GM6419 cells (human diploid fibroblaststhat express wild-type pS3) were exposed to a series of peptidealdehydes that inhibit the activity of the 265 proteasome in culturedcells (27), and p53 levels were determined by Western blot analysis(Fig. 1A). Steady-state levels of p53 were markedly increased inGM6419 cells exposed to the inhibitors MG1O1, MG1 15, and

MG132. In contrast, p53 levels did not change in response to calpaininhibitor II (MG1O2), which is structurally similar to MG1OI, 115,and 132 but which has little effect on proteasome function (27). These

A. I@: GMM19@

@ g@ @,

C E@@

p53.*.@@

B. I GM6419__@fl RKO

C')@@@@@@ @u,@@

C') 0 0 @- C\1 Lt)@ 0 @- c'.j @n (‘.1(-‘C E@@@ E @!

p53-ø@.-

data suggested that inhibition of the proteasome resulted in increasedsteady-state levels of p53.

These findings were extended to other cell types, including RKOcells (a human colon cancer cell line that expresses wild-type p53),AGl52l cells (normal primary human skin fibroblasts, which alsoexpress wild-type pS3), and GM6419 and AG1521 cells, which cxpress the HPV-16 E6 oncoprotein (Figs. lB and 2). Tsang et a!. (29)have shown that expression of HPV-l6 E6 in GM6419 and AG 1521cells can block the accumulation of p53 protein after DNA damageand thus prevent a DNA damage-induced G1 arrest. Cells were cxposed to the indicated concentrations of MG132, and p53 levels weredetermined by Western blotting. Increased levels of p53 were observed in each cell type examined in response to MG132. In theseexperiments, the levels of the p53 increases in cells exposed to 0.025mM MG132 were determined by densitometric scanning and were

approximately 5-fold in GM6419 cells, 3-fold in RKO cells, 15-foldin AG1S21 cells, and 6- and 8-fold in the AG1521(E6) andGM6419(E6) cells, respectively. Thus, proteasome inhibitors cause anincrease in p53 levels in either the presence or absence of HPV-16 E6.These results support the hypothesis that p53 is degraded by theproteasome in vivo and are consistent with in vitro results, whichindicate that the proteasome- and ubiquitin-dependent proteolysis isinvolved in the degradation of p53 in the presence of E6.

Increased Levels of p53 Result from Inhibition of the Proteasome. Not all proteins increase in response to these peptide aldehydes. To demonstrate this specificity, the effect of these inhibitors ona protein with a very long half-life, the retinoblastoma protein pRb,was examined. Transient treatment with the inhibitor is predicted tohave little effect on the level of long-lived proteins. As expected, theoverall level of pRb did not change in response to inhibitor treatment

2650

Fig. 1. A. exponentially growing GM6419 cells were either untreated (no tr.), DMSOtreated (mock), or exposed to a 0.04 msi concentration of the indicated peptide aldehydesfor 4 h, and protein extracts were prepared. One hundred pg of each extract were resolvedon 9% polyacrylamide gels (SDS-PAGE), and p53 levels were determined by Westernblotting using the human-specific anti-p53 Ab-6 (Oncogene Science). Arrow, p53 band.Band above p53 band, nonspecific background band, which was not consistently ohserved. B, Exponentially growing GM6419, RKO, and AGl52l cells were either untreated(no tr.), treated with DMSO (mock), or exposed to the indicated m@ concentration ofMG132 for 4 h, and protein extracts were prepared. p53 levels were determined asdescribed above. An extract from the C33 human cervical cancer cell line, whichexpresses high levels of a mutant form of p53 (7). was used as a positive control for theWestern blot.

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PROTEASOME INHIBITION AFFECTS p53

the presence of proteasome inhibitors (Fig. 4, A and B). This suggeststhat inhibition of the proteasome might also stabilize proteins thataffect p53 ubiquitination, including ubiquitin hydrolases (isopeptidases), which cleave ubiquitin from protein substrates. Indeed, thedata in Fig. SA (below) indicate that ubiquitinated forms of p53 are nolonger detected in proteasome inhibitor-treated cells, supporting thepossibility that proteasome inhibitors may stabilize an isopeptidaseactivity that can deubiquitinate p53. The results of Fig. 4 indicate thatthe increased levels of p53 following inhibitor treatment can beaccounted for in large part by an increase in the half-life of the protein.p53 mRNA levels were unchanged in GM6419 cells after treatmentwith MG132 (0.025 nmi) for 4 h (not shown).

Effect of Proteasome Inhibitors on p53 Ubiquitination. Although ubiquitin-dependent proteolysis has been implicated in thedegradation of p53, ubiquitin-p53 conjugates have never been demonstrated in vivo. Ubiquitin-protein conjugates, however, are difficultto detect, because they are intermediates of degradation and are rarein vivo. In initial experiments, we were unable to detect ubiquitin-p53conjugates by Western blot analysis in untreated cells or in cellsexposed to the proteasome inhibitors. To detect ubiquitinated p53, itwas necessary to first immunoprecipitate p53 from a large number ofcells and then examine the immunoprecipitates for ubiquitinated p53

A. 5

C@) GM6419(E6) AG1521 (E6)C@)‘@ U)

‘- U)

MG132(mM) o c@@ o@

p53@ .@ - ____

Fig. 2. AGl52l and GM6419 cells, which express the HPV-l6 E6 protein, wereexposed to the indicated concentrations (mM)of MG132 for 4 h. Protein extracts wereprepared, and p53 levels were determined by Western blotting using p53 Ab-6. An extractfrom C33 cells serves as a control.

(not shown). To demonstrate that the increased levels of p53 observedin inhibitor-treated cells resulted from inhibition of the proteasome,the relative ability of the proteasome inhibitors to cause an increase inp53 levels was examined. The inhibitors MG1O1, MG11S, andMG132 have different K1sagainst a purified 205 proteasome in vitro(MG1O1, 140 nM;MG! 15, 21 nz@i;and MG132, 4.0 nrs@;Ref. 27). Eachof the inhibitors was tested in a dose-dependent manner for its abilityto increase the levels of p53 in cultured cells. As shown in Fig. 3A,MG132 was a more potent inducer of p53 in cells than was MG1 15,which was a more potent inducer than MG1O1. These results indicatethat the relative efficacy with which the inhibitors affect a p53increase parallels their K1values against the 20S proteasome in vitro,suggesting that the increase in p53 levels is through the inhibition ofproteasome function. The dose effect observed in Fig. 3 is not entirelyconsistent with the K1 values of the inhibitors (i.e., MG! 15 at fivetimes the concentration of MG132 is expected to be equally effectiveat causing a p53 increase, but it is not). This discrepancy may be dueto the fact that the K1s were determined in vitro against the 205proteasome, whereas the data in Fig. 3A were obtained in vivo, inwhich ubiquitinated proteins are degraded by the 26S proteasome,which has the 205 particle as its proteolytic core. The dose dependency for these peptide aldehydes demonstrated for p53 is similar tothat previously shown for MHC class I antigen presentation (27) andthe processing of NF-KB (28).

p53 levels were also examined in cells treated with lactacystin, aStreptomyces metabolite that is the most highly specific proteasomeinhibitor known (31). Treatment with lactacystin also resulted in theaccumulation of p53 protein (Fig. 3B) and did so with an efficiencyequal to that of MG132. These data are consistent with the hypothesisthat the increase in p53 protein in response to the inhibitors resultsfrom an inhibition of the proteasome.

p53 Half-Life Increases in Response to Proteasome Inhibitors.If p53 is degraded by the proteasome in vivo as it is in vitro, theninhibition of the proteasome in cells would be expected to stabilizep53 protein. The half-life of p53 protein in the presence or absence ofinhibitors was therefore determined (Fig. 4). AG1521 and GM6419cells (untreated or treated with the proteasome inhibitor MG132) werepulse labeled in media containing 35S-methionine and 35S-cysteineand then chased in the presence or absence of MG132. p53 wasimmunoprecipitated from labeled cell extracts at various times afterinitiating the chase, and the amount of p53 immunoprecipitated ateach time point was used to determine the half-life of the protein. Inthe absence of the inhibitor, the half-life of p53 protein was less than4 h in both cell types (Fig. 4C). In contrast, the half-life of p53 wasgreater than 15 h in AG1521 and GM6419 cells in the presence ofMG132.Theseresultsindicatedthat the p53 proteinhalf-lifeincreased by at least 4-fold in each cell type in response to the protea

some inhibitors. Interestingly, the radiolabeled, full-length p53 proteindid not accumulate into higher molecular weight species (whichwould be indicative of ubiquitination) when the cells were chased in

I32

115

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0

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>

CDCD

I

1.0 10 25Inhibitorconc. (micromolar)

B. GM64I9 AG1521

p53—ø'.@4 p53

Fig. 3. A. A0l521 cells were exposed for 4 h to the indicated m@ concentrations ofMGlOl, MGll5, and MGI32, and p53 levels were determined by Western blotting andquantitated using a phosphorimager. The level of p53 in untreated cells was given arelative value of 1. These results were repeated in two independent experiments usingAGI52I cells and two independent experiments using GM6419 cells. Comparable resultswere obtained. B, A0l52l and GM64l9 cells were mock treated (—)or exposed to a0.025 msi concentration of lactacystin (+) for 4.5 h, and p53 levels were determined byWestern blotting.

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PROTEASOME INHIBITION AFFECTS p53

and the im.munoprecipitates were examined by Western blot analysiswith monoclonal Abs against either ubiquitin or p53 (Fig. SA). Aladder of high molecular weight protein bands was recognized by theubiquitin monoclonal Ab in the p53 immunoprecipitates from bothuntreated and y irradiated cells. The sizes of these bands, rangingfrom Mr @69,000to —90,000, are consistent with the addition ofbetween two and five ubiquitin moieties to p53. Identical bands wererecognized by the p53 monoclonal Ab 1801 when the blot wasstrip@ (confirmed by re-exposure) and reprobed with the 1801 Ab.This confirms that these bands are ubiquitinated conjugates of p53,providing very strong evidence that wild-type p53 is indeed degradedvia the ubiquitin-proteasome pathway. The levels of p53 as well as thep53-ubiquitin conjugates were consistently more abundant in U2OScells following @‘irradiation (as illustrated in Fig. 5, A and B).Surprisingly, ubiquitin-p53 conjugates were not detected in extractsfrom cells treated with the proteasome inhibitor MG115. Possibleexplanations for these results are discussed below. Western blotanalysis without prior immunoprecipitation showed that p53 levelsincreased in U2OS cells in response to y irradiation and after treatment with the proteasome inhibitor MGI15 (Fig. SB).

p2! Protein and mRNA Levels Increase in Response to Proteasome Inhibitors. Overexpression of p53 is known to activate transcription of a number of cellular genes, including the gene encodingthe cyclin:cdk inhibitor p2! (also called WAFJ, Cipi, and Sdil; Refs.33 and 36—38).Therefore, p21 protein and mRNA levels were also

p53—ø@-@

.@ >, 10U@ I—

Fig. 5. A, U2OS cells were either untreated (no tr.), exposed to 0.025 mat MG1I5, or.y irradiated (X-ray) at a dose of 6 Gy; 6.5 h after treatment, cell extracts were preparedin radioimmunoprecipitation assay buffer, and p53 was immunoprecipitated from —2mgextract using the anti-p53 monoclonal Ab-6 (Oncogene Science). Immunoprecipitateswere resolved through 9% SDS-PAGE gels and transferred to an Immobilon-P membrane.Toavoiddetectionof theAbheavychain(Mr50,00055,000),tbe membranewascutatMr @60,OOO.The top portion of the membrane was examined by Western blot analysiswith the ubiquitin monoclonal Ab M43 (left). The blot was then snipped and reprobedwith the p53 monoclonal Ab 1801 (right), which recognizes an epitope separate from thatrecognized by Ab-6. The sizes (X 1000) of molecular weight protein markers are mdicated. The ladder of bands (p53-Ub@) recognized by both Abs represents ubiquitinatedforms of p53. The same bands were detected when blots were first probed for p53 andsecondarily for ubiquitin (not shown). B, U2OS cells were either treated with DMSO(mock), y irradiated (X-ray), or treated with MG1 15, as in A. Thirty @sgcell extracts wereexamined without prior immunoprecipitation by Western blot analysis with p53 Ab-6.

A.

C')@3 +lnhib. _____@@@@@ 101520 0 2 5 815

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B.I GM6419cells

+ Inhib. - lnhlb.Chase(his.)0 51015 0 51015

p53 -@

C.

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AG1521 cells

Fig. 4. AGI52I (A) or GM6419 (B) cells were grown in media lacking methionine andcysteine for I h and then labeled for 1 h in the presence of 250 gaCi35S-methionineand35S-cysteine(Express labeling mix; DuPont NEN Research Products) and the presence orabsence of M0132 (0.04 mM). The cells were then chased in normal media for theindicated amounts of time, and cell extracts were prepared. MG132 was maintained in themedia of the treated cells during the chase period. Labeled p53 was immunoprecipitatedfrom total cell extracts using anti-p53 Ab-42l. Immunoprecipitates were resolved on 12%SDS-PAGE gels, and the gels were dried and exposed to film. p53 band, p53 immunoprecipitated from C33 cells (which contain high levels of p53) but not from Saos-2 cells(whichlackp53;notshown).Thep53bandcouldalsobe demonstratedby immunoprecipitation using anti-p53 Ab-6 (data not shown). C. half-life of p53 is extended in thepresence of proteasome inhibitor. AG 1521 cells (•and 0) and GM6419 cells (U and U)were labeled and then chased in the presence (•and U) or absence (0 and 0) of 0.04 matMG132. The amount of radiolabeled p53 immunoprecipitated at each time point wasquantitated on a phosphoimager and is plotted. The amount of p53 at the zero time pointis considered 100%. The average of two separate experiments is shown.

0 5 10 15 20

@-Ub@[1

200

@-,— 69 —

92

Time (hrs.)

B.

species. For these experiments, U2OS cells (a human osteosarcomacell line) were used, because they expresses wild-type p53 (34), andbecause the p53 growth arrest pathway in response to DNA damage isfunctionally intact (35). U2OS cells were either untreated, ‘yirradiated, or treated with the proteasome inhibitor MG1 15 for 6.5 h. p53protein was then immunoprecipitated with anti-p53 monoclonal Ab-6,

2652

p53 ip (Ab-6

Ubwestern p53western(1801)

Ip53-Ub@>1 10

.@ @E .@ 0

C X

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PROTEASOME INHIBITION AFFECTS p53

A.

B.

DISCUSSION

The E6 oncoprotein encoded by the cancer-associated HPV typescan complex p53 (8, 9) and target its ubiquitination and subsequentdegradation by the proteasome in vitro (10—12).This activity correlates well with the low steady-state levels and shortened half-life ofp53 usually observed in E6-expressing cells (6, 7). There is evidencethat p53 may be degraded via ubiquitin-mediated proteolysis in normal cells in the absence of E6. Ciechanover et a!. (14), using rabbitreticulocyte lysates, showed that the degradation of p53 in vitro wasATP dependent and involved an El ubiquitin-activating enzyme.Also, Chowdary et a!. (13) demonstrated increased levels and stabilityof p53 in vivo in a mouse cell line in which an El ubiquitin-activatingenzyme was conditionally inactivated. However, it was heretoforeunknown whether or not p53 itself was a direct target for ubiquitinmediated degradation in vivo. In this report, we demonstrate for thefirst time the presence of ubiquitinated p53 protein species in vivo incells expressing wild-type p53. This provides direct evidence thatwild-type p53 is degraded by the ubiquitin-proteasome system.

Ubiquitin-p53 conjugates were detected in U2OS cells (a humanosteosarcoma cell line that expresses wild-type pS3) that were eitheruntreated or DNA damaged by y irradiation. Given these results, itwas surprising that ubiquitin-p53 conjugates were not detected inextracts from cells treated with the proteasome inhibitors. There are anumber of explanations that could account for these results. First, it ispossible that a highly ubiquitinated form of p53 did accumulate inresponse to the proteasome inhibitor, but that this form could not beimmunoprecipitated and was, therefore, not detected in our assay. Wewere able to detect ubiquitinated p53 molecules with migration thatwas consistent with a size (Mr @90'000)that corresponded to theaddition of five ubiquitins to p53. Second, the inability to detectubiquitinated p53 after proteasome inhibitor treatment could be cxplained if the isopeptidase activity that removes ubiquitin from p53were enhanced by the inhibitor. For example, if the isopeptidaseresponsible for removing the ubiquitin moieties from p53 was itself atarget of the proteasome, then the proteasome inhibitors would causea stabilization of the isopeptidase, increasing its activity and resultingin the deubiquitination of p53. This may explain why only the fulllength, non-ubiquitinated form of p53 accumulates in proteasomeinhibitor-treated cells. The proteasome inhibitors have been reportedto have no inhibitory effect on isopeptidase activity (cited in Ref. 27).

Increased p53 levels were observed in a variety of cell linesexposed to different protcasome inhibitors, and this increase resultedin large part from a stabilization of the protein. The relative efficacywith which the proteasome inhibitors caused an increase in p53protein paralleled their K1values against the 205 proteasome in vitro.This suggests that the increase in p53 levels resulted from proteasomeinhibition. Inhibition of the proteasome resulted in increased levels ofp53 in normal cells and in cells expressing the HPV-16 E6 oncoprotein. HPV-16 E6 expression blocks the increase in p53 protein levels,which is normally observed in cells exposed to DNA-damaging agents(3—5,29), but does not prevent the accumulation of p53 in response tothe proteasome inhibitors (Fig. 2). Taken together, these results suggest that the increase in p53 following DNA damage does not resultfrom an inhibition of the proteasome. Furthermore, because E6 doesnot prevent the stabilization of p53 by the proteasome inhibitors, theinhibitors are not stabilizing p53 by acting as a DNA-damaging agent.

A number of cell cycle regulatory proteins are substrates of theubiquitin-proteasome pathway in both mammalian and yeast systems.Among these are the Gl and mitotic cyclins (17, 18, 22, 23), the yeastcdk inhibitor p40(Sicl) (25), and the mammalian cdk inhibitor p27(43). We examined the effect of proteasome inhibitors on the cdkinhibitor p2l, the gene of which is transcriptionally activated by p53.

w

Saos-2

p21-@

p21—.-@@Jl@@#

Fig. 6. A: left, GM6419 cells were mock treated (—)or exposed to a 0.025 matconcentration of MG132 or lactacystin (fact.) for 4 h, and protein extracts were prepared.Onehundred@agof eachextractwereresolvedthrough15%polyacrylamidegels(SDSPAGE), and p2l was detected by Western blotting using the anti-p21 Ab l543lE(PharMingen). Right, Saos-2 cells were mock treated (—)or exposed to a 0.025 matconcentration of MG132 for 4 h, and protein extracts were prepared (duplicate lanes). p21was immunoprecipitated from an equivalent amount of each extract (approximately 800iig) using the 1543lE anti-p2l Ab. Immunoprecipitates were resolved through 15%polyacrylamide gels (SDS-PAGE), and p21 was detected by Western blotting, as described above. B. GM6419 cells and Saos-2 cells were either untreated (no tr.), irradiatedwith 4 Gy from a Cobalt-60 gamma irradiator (4 gy.), or exposed to 0.025 mat MG132 for1, 2, or 4 h. Ten ,sg total RNA were resolved through a 1.2% agarose formaldehyde geland transferred to a Hybond N+ membrane (Amersham). p21 mRNA was detected byprobing the blot with a human p21 cDNA as the probe. The blot on the left (GM6419) wasexposed for 15 h, and the blot on the right (Saos-2) was exposed for 85 h. p21 mRNAlevelswerequantitatedon a phosphoimager.In eachcell type,the levelof p21 mRNAafter treatment with MG132 for 4 h was 2.0—2.5-foldgreater than in the untreated sample.Ethidium bromide staining of the gels prior to transfer showed equal loading of RNA ineach lane (not shown).

2653

Saos-2

c@1 c.*1C') C')I-. @-

(@ (@— —@

— —00C C4@

examined in untreated and inhibitor-treated cells. A large increase inthe level of p21 protein was observed in GM6419 cells exposed tolactacystin or MG132 (Fig. 6A). In contrast, p21 mRNA levels wereincreased by only 2.0—2.5-fold in GM6419 cells after treatment withMG132 for 4 h (Fig. 6B). Similar results were obtained in AGl52lcells (not shown). To determine whether the increase in p21 wasdependent on p53, the effect of the inhibitors on p21 was determinedin Saos-2 cells, an osteosarcoma cell line that lacks p53. Due toextremely low levels of the protein, p21 was undetectable by Westernblot analysis in Saos-2 cells before and after inhibitor treatment (notshown). To detect p21 in Saos-2 cells, it was necessary to firstimmunoprecipitate the protein from a large amount of protein extract,followed by Western blot analysis with an anti-p21 Ab. By thismethod, increased levels of p21 protein were also observed in Saos-2cells treated with the proteasome inhibitor MG132 (Fig. 6A). This isconsistent with several reports that have demonstrated a p53-independent regulation of p21 (39—42). Northern blot analysis showedthat p21 mRNA levels were increased slightly in Saos-2 cells following proteasome inhibitor treatment (Fig. 6B). The increase in p21protein levels following proteasome inhibitor treatment was consistently larger than the increase in p21 mRNA in each cell type exammcd, suggesting that the higher levels of p2! protein did not resultsolely from increased p21 gene transcription. In subsequent experiments, we have found that the half-life of p2! is increased by morethan 5-fold in GM6419 cells treated with MG132 (data not shown).This suggests that p21 may also be degraded via the ubiquitinproteasome pathway.

GM6419

C,,1C')

-

p21-0-@* . .

GM6419

@-

0MG132 C @‘(hrs.) 1 2 4

p21—@ -@

Research. on November 30, 2020. © 1996 American Association for Cancercancerres.aacrjournals.org Downloaded from

PROTEASOME 1NH1B@ON AFFECTS p53

Inhibition of the proteasome caused an increase in the levels of p21protein as well as mRNA. Increased levels of p2 1 were observed inproteasome inhibitor-treated Saos-2 cells, an osteosarcoma cell linethat lacks p53. This indicates that the proteasome inhibitors can causean increase in p21 protein that is independent of p53. p21 is undetectable in Saos-2 cells by Western blot analysis, and we have foundthat p21 levels are generally very low in cells that lack wild-type p53.By Western blotting, steady-state levels of p21 remained undetectablein Saos-2 cells after exposure to the proteasome inhibitors. To detectthe p21 increase in inhibitor-treated Saos-2 cells, it was necessary toimmunoprecipitate p21 from a large amount of protein extract, followed by Western blot analysis for p21 . Recently, Pagano et a!. (43)reported that proteasome inhibitors caused increased levels of the cdkinhibitor p27 in a human osteosarcoma cell line that lacks wild-typep53. In their study, they also observed that by Western blot analysis,the inhibitors had no observable effect on p21.

The gene encoding p21 is transcriptionally activated by increasedlevels of wild-type p53 (36). p21 mRNA levels were increased by2.0—2.5-foldfollowing proteasome inhibitor treatment and stabilizationof p53. This indicates that the stabilized p53 protein may activate p21transcription in proteasome inhibitor-treated cells. However, a slightincrease in p21 mRNA was also observed in cells that lack p53 in

response to the proteasome inhibitors. This suggests that the p21 gene

may be regulated by one or more transcription factors in addition to p53,the levels of which are regulated in a proteasome-dependent manner.Such factors could include NF-KB (28), c-Jun (26), and c-Fos (44), whichhave all been shown to be regulated by the ubiquitin-proteasome system.

ACKNOWLEDGMENTS

We thank Phil Hinds and Karl MOngerfor critical reading of the manuscriptand Jennifer Dowhanick for supplying some needed reagents. We are gratefulto Ross Stein (Myogenics Corp.) for providing the proteasome inhibitors usedin this study.

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1996;56:2649-2654. Cancer Res   Carl G. Maki, Jon M. Huibregtse and Peter M. Howley  p53

Ubiquitination and Proteasome-mediated Degradation ofIn Vivo

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