SUMO-3 enhances androgen receptor transcriptional activity through a sumoylation-independent mechanism in prostate cancer cells.

Abstract

Androgens are important for male sexual development, which depend on the cognate receptor, the androgen receptor. The transcriptional activity of the androgen receptor, like other nuclear receptors, is regulated by accessory proteins that can have either positive or negative effects. Through a yeast functional screen, we have identified SUMO-3 as a regulator of androgen receptor activity in prostate cancer cells. SUMO-3 is one of three eukaryotic proteins that become post-translationally conjugated to their target proteins in a manner analogous to the attachment of ubiquitin. In primary prostate epithelial cells, PrEC, and the prostate cancer cells, PC-3, SUMO-3 has a weak negative effect on androgen receptor transcriptional activity. In contrast, SUMO-3 and it close relative SUMO-2 strongly enhance transactivation by endogenous androgen receptor in LNCaP cells. This positive effect is observed in both androgen-dependent and androgen-independent LNCaP cells. Interestingly, SUMO-1, unlike SUMO-3 and SUMO-2, can inhibit, but not stimulate, androgen receptor activity. Mutational analysis of the androgen receptor and SUMO-3 demonstrates that the SUMO-3-positive activity does not depend on either the sumoylation sites of the androgen receptor or the sumoylation function of SUMO-3. Stable overexpression of SUMO-3 in LNCaP cells significantly enhances the androgen-dependent proliferation of these cells. Additionally, siRNA-mediated repression of SUMO-2 significantly inhibits the growth of both androgen-dependent and -independent LNCaP cells. Collectively, these results suggest (i) a novel mechanism for elevating AR activity through the switch of SUMO-3 from a weak negative regulator in normal prostate cells to a strong positive regulator in prostate cancer cells and (ii) a proliferative role for SUMO-3 and SUMO-2 in the growth of prostate cancer cells that is independent of sumoylation of the androgen receptor.

Full text

Page 1

SUMO-3 Enhances Androgen Receptor Transcriptional
Activity through a Sumoylation-independent
Mechanism in Prostate Cancer Cells*
Receivedforpublication,August22,2005,andNovember21,2005 Published,JBCPapersinPress,December15,2005,DOI10.1074/jbc.M509260200
Zhe Zheng1, Changmeng Cai, Josephat Omwancha, Shao-Yong Chen2, Timour Baslan, and Lirim Shemshedini3
FromtheUniversityofToledo,DepartmentofBiologicalSciences,Toledo, Ohio 43606
Androgens are important for male sexual development, which
depend on the cognate receptor, the androgen receptor. The tran-
scriptional activity of the androgen receptor, like other nuclear
receptors, is regulated by accessory proteins that can have either
positive or negative effects. Through a yeast functional screen, we
haveidentifiedSUMO-3asaregulatorofandrogenreceptoractivity
inprostatecancercells.SUMO-3isoneofthreeeukaryoticproteins
thatbecomepost-translationallyconjugatedtotheirtargetproteins
in a manner analogous to the attachment of ubiquitin. In primary
prostate epithelial cells, PrEC, and the prostate cancer cells, PC-3,
SUMO-3hasaweaknegativeeffectonandrogenreceptortranscrip-
tional activity. In contrast, SUMO-3 and it close relative SUMO-2
stronglyenhancetransactivationbyendogenousandrogenreceptor
in LNCaP cells. This positive effect is observed in both androgen-
dependent and androgen-independent LNCaP cells. Interestingly,
SUMO-1, unlike SUMO-3 and SUMO-2, can inhibit, but not stim-
ulate, androgen receptor activity. Mutational analysis of the andro-
genreceptorandSUMO-3demonstratesthattheSUMO-3-positive
activity does not depend on either the sumoylation sites of the
androgen receptor or the sumoylation function of SUMO-3. Stable
overexpression of SUMO-3 in LNCaP cells significantly enhances
the androgen-dependent proliferation of these cells. Additionally,
siRNA-mediated repression of SUMO-2 significantly inhibits the
growth of both androgen-dependent and -independent LNCaP
cells. Collectively, these results suggest (i) a novel mechanism for
elevating AR activity through the switch of SUMO-3 from a weak
negative regulator in normal prostate cells to a strong positive reg-
ulator in prostate cancer cells and (ii) a proliferative role for
SUMO-3 and SUMO-2 in the growth of prostate cancer cells that is
independent of sumoylation of the androgen receptor.
Thephysiologicalfunctionsoftheandrogenstestosteroneand5?-di-
hydrotestosterone(DHT)4aremediatedbytheandrogenreceptor(AR)
(reviewed in Ref. 1), a member of the nuclear receptor superfamily
(reviewed in Refs. 2–6). Through the regulation of target genes, andro-
gens and AR play an essential role in male sexual development and the
proper development and function of male reproductive organs, such as
prostate and epididymis (7). Patients with 5?-reductase II deficiency,
which results in low levels of DHT, have ambiguous external genitalia
and a highly underdeveloped and impalpable prostate (8, 9). Reduction
orlossofARactivityinmalesresultsinandrogeninsensitivitysyndrome
(10).Inadditiontonormalprostatedevelopment,ARisessentialforthe
initiationandprogressionofprostatecancer.Thebestdemonstrationof
thisistheeffectivenessofanti-androgenandandrogenablationtherapy
ininhibitingthedevelopmentofprostatecancerintheearlystageofthe
disease (11). However, local recurrences and metastases will eventually
develop in most, if not all, patients after therapy, and prostate cancer
becomes androgen-independent (12). Since AR is expressed in both
androgen-dependent and androgen-independent prostate cancer, this
receptorisprobablyinvolvedintheprogressiontoandrogenindepend-
ence. Moreover, it has been determined that about 10–20% of prostate
tumorsharbormutationsintheARgene,andthefrequencyofmutation
generally is higher in androgen-independent, metastatic tumors com-
pared with untreated lower grade primary tumors (13–18).
Like other nuclear receptors, the AR is regulated by multiple post-
translationalmodifications,includingphosphorylation(19),acetylation
(20, 21), and sumoylation (22). Sumoylation represents an important
post-translational modification system that regulates the activity of
many transcriptional regulators (reviewed in Ref. 23). The continually
growing list includes not only AR but also other nuclear receptors and
transcriptional activators, coactivators, and corepressors (reviewed in
Ref. 24). The biological functions of sumoylation include protein sub-
cellular translocation, subnuclear structure formation, and modulation
of transcriptional activity (reviewed in Ref. 25). Sumoylation depends
upontheactivityofsmallubiquitin-relatedmodifier(SUMO),aprotein
moiety that is conjugated to a specific lysine residue on target proteins
(reviewed in Ref. 23). Three SUMO family members exist, SUMO-1/
Smt3C, SUMO-2/Smt3A, and SUMO-3/Smt3B, and all are ubiqui-
touslyexpressedinmammals(27,28).Attheaminoacidlevel,SUMO-2
and SUMO-3 are 87% identical but only ?50% identical to SUMO-1
(27). Although they exhibit low homology in amino acid sequence,
SUMO-1 and ubiquitin are structurally related and share significant
similarity in secondary and tertiary structures (29). Therefore, it is not
surprising that the processes of sumoylation and ubiquitination are
mechanistically similar (reviewed in Ref. 24). Like ubiquitination, the
conjugation of SUMO is mediated by a series of enzymatic reactions
catalyzed by E1, E2, and E3 enzymes that are distinct from those
enzymes that catalyze ubiquitination (27). The SUMO E1 enzymes
SAE1(SUMO-activatingenzyme)andSAE2activateSUMOandtrans-
fer it to the E2 enzyme Ubc9, which then directs the conjugated SUMO
to its target substrates (27). In vitro evidence has indicated that Ubc9 is
* This work was supported by grants from the National Institutes of Health (DK51274)
and Ohio Cancer Research Associates. 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.
1Present address: Medical University of Ohio, 3000 Arlington Ave., Toledo, OH 43614.
2Present address: Harvard Medical School, 330 Brookline Ave., Boston, MA 02215.
3To whom correspondence should be addressed: Dept. of Biological Sciences, Univer-
sity of Toledo, Toledo, OH 43606. Tel.: 419-530-1553; Fax: 419-530-7737; E-mail:
lshemsh@uoft02.utoledo.edu.
4The abbreviations used are: DHT, dihydrotestosterone; SUMO, small ubiquitin-like
modifier1;AR,humanandrogenreceptor;MMTV,mousemammarytumorvirus;CAT,
chloramphenicol acetyltransferase; SC, synergy control; GR, glucocorticoid receptor;
PIAS,proteininhibitorofactivatedSTAT;MTT,3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide; FBS, fetal bovine serum; siRNA, small interfering RNA; RT,
reverse transcription; X-gal, 5-bromo-4-chloro-3-indolyl-?-D-galactopyranoside; E1,
ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein
isopeptide ligase; STAT, signal transducers and activators of transcription.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 7, pp. 4002–4012, February 17, 2006
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
4002 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 281•NUMBER 7•FEBRUARY 17, 2006
at University of Michigan on September 28, 2006
www.jbc.org
Downloaded from

Page 2

sufficient for binding to the SUMO acceptor site and efficiently trans-
ferringSUMOtoselectedtargets(27).However,recentevidenceshows
that a specific E3 ligase might be required for efficient sumoylation in
vivo. Three classes of proteins have been identified to have SUMO E3
ligase activity: the protein inhibitor of activated STAT (PIAS) family
proteins (30, 31), the polycomb protein Pc2 (32), and RanBP2 (Ran-
binding protein 2) (33). The PIAS proteins are reported to act as
SUMO-E3 ligases for the SUMO-1 conjugation to AR in vivo and in
vitro (34), resulting in inhibition of AR transcriptional activity (22).
Interestingly, several recent studies have shown that the PIAS pro-
teins can have multiple effects on AR activity, depending on the type of
PIASprotein,promoter,andcells(reviewedinRef.35).ThePIASfamily
is composed of several homologous proteins, including PIAS1, PIAS3,
PIASx?, PIASx?, and PIASy. Nishida et al. (34) showed that AR-de-
pendent transcription is either repressed by PIAS1 and PIASx? in the
presence of exogenous SUMO-1 and PIAS RING finger-like domain or
enhanced in the absence of sumoylation. PIAS3 inhibits AR transacti-
vationinLNCaPandHeLacellsbutenhancesARactivityinHepG2and
AR-overexpressing LNCaP cells (36–38). Moreover, Ubc9, the SUMO
E2 enzyme, can stimulate AR transcriptional activity that is independ-
ent of its ability to catalyze SUMO-1 conjugation (39). These results
demonstrate that the enzymes of the sumoylation pathway can have
diverse effects on AR activity. We add to this diversity with the current
study, in which we show that SUMO-3 can have a negative or strongly
positive effect on AR, depending on the type of prostate cancer cells.
Further, the positive effect does not depend on either the sumoylation
sitesofARorthesumoylationfunctionofSUMO-3.Finally,SUMO-1is
different from either SUMO-3 or SUMO-2, because it is unable to
enhance AR activity.
MATERIALS AND METHODS
Plasmids—For mammalian expression, AR and c-Jun in pSG-5 have
been described (40). SUMO-3, SUMO-2, and SUMO-1 (generous gifts
from Dr. T. Nishimoto) were expressed from the mammalian expres-
sion plasmid pCMV-FLAG (41). p5HB-AR and a mutant of p5HB-AR
(K385E/K519E) were generous gifts from Dr. J. In ˜iguez-Lluhı ´ (42).
pSRC-1 and pSRC-3 were generous gifts from Dr. B. Rowan (43). TIF-2
(kindly provided by Dr. P. Chambon) has been described (44). FLAG-
SUMO3/pCI-Neo was constructed by first excising SUMO-3 from
FLAG-SUMO3/pCMV with EcoRI/SmaI and inserting it into the pCI-
Neo vector digested with EcoRI/SmaI. Then the FLAG segment was
inserted into SUMO-3/pCI-Neo digested with EcoRI/XbaI. The FLAG
segment was the annealed oligonucleotides 5?-AATTCATGGACTA-
CAAAGACGATGACGACAAG-3?
CATCGTCTTTGTAGTCCAT-3?. SUMO-3(?GG) was constructed
by annealing the oligonucleotides 5?-AATTGATGTGTTCCAACAG-
CAGACGTGACCC-3? and 5?-GGGTCACGTCTGCTGTTGGAA-
CACATC-3? (from Integrated DNA Technology) and inserting into
FLAG-SUMO-3 digested with SmaI and MfeI. The reporter plasmids
used in mammalian cells have the gene for chloramphenicol acetyl-
transferase (CAT) driven by different promoters. The AR-inducible
reporterplasmidMMTV-CAT(40)andARE3-E1B-CAT(generousgift
from Dr. E. Sanchez) (45) are described previously. For the yeast func-
tional screen, LexA-AR(AB) was constructed by digestion of the AB
region out of AR(AB)/pTL1NLS (46) with EcoRI and BglI and inserting
into the EcoRI and BamHI sites of pEG202 (47). All newly generated
constructswereconfirmedbyDNAsequencing(donebytheOhioState
University Plant Microbe Genomics Facility).
Cell Transfection and CAT Assays—PC-3 cells were maintained in
F12Kmedium(Sigma)supplementedwith10%fetalbovineserum(FBS)
and5?-CTAGACTTGTCGT-
(Hyclone). LNCaP (androgen-dependent and -independent cells) and
DU145 cells were maintained in RPMI1640 medium (Sigma) supple-
mented with 10% FBS. PrEC cells were maintained in the PrEGM
medium(Clonetics).C33andC81cells(kindlyprovidedbyDr.Lin)(48)
weregrowninRPMI1640mediumwith5%FBS,1%glutamine,and0.5%
gentamycin. All cells were cultured with 5% CO2at 37 °C. These cell
lines were grown in RPMI1640 medium with 10% FBS and 0.1 mg/ml
neomycin. ?-Galactosidase assay and CAT assay were described previ-
ously (40). For transient transfection of LNCaP and DU145 cells, cells
were grown to 80–90% confluence in RPMI1640 complete medium.
Four hours before transfection, medium was changed to RPMI1640
with 5% FBS (dextran-coated charcoal-treated). Transient transfection
was performed with FuGene6 reagent (Roche Applied Science). DHT
was added 24 h after transfection. LNCaP and DU145 cells were incu-
bated for 24 h in RPMI1640 with 5% FBS (dextran-coated charcoal-
treated) with or without DHT, respectively. Reporter analysis (?-galac-
tosidaseassayandCATassay)weredoneaftertheincubation.ForPC-3
cells, transient transfection was performed with the calcium phosphate
precipitation(CaPO4)method(49).InPrECcells,transienttransfection
was performed with FuGene6 in PrEGM medium. Transfection effi-
ciency was standardized by measuring ?-galactosidase activity, origi-
natingfromtheco-transfectedplasmidpCH110(2?g).Notethatforall
transfections, empty expression plasmid was used to bring the final
plasmid amount to 9 ?g/transfection.
CAT activity was quantified by scanning with the Bio-Rad Molecular
Imager FX of autoradiograms of three independent replicates for each
transfection.Thus,eachCATvaluerepresentstheaverageofthreerep-
etitions plus the S.D.
siRNA Transfections—LNCaP and C81 cells were transfected with a
SUMO-2 siRNA (5?-GGGAUGAAUCUGUAACUUAtt-3? and anti-
sense oligonucleotide) (purchased from Ambion). A luciferase siRNA
with 42% GC content was used as control siRNA (GL3 siRNA from
Dharmacon). The X-tremeGENE siRNA transfection reagent was used
to transfect siRNA into cells following the prescribed protocol (Roche
Applied Science).
GenerationofStableCellLines—LNCaPcellsweregrownin100-mm
dishes with 10 ml of RPMI1640 complete medium until 60–70% con-
fluence, and then cells were transfected with 2 mg of FLAG-SUMO3/
pCI-Neo. The transfection was done with FuGene6 described above.
After a 48-h incubation, the LNCaP cells were selected in RPMI1640
complete medium containing 0.9 mg/ml neomycin. The medium was
refreshed every 4 days until individual colonies appeared.
ThegenerationofstableLNCaPcelllinesC14andAJ6andPC-3lines
C-3, A-103, and V-28 has been described (50). The AJ6 LNCaP cells
stably express antisense c-Jun. C14 is a control LNCaP cell line stably
transfected with an empty pCI-Neo vector. A-103 cells stably express
AR, V-28 cells express a fusion protein containing AR and the VP16
activation domain (51), and C-3 cells were transfected with empty PCI-
neo vector.
Cellular Proliferation Assay—8 ? 104LNCaP cells were seeded in
6-well plates with 5 ml of RPMI1640 medium containing 2% dextran-
coated charcoal-treated FBS. After a 2-day incubation, either ethanol (ve-
hiclecontrol)or100nMDHTwasaddedintothewells.Afteranadditional
2, 4, or 6 days of incubation, the MTT assay was done according to the
manufacturer’sinstructions(Sigma).Cellnumberwasquantifiedbymeas-
uringabsorbanceatawavelengthof570nm.Notethatbargraphsrepresent
theaveragesoftheeindependentexperimentsplusS.D.
Western Blot Analysis—For Western blot analysis, COS and LNCaP
cells were grown in 100-mm dishes and subjected to transfection.
Whole-cell extracts were prepared and subjected to SDS-PAGE and
SUMO-3ActivatesAR-dependentTranscription
FEBRUARY 17, 2006•VOLUME 281•NUMBER 7 JOURNAL OF BIOLOGICAL CHEMISTRY 4003
at University of Michigan on September 28, 2006
www.jbc.org
Downloaded from

Page 3

Western blot analysis as described (52). The nitrocellulose blots were
probed with the anti-FLAG antibody M2 (Sigma), anti-AR antibody
PA1-111A (Affinity Bioreagents), or anti-?-actin AC-15 (Abcam). The
ECL chemiluminescence detection kit (Amersham Biosciences) was
used to develop the Western blots.
Northern Blot Analysis—A multiple human tissue blot was obtained
from Clontech (catalog no. 7761-1) and probed for SUMO-3 mRNA
according to the manufacturer’s instructions.
SemiquantitativeReverseTranscription(RT)-PCR—ToprepareRNA
for the RT reaction, total RNA from LNCaP, PC-3, or PrEC cultured
cells was extracted using the Trizol reagent (Invitrogen). For the RT-
PCR (reverse transcription-PCR) assays, cDNA was prepared from the
isolated RNA using the Moloney murine leukemia virus reverse tran-
scriptase, according to the manufacturer’s instructions (Fisher). The
PCRs were carried out using primers specific for the mRNA: the
upstream primer 5?-GGGCAACCAATCAATGAAAC-3? and the
downstream primer 5?-AGTCAGGATGTGGTGGAACC-3? (SUMO-
3), the upstream primer 5?-CTGGCCCTCAAGCATGTAAC-3? and
the downstream primer 5?-AAATCTGAGGCCACAACACC-3? (SU-
MO-2), the upstream primer 5?-ACCGTCATCATGTCTGACCA-3?
and the downstream primer 5?-TGGAACACCCTGTCTTTGAC-3?
(SUMO-1); the upstream primer 5?-TCATAAGCAGCGACCTTGT-
G-3? and the downstream primer 5?-ACCGAAGGAAGAGACCCTG-
T-3? (Ubc9); the upstream primer 5?-CTTCTTGTCGGCTTGAAAG-
G-3? and the downstream primer 5?-ACCATGGGGTTGAGATTCT-
G-3? (SAE1); the upstream primer 5?- GACAGAGCTGACCCTGAA-
GC-3? and the downstream primer 5?-TTTTCCGCCATAGTTTGT-
CC-3? (SAE2). Glyceraldehyde-3-phosphate dehydrogenase-specific
primers (upstream primer 5?-CGACCACTTTGTCAAGCTCA-3? and
thedownstreamprimer5?-AGGGGAGATTCAGTGTGGTG-3?)were
used in the RT-PCR as a control. RT-PCR was carried out for 30 cycles
usingthefollowingconditions:denaturationat94 °Cfor30s,annealing
at 60 °C for 30 s, and extension at 72 °C for 1 min. The PCR products
were electrophoretically separated on a 2% agarose gel and stained with
ethidium bromide.
Yeast Transformation for the Functional Screen—The yeast transfor-
mation protocol was described previously (53). A 20-ml culture of
YPH499/pSH18–34 (47) was grown and transformed with AR(AB)/
pEG202 in Glu/CM?Ura?His liquid dropout medium overnight at
30 °C. The culture was diluted into a 300-ml Glu/CM?Ura?His liquid
dropoutmediumwith2?106cells/mlandincubatedat30 °CuntilA600
reading reached 0.5, after which it was centrifuged for 5 min at 1000–
1500 ? g. The liquid was discarded, and the cells were resuspended in
1.5mlofTEbuffer,0.1 Mlithiumacetate.1?gofP19library(54)and50
?g of high quality sheared salmon sperm carrier DNA were added to
eachof30sterile1.5-mlmicrocentrifugetubes.50?loftheresuspended
yeast cells were added to each tube, and they were incubated for 30 min
at 30 °C, and Me2SO was then added to 10%. The samples were heat-
shocked for 10 min at 42 °C. For 28 tubes, the complete content of one
tubewasaddedper24?24-cmGlu/CM?Ura?His?Leu/X-Galdrop-
out plate and incubated at 30 °C. For the remaining two tubes, 360 ?l of
each tube was spread on 24 ? 24-cm Glu/CM?Ura?His?Leu/X-Gal
dropout plate. The remaining 40 ?l from each tube was used to make a
seriesof1:10dilutioninsterilewater.Dilutionswereplatedon100-mm
Glu/CM?Ura?His?Leu dropout plates. All plates were incubated at
30 °C until colonies appeared (2–3 days). Colonies were monitored for
color (blue or white). Among 300,000 transformed colonies, nine white
coloniesappearedontheX-galmedium.Plasmidwasisolatedfromeach
of the white colonies and used for retransformation. Only four of the
isolated plasmids were able to cause a white phenotype upon retrans-
formation. DNA sequencing showed that one of the four plasmids
matched the open reading frame of mouse SUMO-3, as well as the part
of the 5?- and 3?-untranslated region.
YeastLiquid?-GalactosidaseAssay—Asingleyeastcolonywasinoc-
ulated in 3 ml of YPD (or appropriate selective) medium and incubated
overnightat30 °C.20–50?lofeachovernightculturewasinoculatedin
4mlofYPDmedium(orappropriateselectivemediumand/orinducing
conditions)andgrowntomiddleorlatelogphase.Thiswassubjectedto
a liquid ?-galactosidase assay using 2-nitrophenyl ?-D-galactopyrano-
side as described (55).
DNA Sequencing Analysis—cDNA fragment was isolated from the
positiveclonesusingtheYeastmakerTMYeastPlasmidIsolationKit(BD
Biosciences) and amplified using the upstream oligonucleotide
5?-GTTTTTCAAGTTCTTAGATG-3? and the downstream oligonu-
cleotide5?-CTGGCAATTCCTTACCTTCC-3?
Technology). DNA sequencing for the nine putative cDNA clones was
carried out using the same primers (sequencing done by Ohio State
University Plant Microbe Genomics Facility).
(IntegratedDNA
RESULTS
SUMO-3 Was Identified Using a Yeast Functional Screen—In an
effort to identify novel repressors of AR, we developed a modified yeast
two-hybridsystemthatwecalla“yeastfunctionalscreen.”Liketheyeast
two-hybrid system, the yeast functional screen depends on a fusion
proteincontainingtheLexADNA-bindingdomainfusedtoaproteinof
interest and the LexA-responsive LacZ reporter (47). Unlike the yeast
two-hybrid system, the protein of interest must have strong transcrip-
tionalactivitysothatallyeastcoloniesexpressingthisfusionproteinwill
become blue when grown on X-gal plates. Expression of a protein that
blocks the transcriptional activity of the protein of interest will cause
yeast colonies to remain white (Fig. 1A). In the present study, the
AR(AB) region acted as a bait protein and exhibited strong transcrip-
tional activity in yeast, compared with the activity of LexA alone (Fig.
1B). The AB region harbors most of the AR transcriptional activity (46,
56) and is the target of multiple coactivators (44, 46, 56–63). All yeast
transformants expressing LexA-AR(AB) formed blue colonies on
X-containing medium.5Co-transformation of a cDNA expression
library from P19 embryocarcinoma cells (54) resulted in nine white
colonies among a total of 300,000 transformants. From these colonies,
useful DNA sequence data were obtained for only four, one of which
was SUMO-3. The SUMO-3 cDNA clone matched the open reading
5Z. Zheng and L. Shemshedini, unpublished results.
FIGURE1.AyeastfunctionalscreenforisolatingrepressorsofARtransactivation.A,
a schematic diagram of the yeast functional screen for repressors of AR(AB)-dependent
transcription. LexA-AB(AB) can activate the LacZ reporter and cause yeast colonies to
becomeblueonX-galmedium.ExpressionofaproteinthatcanrepressesAR(AB)trans-
activation disrupts LacZ expression, and yeast colonies remain white. B, yeast YPH499
wastransformedwith1?gofLexAorLexA-AR(AB)and1?gofreporterplasmidpSH18–
34. Transcriptional activity was measured by liquid ?-galactosidase assay.
SUMO-3ActivatesAR-dependentTranscription
4004 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281•NUMBER 7•FEBRUARY 17, 2006
at University of Michigan on September 28, 2006
www.jbc.org
Downloaded from

Page 4

frame of mouse SUMO-3 as well as the part of the 5?- and 3?-untrans-
lated region.
SUMO-3 Has Different Effects on AR Transactivation in Prostate
Cancer Cell Lines—To verify the negative effect by SUMO-3 on AR
transactivation observed in the yeast functional screen, we transiently
transfected SUMO-3 and two other members of this gene family into
three prostate cancer cell lines (PC-3, LNCaP, and DU145) and PrEC
cells (Fig. 2). PC-3 cells were derived from the bone metastasis of a
prostate cancer patient and lack endogenous AR (64). Transfected
SUMO-3 exhibited a weak but reproducible negative effect (?20%) on
ARtransactivationinPC-3cells(Fig.2A).SUMO-2hadnoeffectonAR
transactivation,despitethefactthatitsstructureisverysimilartothatof
SUMO-3 (27). SUMO-1 also showed a weak negative effect on AR
transactivation, consistent with other reports showing that SUMO-1
can repress several different transcription factors (reviewed in Ref. 65).
To determine whether SUMO-3 can affect the activity of stably
expressed AR, we used the PC-3 stable cell lines A103 and V28. A103
cells stably express AR and V28 cells stably express a VP16-AR fusion
protein. These two lines exhibited DHT-induced AR transactivation,
and VP16-AR has about 10-fold higher transcriptional activity than AR
(50). SUMO-3 also had a weak negative effect on AR transactivation in
A103 cells (Fig. 2B). In V28 cells, the negative effect was more pro-
nounced (Fig. 2B). These results demonstrate that the weak negative
effect of SUMO-3 can be seen with both transiently and stably
expressed AR.
To study a potential biological role for SUMO-3 on AR activity, we
used LNCaP cells. These cells express endogenous AR and exhibit
androgen-dependent gene expression and cellular proliferation
(reviewedinRef.11).Surprisingly,transfectedSUMO-3significantly
enhanced (3.5-fold) AR transactivation in LNCaP cells (Fig. 2C).
SUMO-2 also stimulated AR activity, but to a lesser degree than did
SUMO-3, whereas SUMO-1 had no effect (Fig. 2C). The endogenous
AR gene in LNCaP cells possesses a mutation that replaces residue
Thr877with Ala, which broadens the AR ligand specificity (15). To
study the possibility that this mutation is responsible for the positive
effect of SUMO-3 in LNCaP cells, we studied LNCaP cells that were
transiently transfected with wild-type AR. Transfected AR has a
3–5-fold higher transcriptional activity than endogenous AR (see
Fig. 3A), making it possible to distinguish the activity of transfected
AR. SUMO-3 has a similar positive effect on transfected wild-type
AR as on endogenous mutant AR in LNCaP cells (compare Figs. 2C
and 3, B and C), excluding the possibility that the T877A mutation is
responsible for the SUMO-3-positive effect. In further support of
this, we observed a SUMO-3 enhancement of transfected AR activity
alsoinDU-145cells(Fig.2D),aprostatecancercelllinederivedfrom
a brain metastasis that, like PC-3 cells, lacks endogenous AR (66).
ThesedatatogethershowthatthedirectionoftheSUMO-3effecton
AR depends on the prostate cancer cell context.
To study the role of SUMO-3 on AR transactivation in normal pros-
tate cells, we used PrEC cells. These cells are normal human prostate
epithelial cells that have been widely used as the normal counterpart cell
line to prostate cancer cell lines (67). In our experiments, SUMO-3 had a
similarlyweaknegativeeffectontransfectedARinPrECcells(Fig.2E)that
was observed in PC-3 cells (see Fig. 2A). Together, these results suggest a
novel mechanism of elevating AR activity through the switch of SUMO-3
from a weak negative regulator of AR in normal prostate cells (PrEC) to a
strongpositiveregulatorofARinprostatecancercells(LNCaPcells).Since
enhanced AR activity is probably involved in the development of prostate
cancer(reviewedinRef.68),SUMO-3mayprovideanovelmechanismfor
up-regulatingARactivityinprostatecancer.
SUMO-3StimulatesARTransactivationIndependentofthePromoter—
OurpreliminaryexperimentsontheSUMO-3effectonARtransactivation
in LNCaP cells were conducted using the MMTV promoter (see Fig. 2C).
To determine if this effect is unique to the MMTV promoter, we studied
the ARE3-E1B-CAT reporter, which harbors three AREs and an E1B pro-
moter (5). Since transactivation by endogenous AR of ARE3-E1B-CAT is
very low in LNCaP cells,5exogenous AR was expressed in LNCaP cells
together with SUMO-3 (Fig. 3). As is shown in Fig. 3A, transfected AR
yielded significantly higher transcriptional activity than did endogenous
AR. Importantly, SUMO-3 had a similar positive effect on exogenous AR
transactivation of MMTV-CAT (Fig. 3B) and ARE3-E1B-CAT (Fig. 3C),
demonstratingthatSUMO-3up-regulationofARtranscriptionalactivityis
notpromoter-specific.
The SUMO-3-positive Effect on AR Does Not Depend on the Putative
AR Sumoylation Sites—SUMO proteins are known to influence the
activities of other proteins by direct conjugation to these proteins.
FIGURE 2. SUMO-3 can inhibit or stimulate AR
transactivationindifferentprostatecancercell
lines. A, PC-3 cells were transiently transfected
with 1 ?g of MMTV-CAT, 1 ?g of AR, and 5 ?g of
SUMO-3, SUMO-2, or SUMO-1. B, PC-3 stable cell
lines were transiently transfected with 1 ?g of
MMTV-CAT and 5 ?g of SUMO-3, SUMO-2, or
SUMO-1. Note that A-103 cells express AR, V-28
express a VP16-AR fusion protein, and C-3 repre-
sents a stable transfection with the empty vector
(pCI-neo). C, LNCaP cells were transiently trans-
fected with 2 ?g of MMTV-CAT and 2 ?g of
SUMO-3,SUMO-2,orSUMO-1.D,DU145cellswere
transiently transfected with 2 ?g of MMTV-CAT, 2
?g of AR, and 2 ?g of SUMO-3. E, PrEC cells were
transiently transfected with 2 ?g of MMTV-CAT, 2
?g of AR, and 2 ?g of SUMO-3, SUMO-2, or
SUMO-1. Note that all cells were treated with
either ethanol (open bars) or 100 nM DHT (gray
bars).Allactivitiesarerelativetotheactivityinthe
absence of 100 nM DHT and transfected SUMO,
and this activity was set to 1.
SUMO-3ActivatesAR-dependentTranscription
FEBRUARY 17, 2006•VOLUME 281•NUMBER 7 JOURNAL OF BIOLOGICAL CHEMISTRY 4005
at University of Michigan on September 28, 2006
www.jbc.org
Downloaded from

Page 5

Therefore,itispossiblethatSUMOconjugationtosomefactor(s)isthe
mechanism underlying the action of SUMO-3 on AR. This factor may
be AR itself or another factor, such as a coactivator. It was recently
reported that SUMO-1 can covalently attach to two consensus sumoy-
lation sites found on AR (K385/K519) and inhibit this receptor’s tran-
scriptionalactivity(22).TotestifSUMO-3conjugationtoARisrespon-
sible for this protein’s positive activity on AR, we obtained a plasmid
expressing an AR mutant that has both sumoylation sites disrupted
(K385E/K519E). Since the consensus sumoylation site for all three
SUMO proteins is the same (27), mutation of Lys385and Lys519on AR
should disrupt any potential SUMO-3 conjugation to AR and possibly
block the SUMO-3-positive effect. Surprisingly, in LNCaP cells,
SUMO-3 can enhance the transcriptional activity of not only wild-type
ARbutalsotheARmutantK385E/K519E,usingeitherMMTV-CATor
ARE3-E1B-CATasthereporterplasmid(Fig.4A).Weareconfidentthat
the transcriptional activity measured above came from transfected AR,
since exogenous AR in the experiments yielded significantly higher
transcriptionalactivitythandoesendogenousAR(seeFig.3A).Tocon-
firmthesedatafromLNCaPcells,weusedDU145cellstransfectedwith
either wild-type AR or the AR mutant K385E/K519E. As shown in Fig.
4B, both AR proteins responded positively to co-transfected SUMO-3.
Together,theresultsaboveindicatethattheSUMO-3-positiveeffecton
AR does not depend on the AR putative sumoylation sites, suggesting
thattheeffectismediatedviaeitherothersitesonARorotherproteins.
Mutation of AR Sumoylation Sites Alters AR Transactivation—The
consensusSUMOacceptorsite,?KXE,overlapswithasynergycontrol
(SC)motifinGRandSp3,whichwasdemonstratedtorepresssynergis-
tictranscriptionfrompromoterscontainingmultiplebindingsites(42).
Our results show that mutations in the two SC motifs on the AR N-ter-
minalregion(K385E/K519E)ledtoenhancementofARtransactivation
in LNCaP and DU145 cells (Fig. 4, A and B), consistent with previous
observations made in COS cells (22). The same result was obtained in
PC-3 cells (Fig. 4C). However, in PrEC cells, the AR mutant had lower,
not higher, transcriptional activity than did the wild-type AR (Fig. 4D).
These data provide the first evidence that mutations of the SC motifs
can either enhance or compromise AR transactivation, depending on
the type of prostate cancer cell line.
Endogenous c-Jun Is Required for Full Enhancement by SUMO-3 of
AR Transactivation—Our published data show that c-Jun can enhance
ARtransactivationindependentofpromoterorcelltypeandtargetsthe
AR N terminus (40, 46, 52, 69). In view of these results and the finding
that c-Jun is a substrate for sumoylation (70), it is possible that SUMO
modification of this protein may be involved in the SUMO-3-positive
effect on AR in LNCaP cells. To address this possibility, we used an
LNCaP-stable cell line expressing an antisense c-Jun transcript. These
cells, called AJ6, exhibit reduced endogenous c-Jun levels, AR transac-
tivation, and androgen-dependent proliferation (50). As expected, AR
transactivation in AJ6 cells is significantly lower than in control C14
cells (Fig. 5A), which are transfected with empty expression vector.
WhenanalyzedforaSUMO-3effect,AJ6cellsexhibitagreatlyreduced
responsetotransfectedSUMO-3onARtransactivation,suggestingthat
endogenous c-Jun is required for full enhancement by SUMO-3 of AR
transactivation.
TIF-2 Represses SUMO-3-positive Effect on AR Transactivation in
LNCaP Cells—The SRC family proteins represent another group of AR
coactivators that act by interacting with the N and C termini of the
receptor (57, 58) and recruiting additional cofactors such as CBP/p300
and protein with acetyltransferase or methyltransferase activity (71).
Recently,SRC-1(72)andTIF-2(73)havebeenshowntobesumoylated
bySUMO-1.Furthermore,mutationoftheconsensussumoylationsites
found on TIF-2 can impair its coactivation ability on AR (73). Based on
these observations, SUMO modification of SRC family proteins may be
involvedintheSUMO-3-positiveeffectonARinLNCaPcells.Transfected
SRC-1, SRC-3, and TIF-2 were analyzed for modulatory effects on the
SUMO-3stimulationofARactivityinLNCaPcells.Intheabsenceoftrans-
fectedSUMO-3,SRC-3andSRC-1stimulatedARtransactivation,whereas
TIF-2, surprisingly, had no measurable effect (Fig. 5B). By contrast, in the
presenceoftransfectedSUMO-3,TIF-2hadthestrongesteffect,markedly
impairingthepositiveeffectofSUMO-3onARactivity(Fig.7B);SRC-1and
SRC-3hadanappreciablyweaker,ifany,effect(Fig.5B).Theseresultsshow
that TIF-2 can down-modulate the SUMO-3-positive effect on AR and
thereforemaybeinvolvedinthiseffect.
AConjugation-deficientSUMO-3MutantCanEnhanceARTransac-
tivation in LNCaP Cells—The double glycine residues on the C termi-
nus of SUMO proteins have been shown to be critical for the sumoyla-
tionpathway(74,75).DeletionofthesetwoglycineresiduesinSUMO-1
leads to the loss of conjugation to AR (22), a finding confirmed by our
results with the SUMO-3(?GG) mutant (Fig. 6A). Transfection into
COScellsofwild-typeSUMO-3yieldsmultiplebandsonaWesternblot
probed with an anti-SUMO-3 antibody; these multiple bands include
unconjugated monomeric SUMO-3 and conjugated SUMO-3 in the
upper half of the blot (Fig. 6B). In contrast, only the monomeric form is
FIGURE 3. SUMO-3 can up-regulate AR-dependent transactivation independent of
promoter specificity. A, LNCaP cells were transiently transfected 2 ?g of MMTV-CAT
withorwithout0.5?gAR,asindicated.BandC,LNCaPcellsweretransientlytransfected
with 2 ?g of SUMO-3, 0.5 ?g of AR, and 2 ?g of MMTV-CAT (B) or 3 ?g of ARE3-E1B-CAT
(C). Note that all cells were treated with either ethanol (open bars) or 100 nM DHT (gray
bars). All activities are relative to the activity of LNCaP cells in the absence of DHT and
transfected AR (A) or SUMO-3 (B and C), and this activity was set to 1.
SUMO-3ActivatesAR-dependentTranscription
4006 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 281•NUMBER 7•FEBRUARY 17, 2006
at University of Michigan on September 28, 2006
www.jbc.org
Downloaded from

Page 6

observedwithtransfectedSUMO-3(?GG)(Fig.5B),indicatingthatthis
mutant protein is deficient in conjugation to target substrates. Impor-
tantly,however,thisSUMO-3mutantstillcanenhanceARtransactiva-
tion in LNCaP cells, although somewhat more weakly than wild-type
protein(Fig.6C),suggestingthatSUMO-3enhancesARtransactivation
via a conjugation-independent mechanism.
Stable Expression of SUMO-3 in LNCaP Cells Stimulates Cell
Proliferation—Our data above show that transient transfection of
SUMO-3canenhanceARtransactivationinLNCaPcells.Todetermineif
this SUMO-3 regulation of AR can modulate a biological function of this
receptor,weoptedtoincreasetheendogenouslevelsofSUMO-3inLNCaP
cellsandmonitorandrogen-dependentcellularproliferation.TwoLNCaP
celllineswereselectedthatstablyexpressFLAG-SUMO-3(Fig.7A).Stable
expression of SUMO-3 does not affect the expression of AR protein (Fig.
7A)ormRNA(Fig.7B).Importantly,thesetwocelllinesalsodemonstrated
enhancedandrogen-dependentcellularproliferation,whencomparedwith
control C14 cells (Fig. 7C). These data support the hypothesis that
SUMO-3canregulateabiologicalactivityofAR.
siRNA-mediated Repression of SUMO-2 Inhibits the Proliferation of
Androgen-dependent LNCaP Cells—To study the role of endogenous
SUMO-3 in cellular proliferation, we used siRNA-mediated down-reg-
ulation. Unfortunately, no SUMO-3 commercial siRNA oligonucleo-
tidesareavailable,noristheSUMO-3nucleotidesequenceamenableto
custom-designed oligonucleotides. Therefore, we opted to use com-
mercial siRNA oligonucleotides for SUMO-2, a nearly identical protein
toSUMO-3inbothaminoacidsequence(27)andenhancingactivityon
AR (see Fig. 2C). Transfection of SUMO-2 siRNA strongly reduces the
expression of SUMO-2 mRNA, whereas a control siRNA did not (Fig.
8A).TheSUMO-2siRNAhadnoeffectonSUMO-3mRNAexpression
(Fig. 8A). Significantly, siRNA-mediated reduction of SUMO-2 expres-
sion resulted in a marked decrease in androgen-induced LNCaP prolif-
eration, whereas control siRNA had no effect (Fig. 8B). These results
demonstrateadirectcorrelationbetweenendogenousSUMO-2protein
levelsandLNCaPcellularproliferation,stronglysuggestinganessential
role for SUMO-2, and probably SUMO-3, protein.
siRNA-mediated Repression of SUMO-2 Inhibits the Proliferation of
Androgen-independent LNCaP Cells—Prostate cancer can become
androgen-independent during disease progression or androgen depri-
vation. Numerous molecules have been shown to be involved in regu-
lating cell growth in androgen-independent prostate cancer, including
FIGURE4.DisruptionoftheARputativesumoy-
lation sites does not alter the SUMO-3 effects
on AR. A, LNCaP cells were transiently transfected
with 2 ?g of MMTV-CAT or ARE3-E1B-CAT, 2 ?g of
SUMO-3, and 0.5 ?g of AR or AR(K385E/K519E). B,
C,andD,2?gofMMTV-CAT,2?gofSUMO-3,and
0.5?gofARorAR(K385E/K519E)weretransiently
transfected into DU145 (B), PC-3 (C), or PrEC (D)
cells. Note that all cells were treated with either
ethanol (open bars) or 100 nM DHT (gray bars). All
activitiesarerelativetotheactivity(AandD)inthe
absence of DHT and transfected SUMO-3, in the
presence of DHT and absence of transfected
SUMO-3(B),orintheabsenceofDHTandpresence
oftransfectedSUMO-3(C),andthisactivitywasset
to 1.
FIGURE 5. AR coactivators have different effects on the SUMO-3 stimulation of AR-
dependenttransactivation.A,stableLNCaPlinesweretransfectedwith1?gofMMTV-
CATand1?gofSUMO-3.NotethatAJ6expressesanti-c-Jun,andC14representsastable
transfectionwiththeemptyvector(pCI-neo).B,LNCaPcellsweretransientlytransfected
with1?gofMMTV-CAT,0.5?gofAR,2?gofSUMO3,and2?gofTIF-2,SRC-1,orSRC-3.
Cellsweretreatedwitheitherethanol(openbars)or100nMDHT(graybars).Allactivities
arerelativetotheactivityofC14cellsintheabsenceofDHTandtransfectedSUMO-3(A)
or in the presence of DHT and the absence of transfected SUMO-3 and SRC protein (B),
and this activity was set to 1.
SUMO-3ActivatesAR-dependentTranscription
FEBRUARY 17, 2006•VOLUME 281•NUMBER 7JOURNAL OF BIOLOGICAL CHEMISTRY 4007
at University of Michigan on September 28, 2006
www.jbc.org
Downloaded from

Page 7

AR,p53,Bcl2,IGF1,andseveralotherfactors(reviewedinRef.76).The
positive effect of SUMO-3 on AR activity may also contribute to AR
transcriptional activity in androgen-independent prostate cancer cells
that express endogenous AR. To address this issue, we have compared
the effect of transfected SUMO-3 on AR in androgen-dependent and
androgen-independent prostate cancer cells. C81 represents LNCaP
cellsthatwereculturedforalongtimetoexhibitandrogen-independent
growth, whereas C33 cells are the androgen-dependent parental line
from which C81 was derived (48). AR expression in these androgen-
independent LNCaP cells is similar to expression in androgen-depend-
ent LNCaP cells (48). Transfected SUMO-3 can enhance androgen-
induced AR transactivation in C33 cells (Fig. 9A). Interestingly,
SUMO-3 also had a positive effect on the endogenous AR in C81 cells,
but the magnitude of the effect was attenuated (Fig. 9A). This reduced
SUMO-3 effect may reflect the lower androgen responsiveness of AR
transactivation in androgen-independent LNCaP cells (Fig. 9A).
To determine if endogenous SUMO proteins are involved in the
growth of androgen-independent LNCaP cells, we utilized again
siRNA-mediatedrepressionofSUMO-2.AsshowninFig.9B,SUMO-2
and SUMO-3 mRNAs are both expressed in C81 cells to slightly higher
levels than in androgen-dependent LNCaP cells. Similarly, transfection
of SUMO-2 siRNA results in a marked decrease in SUMO-2 mRNA
expression in C81 cells (Fig. 9C). These siRNA-transfected cells were
thenstudiedforgrowth,whichexpectedlyislargelyandrogen-indepen-
dent (Fig. 9D). More importantly, the androgen-independent prolifera-
tion of C81 cells is dramatically reduced by SUMO-2 siRNA treatment,
incomparisonwithcontrolsiRNAtreatment(Fig.9D).Thesedataillus-
trateanimportantroleforendogenousSUMO-2inandrogen-indepen-
dent growth of LNCaP cells.
SUMO-3 mRNA Is Highly Expressed in Human Prostate and Testis
and Prostate Cancer Cell Lines—To detect the expression of SUMO-3
inhumantissues,Northernblotanalysiswascarriedoutusingahuman
FIGURE 6. SUMO-3 can stimulate AR-dependent transactivation in a sumoylation-independent manner. A, a schematic diagram showing the conjugation-deficient mutant of
SUMO-3(SUMO-3(?GG))lackingtheC-terminaldoubleglycineresidues.B,COScellsweretransfectedwith5?gofFLAG-SUMO-3andFLAG-SUMO3(?GG).Cellextractswereanalyzed
by Western blot analysis using an anti-FLAG antibody. C, LNCaP cells were transiently transfected with 2 ?g of MMTV-CAT and 2 ?g of SUMO-3 or SUMO-3(?GG). All activities are
relative to the activity of LNCaP cells in the absence of DHT and transfected SUMO-3, and this activity was set to 1.
FIGURE 7. Stable overexpression of SUMO-3 enhances androgen-dependent proliferation of LNCaP cells. A and B, LNCaP cells were transfected with FLAG-SUMO-3/pCIneo
plasmid,andtwostablecelllines(FS11andFS31)wereisolated.Whole-cellextractsweresubjectedtoWesternblotanalysisusinganti-FLAGM2antibody(Sigma)oranti-ARantibody
(PA1–111A from Affinity Bioreagents) (A) or semiquantitative RT-PCR (B). Note that C14 represents LNCaP cells stably transfected with the empty pCI-neo-FLAG plasmid. C, LNCaP-
stablecelllinesweremonitoredforgrowthfor0–6daysinthepresenceofethanol(openbar)or100nMDHT(graybar).ThecellnumberwasquantifiedusingtheMTTassay(Sigma).
GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
SUMO-3ActivatesAR-dependentTranscription
4008 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 281•NUMBER 7•FEBRUARY 17, 2006
at University of Michigan on September 28, 2006
www.jbc.org
Downloaded from

Page 8

multipletissueblot(fromClontech).SUMO-3mRNAwasubiquitously
expressed in all human tissues studied, with the highest expression
found in prostate, testis, and thymus (Fig. 10A).
RT-PCRwasemployedtomeasuretheexpressionofthethreeSUMO
genesinLNCaP,PC-3,andPrECcells(Fig.10B).SUMO-3wasdetected
inallthreecelltypes.SUMO-2andSUMO-1arealsoexpressedinthese
cells,suggestingthatthesumoylationpathwayisabundantand/orthree
SUMO proteins act on variety of targets. In the sumoylation pathway,
theE1enzymesSAE1andSAE2activateSUMOandtransferittotheE2
enzyme Ubc9 (27). Ubc9 conjugates to SUMO and directs it to target
substrates (27). If sumoylation is a biological regulator of AR in LNCaP
cells, then the SUMO E1 and E2 enzymes must be involved, and the
genes encoding these enzymes must be expressed in these cells. As
showninFig.9B,SAE1,andSAE2,andUbc9areallexpressedinLNCaP
cells.
DISCUSSION
Sumoylation is a novel pathway for post-translational modification (re-
viewed in Ref. 23). In mammalian cells, many transcription factors and
cofactors have been identified as targets of sumoylation (reviewed in Refs.
24 and 77). SUMO modification can either enhance or repress the tran-
scriptionalactivityofthesefactors.Inthecaseofnuclearreceptors,exoge-
nous SUMO-1 significantly increases glucocorticoid receptor (GR) trans-
activation of a glucocorticoid-responsive reporter (78). Similarly,
transactivationbyeithertheprogesteronereceptorandestrogenreceptor?
can be up-regulated by SUMO-1 (72). Mutations that prevent SUMO-1
from binding to AR can enhance AR transactivation, indicating a negative
role for SUMO-1 on AR (22). To date, few studies have focused on
SUMO-3.Recently,Li etal.(79)foundthatSUMO-3caneitherreduceor
increasethegenerationofamyloid?peptide,criticaltoAlzheimerdisease.
FIGURE8.SUMO-2siRNAinhibitstheproliferationofandrogen-dependentLNCaPcells.AandB,androgen-dependentLNCaPcellsweretransfectedwitheitherSUMO-2siRNA
orcontrolsiRNAandsubjectedtosemiquantitativeRT-PCR(A)orcellproliferationassay(B).Cellsmonitoredforproliferationweregrownfor0–6daysinthepresenceofethanol(open
bar) or 100 nM DHT (gray bar). The cell number was quantified using the MTT assay (Sigma).
FIGURE 9. SUMO-3 stimulates AR-dependent
transcriptioninandSUMO-2siRNAinhibitsthe
proliferation of androgen-independent LNCaP
cells. A, different LNCaP cell lines were transiently
transfected with 2 ?g of MMTV-CAT and 2 ?g of
SUMO-3. Cells were treated with either ethanol
(open bars) or 100 nM DHT (gray bars). All activities
are relative to the activity of LNCaP cells in the
presence of DHT and absence of transfected
SUMO-3, and this was set to 1. Note that C33 rep-
resents the parental androgen-dependent LNCaP
cell lines from which were derived C81 cells, an
androgen-independent cell line. B, LNCaP or C81
cells were subjected to semiquantitative RT-PCR
to measure expression of SUMO-2, SUMO-3, or
glyceraldehyde-3-phosphate
(GAPDH). C and D, C81 cells were transfected with
either SUMO-2 siRNA or control siRNA and sub-
jectedtosemiquantitativeRT-PCR(C)oracellpro-
liferation assay (D). Cells monitored for prolifera-
tion were grown for 0–6 days in the presence of
ethanol (open bar) or 100 nM DHT (gray bar). The
cell number was quantified using the MTT assay
(Sigma).
dehydrogenase
SUMO-3ActivatesAR-dependentTranscription
FEBRUARY 17, 2006•VOLUME 281•NUMBER 7JOURNAL OF BIOLOGICAL CHEMISTRY 4009
at University of Michigan on September 28, 2006
www.jbc.org
Downloaded from

Page 9

In our study, overexpression of SUMO-3 or SUMO-2 markedly
enhanced AR transactivation in LNCaP and DU145 cells. However,
importantly,SUMO-3hasnosignificanteffectinnormalprimarypros-
tateepithelialcells,suggestingthattheSUMO-3effectonARmaybeat
least partially responsible for the elevated AR activity that is commonly
observed in prostate cancer. Additionally, the SUMO-3-positive effect
on AR is not observed in PC-3 cells, a cell response difference that may
reflect the cellular heterogeneity of prostate tumors.
What causes the different effects of SUMO-3 on AR transactivation?
LNCaP cells contain a mutated AR, which leads to low ligand binding
specificity (15). Our data show that wild-type AR also can be up-regu-
lated by SUMO-3 in both LNCaP and DU145 cells, demonstrating that
themutationofARisnotresponsibleforthepositiveeffectofSUMO-3
on AR transactivation. Another possible explanation comes from evi-
dence showing that the family of PIAS proteins may act as E3 ligases in
theSUMOpathway,especiallyfornuclearreceptors.WhereasthePIAS
proteinswereoriginallyidentifiedasaninhibitorofSTATtranscription
factors (80), recent studies have shown that PIAS1, PIAS3, and PIASx?
regulateARtransactivationinacell-andpromoter-specificmanner(34,
36, 81, 82). Moreover, PIAS1 and PIASx? increase the sumoylation of
AR (34). Therefore, differences in expression levels of the various PIAS
proteins in prostate cancer cell lines may contribute to the cell-specific
effects of SUMO-3 on AR transactivation. Interestingly, the positive
effect of SUMO-3 on AR in LNCaP cells was observed on both the
natural promoter MMTV and the artificial androgen-inducible pro-
moter ARE3-E1B, suggesting that this action, unlike the activities of the
PIAS proteins, is not dependent on the promoter context. A third pos-
sibilityisthatanunknownfactorinLNCaPcellsmediatestheSUMO-3-
positive effect on AR transactivation, and this factor is not expressed in
PC-3 or PrEC cells. Such a factor may be a PIAS protein or one of many
known AR coactivators.
Ourpreviousdatashowedthatc-JuntargetstheNterminusofARto
enhance androgen-dependent transactivation (46). In this study, we
observedthatintheanti-c-JunstableLNCaPcelllineAJ6,SUMO-3still
possessed a positive effect on AR transactivation, although the magni-
tude was lower than in control cells. This suggests that endogenous
c-Jun is required for full enhancement by SUMO-3 of AR transactiva-
tion. However, since there are no c-Jun expression differences among
thevariousprostatecancercelllinesusedinthisstudy,5itisunlikelythat
c-Jun is directly responsible for the cell-specific effects of SUMO-3 on
AR.
As a coactivator, TIF-2 can increase AR-mediated transactivation of
theprostate-specificantigenpromoterinLNCaPcells(83).TIF-2alsois
modifiedbySUMO-1(37).Mutationoftheconsensussumoylationsites
on TIF-2 impaired its coactivation function and cooperation with
PIAS1, a SUMO E3 ligase, on AR transactivation in COS cells (37). In
culturedcells,themRNAlevelsofTIF-2aresignificantlyhigherinPC-3
cellsthaninLNCaPcells(84).Furthermore,usingWesternblotanalysis
of human prostate tissues, Gregory et al. (85) reported that TIF-2 was
not detected in BPH and androgen-dependent tumors but was overex-
pressed in recurrent prostate tumors. Our results showed that exoge-
nous TIF-2 can partially repress the SUMO-3-positive effect in LNCaP
cells.ThesedatasupportthepossibilitythatthelowexpressionofTIF-2
in LNCaP cells may contribute to the positive effect of SUMO-3 on AR
transactivation.ThespecificmechanismofhowTIF-2isinvolvedinthe
SUMO-3 effect is unclear. Since TIF-2 is a known target of SUMO-1
conjugation (37), it is possible that sumoylation of TIF-2 titrates
SUMO-3awayfromthepathwaythatleadstothepositiveeffectonAR.
Moreover, HDACs, PIAS proteins, and SRC-1 can all be modified by
SUMO-1 (reviewed in Ref. 24). Since SUMO-3 is a functionally homol-
ogous protein to SUMO-1, it is possible that SUMO-3 acts on AR by
modifying these AR coregulators.
TheconsensusSUMOacceptorsite,?KXE,foundinGRandSp3has
beenreferredtoasanSCmotif,mutationofwhichabolishesthesumoy-
lation of GR and Sp3 and significantly increases GR and Sp3 transacti-
vation on promoters containing multiple responsive elements (42).
Replacement of lysine with arginine in this motif abolishes SUMO-1
conjugationofARandmarkedlyincreasesARtransactivation(22).Our
results support this earlier finding, since mutation of the two SC motifs
found on the N-terminal region (K385E/K519E) of AR led to enhance-
mentofARtransactivationinLNCaP,DU145,andPC-3cells.However,
surprisingly, in the primary prostate epithelial cells, PrEC, the AR
K385E/K519E mutant had lower activity than the wild-type AR. These
dataprovidethefirstevidencethatmutationoftheSCmotifscaneither
enhanceorcompromiseARtransactivation,dependingonprostatecell
type.
More importantly, we observed that mutation of the AR SC motifs
didnotalterthepositiveSUMO-3effectonARtransactivationineither
LNCaPorDU145cells.ThisfindingsuggeststhattheSUMO-3-positive
effectonARismediatedeitherbyotherunknownARsumoylationsites
oranotherfactoractingonAR.Ourdatasupportthesecondpossibility.
Using immunoprecipitation and Western blot studies, we have been
unable to obtain evidence for either sumoylated forms of AR or AR
physical interaction with SUMO-3 in LNCaP, PC-3, and COS cells.5
ThisincludesthestableLNCaPcelllinesexpressingSUMO-3(FS11and
FS31 cells). In all these cell lines, high molecular weight, SUMO-3-
containing complexes can be obtained upon transfection of SUMO-3;
however, no SUMO-3-modified AR was detected.5Support for an indi-
FIGURE 10. The mRNAs for SUMO-3 and other sumoylation-associated genes are
ubiquitously expressed in human tissues and prostate cancer cells. A, human mul-
tipletissueNorthernblot(Clontech)wasprobedforSUMO-3,usingrandomprimelabel-
ing to synthesize the probe. B, total RNA was isolated from LNCaP, PC-3, and PrEC cells
andsubjectedtoRT-PCRusingprimersforSUMO-1,SUMO-2,SUMO-3,SAE1,SAE2,Ubc9,
or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a control. Note that RT-mi-
nus control templates gave no PCR products.5
SUMO-3ActivatesAR-dependentTranscription
4010 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 281•NUMBER 7•FEBRUARY 17, 2006
at University of Michigan on September 28, 2006
www.jbc.org
Downloaded from

Page 10

rect effect of SUMO proteins on nuclear receptors comes from several
studies. Mutation of the consensus sumoylation site on progesterone
receptor does not impair the effect of SUMO-1 on this receptor (72).
Moreover, estrogen receptor ? transactivation was up-regulated by
SUMO-1,althoughithasnoconsensussumoylationsites(42,72).Fora
nonnuclear receptor protein, Li et al. (79) recently showed that
SUMO-3 indirectly regulates the processing of amyloid precursor pro-
tein (APP), since the APP sequence does not contain the consensus
sumoylationsites,andnosumoylatedAPPwasdetectedinimmunopre-
cipitation analysis.
IfSUMOconjugationtoARisnotinvolvedintheSUMO-3enhance-
ment of AR activity, then it is possible that SUMO-3 conjugation to
another factor is involved. Whereas this cannot be excluded, our data
demonstratethatthesumoylationactivityofSUMOisnotnecessaryfor
mediating AR activity. Indeed, deletion of the two C-terminal glycine
residues, SUMO-3(?GG), which renders SUMO-3 deficient in conju-
gationtotargetproteins,doesnotdisrupttheSUMO-3enhancementof
AR transactivation. These results strongly argue that SUMO-3 can
mediate AR-dependent transcription in a sumoylation-independent
manner, making it mechanistically distinct from the SUMO-1-depend-
ent repression of AR activity (22). Moreover, our findings suggest that
SUMO-3 (and perhaps other SUMO proteins) harbors conjugation-
independent activities. This possibility is supported by a recent obser-
vation that SUMO-3 can coactivate of EBNA2 (Epstein-Barr virus
nuclear antigen 2) in the absence of direct conjugation to EBNA2 (86).
Prostate cancer often becomes hormone-refractory after androgen
ablation therapy (reviewed in Ref. 87). In this stage, cell proliferation
becomes androgen-independent and does not respond to currently
used androgen antagonists. Despite this, AR transactivation remains
intactinmostandrogen-independenttumorsasindicatedbyexpression
of prostate-specific antigen and other AR target genes (87). This tran-
sition from androgen-dependent to androgen-independent prostate
tumors has been reproduced in cultured LNCaP cells (48), resulting in
the generation of the C81 cell line. C81 cells are androgen-independent
LNCaP cells that have similar AR protein expression to the parental
cells,C33(48).Weobservedthatandrogen-inducedARtransactivation
inC81cellsisweakerthaninC33cells.Indeed,Igawaetal.(48)reported
that androgen-induced cell proliferation and expression of prostate-
specific antigen is weak in C81 cells. Additionally, our results demon-
strate that the SUMO-3-positive effect on AR is intact in C81 cells,
although the magnitude is lower than in C33 cells. More significantly,
reducing the expression by siRNA of SUMO-2 significantly compro-
mises the growth of both androgen-dependent and androgen-indepen-
dent LNCaP cells. Because of the high amino acid sequence similarity
between SUMO-2 and SUMO-3, these two proteins are believed to be
functional homologues (27). Indeed, our data here clearly show that
both SUMO-3 and SUMO-2 can enhance AR activity. Thus, our
SUMO-2 siRNA results suggest that the enhancing activity of either
SUMO-2orSUMO-3onARtransactivationisinvolvedinthegrowthof
prostate cancer cells, implicating a potentially significant role for these
two proteins in androgen-independent prostate tumors.
Althoughsumoylationappearstorepresentawidespreadmechanism
ofregulationofproteinactivityandsubcellularlocalization(reviewedin
Ref. 25), the biological consequence of this post-translational modifica-
tion is not fully understood. Recently, Steffan et al. (26) reported that
SUMO-1 can modify the pathogenic fragment of protein Huntington
(Httex1p),whichisaccumulatedinHuntingtondisease.InaDrosophila
model of Huntington disease, unsumoylated Httex1p significantly
reduced the cytotoxicity of this protein, suggesting that sumoylation
may aggravate neurodegeneration disease (26). In this study, we deter-
mined that SUMO-3 is ubiquitously expressed in human tissues, espe-
cially high in prostate, testis, and thyroid. Importantly, overexpression
of SUMO-3 in LNCaP cells leads to a significant increase in androgen-
inducedcellularproliferation.Thisfindingsupportsthecontentionthat
SUMO-3 is a biological regulator of AR transactivation and, therefore,
suggests that SUMO-3 may have a role in prostate carcinogenesis.
Acknowledgments—We thank Dr. T. Nishimoto for providing SUMO-1,
SUMO-2, and SUMO-3; Dr. J. Iniguez-Lluhi for p5HB-AR and p5HB-
AR(K385E/K519E); Dr. B. Rowan for SRC-1 and SRC-3; Dr. P. Chambon for
TIF2; Dr. E. Sanchez for PRE30E1B-CAT; and Dr. M. Lin for C81 and C33
cells. We also thank Dr. S. Leisner for critical reading of the manuscript.
REFERENCES
1. Kokontis, J.M., and Liao, S. (1999) Vitam. Horm. 55, 219–307
2. Mangelsdorf, D. J., Thummel, C., Beato, M., Herrlich, P., Schutz, G., Umesono, K.,
Blumberg, B., Kastner, P., Mark, M., Chambon, P., and Evans, R. M. (1995) Cell 83,
835–839
3. Mangelsdorf, D. J., and Evans, R. M. (1995) Cell 83, 841–850
4. Beato, M., Herrlich, P., and Schutz, G. (1995) Cell 83, 851–857
5. Kastner, P., Mark, M., and Chambon, P. (1995) Cell 83, 859–869
6. Thummel, C. (1995) Cell 83, 571–577
7. Linzey, J., Kumar, M. J., Grossman, M., and Tindall, D. J. (1994) Vitam. Horm. 49,
383–432
8. Thigpen, A. E., Davis, D. L., and Milatovich, A. (1992) J. Clin. Invest. 90, 799–809
9. Wilson, J. D., Griffin, J. E., and Russell, D. W. (1993) Endocr. Rev. 14, 577–593
10. Quigley, C. A., Belli, D., Marschke, A., el-Awady, K. B., and French, F. S. (1995)
Endocr. Rev. 16, 271–321
11. Suzuki, H., Ueda, T., Ichikawa, T., and Ito, H. (2003) Endocr. Cancer 10, 209–216
12. Savarese, D. M., Halabi, S., Hars, V., Akerley, W. L., Taplin, M. E., Godley, P.A.,
Hussain, A., Small, E. J., and Vogelzang, N. J. (2001) J. Clin. Oncol. 19, 2509–2516
13. Suzuki,H.,Sato,N.,Watabe,Y.,Seino,S.,andShimazaki,J.(1993)J.SteroidBiochem.
Mol. Biol. 46, 759–765
14. Suzuki, H., Akakura, K., Komiya, A., Aida, S., Akimoto, S., and Shimazaki, J. (1996)
Prostate 29, 153–158
15. Gaddipati,J.P.,McLeod,D.G.,Heidenberg,H.B.,Sesterhenn,I.A.,Finger,M.J.,and
Moul, J. W. (1994) Cancer Res. 54, 2861–2864
16. Taplin, M. E., Bubley, G. J., Shuster, T. D., Frantz, M. E., Spooner, A. E., and Ogata,
G. K. (1995) N. Engl. J. Med. 332, 1393–1398
17. Taplin,M.E.,Bubley,G.J.,Ko,Y-J.,Small,E.J.,Barur,U.,andRajeshkumar,B.(1999)
Cancer Res. 59, 2511–2515
18. Marcelli, M., Ittmann, M., Mariani, S., Sutherland, R., Nigam, R., and Murthy, L.
(2000) Cancer Res. 60, 944–949
19. Wen, Y., Hu, M. C., Makino, K., Spohn, B., Bartholomeusz, G., Yan, D. H., and Hung,
M. C. (2000) Cancer Res. 60, 6841–6845
20. Fu,M.,Wang,C.,Reutens,A.T.,Angelletti,R.,Siconolfi-Baez,L.,Ogryzko,V.,Avan-
taggiati, M. L., and Pestell, R. G. (2000) J. Biol. Chem. 275, 20853–20860
21. Fu,M.F.,Wang,C.G.,Wang,J.,Zhang,X.P.,Sakamaki,T.,Yeung,Y.G.,Chang,C.S.,
Hopp,T.,Fuqua,S.A.W.,Jaffray,E.,Hay,R.T.,Palvimo,J.J.,Ja ¨nne,O.A.,andPestell,
R. G. (2002) Mol. Cell. Biol. 22, 3373–3388
22. Poukka, H., Karvonen, U., Janne, O. A., and Palvimo, J. J. (2000) Proc. Natl. Acad. Sci.
U. S. A. 97, 14145–14150
23. Weissman, A. M. (2001) Nat. Rev. Mol. Cell. Biol. 2, 169–178
24. Verger, A., Perdomo, J., and Crossley, M. (2003) EMBO Rep. 4, 137–142
25. Hochstrasser, M. (2001) Cell 107, 5–8
26. Steffan,J.S.,Agrawal,N.,Pallos,J.,Rockabrand,E.,Trotman,L.C.,Slepko,N.,Illes,K.,
Lukacsovich,T.,Zhu,Y.Z.,Cattaneo,E.,Pandolfi,P.P.,Thompson,L.M.,andMarsh,
J. L. (2004) Science 304, 100–104
27. Melchior, F. (2000) Annu. Rev. Cell Dev. Biol. 16, 591–626
28. Muller, S., Hoege, C., Pyrowolakis, G., and Jentsch, S. (2001) Nat. Rev. Mol. Cell. Biol.
2, 202–210
29. Hay, R. T. (2001) Trends Biochem. Sci. 26, 332–334
30. Sachdev, S., Bruhn, L., Sieber, H., Pichler, A., Melchior, F., and Grosschedl, R. (2001)
Genes Dev., 15, 3088–3103
31. Kahyo, T., Nishida, T., and Yasuda, H. (2001) Mol. Cell 8, 713–718
32. Kagey, M. H, Melhuish, T. A., and Wotton, D. (2003) Cell 113, 127–137
33. Pichler, A., Gast, A., Seeler, J. S., Dejean, A., and Melchior, F. (2002) Cell 108,
109–120
34. Nishida, T., and Yasuda, H. (2002) J. Biol. Chem. 277, 41311–41317
35. Heinlein, C. A., and Chang, C. (2002) Endocr. Rev. 23, 175–200
36. Gross, M., Liu, B., Tan, J., French, F. S., Carey, M., and Shuai, K., (2001) Oncogene 20,
3880–3887
SUMO-3ActivatesAR-dependentTranscription
FEBRUARY 17, 2006•VOLUME 281•NUMBER 7JOURNAL OF BIOLOGICAL CHEMISTRY 4011
at University of Michigan on September 28, 2006
www.jbc.org
Downloaded from

Page 11

37. Kotaja,N.,Aittomaki,S.,Silvennoinen,O.,Palvimo,J.J.,andJanne,O.A.,(2000)Mol.
Endocrinol., 14, 1986–2000
38. Junicho, A., Matsuda, T., Yamamoto, T., Kishi, H., Korkmaz, K., Saatcioglu, F., Fuse,
H., and Muraguchi, A. (2000) Biochem. Biophys. Res. Commun. 278, 9–13
39. Poukka,H.,Aarnisalo,P.,Karvonen,U.,Palvimo,J.J.,Janne,O.A.(1999)J.Biol.Chem.
274, 19441–19446
40. Shemshedini, L., Knauthe, R., Sassone-Corsi, P., Pornon, A., and Gronemeyer, H.
(1991) EMBO J. 10, 3839–38349
41. Saitoh, H., Sparrow, D. B., Shiomi, T., Pu, R. T., Nishimoto, T., Mohun, T. J., and
Dasso, M. (1998) Curr. Biol. 8, 121–124
42. Iniguiez-Lluhi, J. A., and Pearce, D. (2000) Mol. Cell. Biol. 20, 6040–6050
43. Rowan,B.G.,Weigel,N.L.,andO’Malley,B.W.(2000)J.Biol.Chem.275,4475–4483
44. Voegel,J.J.,Heine,M.J.,Zechel,C.,Chambon,P.,andGronemeyer,H.(1996)EMBO
J. 15, 3667–3675
45. Sanchez, E. R., Hu, J. L., Zhong, S., Shen, P., Greene, M. J., and Housley, P. R. (1994)
Mol. Endocr. 8, 408–421
46. Bubulya, A., Wise, S. C., Shen, X. Q., Burmeister, L. A., and Shemshedini, L. (2000)
Endocrine 13, 55–62
47. Gyuris, J., Golemis, E., Chertkov, H., and Brent, R. (1993) Cell 75, 791–803
48. Igawa,T.,Lin,F.F.,Lee,M-S.,Karan,D.,Batra,S.K.,andLin,M-F.(2002)Prostate50,
222–235
49. Shenk, J. L., Fisher, C. J., Chen, S. Y., Zhou, X. F., Tillman, K., and Shemshedini, L.
(2001) J. Biol. Chem. 276, 38472–38479
50. Chen, S. Y. (2002) The Role of Human Androgen Receptor in the Growth and Survival
of Prostate Cancer Cells, Ph.D. dissertation, University of Toledo, Toledo, OH
51. Chasman, D. I., Leatherwood, J., Carey, M., Ptashne, M., and Kornberg, R. D. (1989)
Mol. Cell. Biol. 9, 4746–4749
52. Bubulya, A, Chen, S. Y., Fisher, C. J., Zheng, Z., Shen, X. Q., and Shemshedini, L.
(2001) J. Biol. Chem. 276, 44704–44711
53. Shen, X. Q., Bubulya, A., Zhou, X. F., Khazak, V., Golemis, E. A., and Shemshedini, L.
(1999) Endocrine 10, 281–289
54. Howell,B.W.,Afar,D.E.,Lew,J.,Douville,E.M.,Icely,P.L.,Gray,D.A.,andBell,J.C.
(1991) Mol. Cell. Biol. 11, 568–572
55. Ausubel,F.M.,Brent,R.,Kingston,R.E.,Moore,D.D.,Seidman,J.G.,Smith,J.A.,and
Struhl,K.(1998)CurrentProtocolsinMolecularBiology,Vol.2,JohnWileyandSons,
Inc., New York
56. Jenster, G., van der Korput, H. A. G. M., Trapman, J., and Brinkman, A. O. (1995)
J. Biol. Chem. 270, 7341–7346
57. McEwan, I. J., and Gustafsson, J.-A. (1997) Proc. Natl. Acad. Sci. U. S. A. 94,
8485–8490
58. Berrevoets, C. A., Doesburg, P., Steketee, K., Trapman, J., and Brinkmann, A. O.
(1998) Mol. Endocrinol. 12, 1172–1183
59. Alen,P.,Claessens,F.,Verhoeven,G.,Rombauts,W.,andPeeters,B.(1999)Mol.Cell.
Biol. 19, 6085–6097
60. Aarnisalo, P., Palvimo, J. J., and Janne, O. A., (1998) Proc. Natl. Acad. Sci. U. S. A. 95,
2122–2127
61. Verrijzer, C. P., and Tjian, R. (1996) Trends Biochem. Sci. 21, 338–342
62. Yeh, S., and Chang, C. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5517–5521
63. Hsiao, P. W., and Chang, C. (1999) J. Biol. Chem. 274, 22373–22379
64. Yuan, S., Trachtenberg, J., Mills, G. B., Brown, T. J., Xu, F., and Keating, A. (1993)
Cancer Res. 53, 1304–1311
65. Johnson, E. S. (2004) Annu. Rev. Biochem. 73, 355–382
66. Jarrard, D. F., Kinoshita, H., Shi, Y., Sandefur, C., Hoff, D., Meisner, L. M., Chang, C.,
Herman, J. G., Isaacs, W. B., and Nassif, N. (1998) Cancer Res. 58, 5310–5314
67. Tran,C.P.,Lin,C.,Yamashiro,J.,andReiter,R.E.(2002)Mol.CancerRes.1,113–121
68. Culig, Z. (2003) Urology 62, 21–26
69. Bubulya, A., Wise, S. C., Shen, X. Q., Burmeister, L. A., Shemshedini, L. (1996) J. Biol.
Chem. 271, 24583–24589
70. Muller, S., Berger, M., Lehembre, F., Seeler, J-S., Haupt, Y., and Dejean, A. (2000)
J. Biol. Chem. 275, 13321–13329
71. Yao, T. P., Ku, G., Zhou, N., Scully, R., and Livingston, D. M. (1996) Proc. Natl. Acad.
Sci. U. S. A. 93, 10626–10631
72. Chauchereau, A., Amazit, L., Quesne, M., Guiochon-Mantel, A., and Milgrom, E.
(2003) J. Biol. Chem. 278, 12335–12343
73. Kotaja, N., Karvonen, U., Janne, O. A., and Palvimo, J. J. (2002) J. Biol. Chem. 277,
30283–30288
74. Hershko, A., and Ciechanover, A. (1992) Annu. Rev. Biochem. 61, 761–807
75. Jentsch, S. (1992) Annu. Rev. Genet. 26, 179–207
76. Abate-Chen, C., and Shen, M. M. (2000) Genes Dev. 14, 2410–2434
77. Seeler, J. S., and Dejean, A. (2003) Nat. Rev. Mol. Cell. Biol. 4, 690–699
78. Le Drean, L., Mincheneau, N., Le Goff, P., and Michel, D. (2002) Endocrinology 143,
3482–3489
79. Li, Y., Wang, H., Wang, S., Quon, D., Liu, Y., and Cordell, B. (2003) Proc. Natl. Acad.
Sci. U. S. A. 100, 259–264
80. Shuai, K. (2000) Oncogene 19, 2638–2644
81. Tan, J., Hall, S. H., Hamil, K. G., Grossman, G., Petrusz, P., Liao, J., and French, F. S.
(2000) Mol. Endocr. 14, 14–26
82. Moilanen, A-M., Karonen, U., Poukka, H., Yan, W., Toppari, J., Janne, O. A., and
Palvimo, J. J. (1999) J. Biol. Chem. 274, 3700–3704
83. Shang, Y., Myers, M., and Brown, M. (2002) Mol. Cell 9, 601–610
84. Fujimoto, N., Mizokami, A., Harada, S., and Matsumoto, T. (2001) Urology 58,
289–294
85. Gregory, C. W., He, B., Johnson, R. T., Ford, O. H., Mohler, J. L., French, F. S., and
Wilson, E. M. (2001) Cancer Res. 61, 4315–4319
86. Rosendorff,A.,Illanes,D.,David,G.,Lin,J.,Kieff,E.,andJohannsen,E.(2004)J.Virol.
78, 367–377
87. Taplin, M. E., and Balk, S. P. (2004) Prostate 91, 483–490
SUMO-3ActivatesAR-dependentTranscription
4012 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281•NUMBER 7•FEBRUARY 17, 2006
at University of Michigan on September 28, 2006
www.jbc.org
Downloaded from