TOPORS Functions As A SUMO-1 E3 Ligase for Chromatin-Modifying
Proteins
Pooja Pungaliya, Diptee Kulkarni, Hye-Jin Park, Henderson Marshall, Haiyan Zheng,
Henry Lackland, Ahamed Saleem, and Eric H. Rubin*
Departments of Pharmacology and Medicine, The Cancer Institute of New Jersey, Robert Wood Johnson
Medical School, University of Medicine and Dentistry of New Jersey, 195 Little Albany Street,
New Brunswick, New Jersey 08901
Received June 13, 2007
TOPORS is the first example of a protein with both ubiquitin and SUMO-1 E3 ligase activity and has
been implicated as a tumor suppressor in several different malignancies. To gain insight into the cellular
role of TOPORS, a proteomic screen was performed to identify candidate sumoylation substrates. The
results indicate that many of the putative substrates are involved in chromatin modification or
transcriptional regulation. Transfection studies confirmed mammalian Sin3A as a sumoylation substrate
for TOPORS. These findings suggest that TOPORS may function as a tumor suppressor by regulating
mSin3A and other proteins involved in chromatin modification.
Keywords: TOPORS • SUMO • ubiquitin • E3 ligase • chromatin • Sin3A
Introduction
TOPORS is a nuclear protein that was identified originally
as a topoisomerase I-binding protein and was shown to
function as a RING-dependent E3 ubiquitin ligase for p53.1
Expression studies implicate TOPORS as a tumor suppressor
in colon, lung, and brain malignancies.2-4 Human TOPORS is
located on chromosome 9p21, with loss-of-heterozygosity in
this region frequently observed in several different malignancies. Forced expression of TOPORS inhibits cellular proliferation
and is associated with cell cycle arrest in the G0/G1 phase.2
Notably, the TOPORS RING domain is not required for this antiproliferative effect.2 Collectively, these results suggest that
although TOPORS may be a tumor suppressor, direct ubiquitination activity is not required for this function.
More recent studies demonstrated that TOPORS also functions as a SUMO-1 E3 ligase for p53.5,6 Furthermore, the
TOPORS RING domain is not required for sumoylation activity
(ref 6 and Kulkarni, et al., submitted). Although recent work
implicates dual ubiquitin/SUMO-1 ligase activity for a multiprotein complex involved in DNA repair,7 TOPORS is the first
example of a single protein that functions as both a ubiquitin
and SUMO-1 ligase. In contrast to ubiquitination, sumoylation
does not target a protein for degradation but may regulate
protein localization,8 transcription factor activity,9 or oppose
protein ubiquitination.10 Similar to ubiquitination, alterations
in sumoylation pathways have been proposed as etiologic in
the development of cancer.11
To gain insight into the cellular role of TOPORS, we
performed a proteomic screen for sumoylation substrates of
* To whom correspondence should be addressed. Eric H. Rubin, The
Cancer Institute of New Jersey, 195 Little Albany St., New Brunswick, NJ
08901; Tel, 732-235-7955; E-Mail, [email protected].
3918
Journal of Proteome Research 2007, 6, 3918-3923
Published on Web 09/06/2007
TOPORS in HeLa cell nuclear extract. Several proteins involved
in chromatin modification or transcriptional regulation were
identified as potential substrates. Using transfection studies,
mSin3A was confirmed as a cellular sumoylation substrate for
TOPORS.
Experimental Procedures
Expression Plasmids. A eukaryotic expression plasmid for
polyhistidine-tagged TOPORS was constructed by digestion of
pGEX-TOPORS1 with EcoRI and NotI, with subsequent ligation
into pcDNA4HisMax (Invitrogen). Eukaryotic expression plasmids for GFP-SUMO-1 and HA-SENP2 were kindly provided
by Hongtao Yu (University of Texas Southwestern Medical
Center).12 An expression plasmid for MYC epitope-tagged
mSin3A was provided by Robert Eisenman and Carol Laherty
(Fred Hutchinson Cancer Research Center, Seattle, WA).13
Purification of TOPORS-Binding Proteins. Expression and
purification of recombinant GST-TOPORS fusion proteins were
performed as described.1 In certain experiments, dimethylpimelimidate dihydrochloride (Pierce) was used to covalently
link GST or GST-TOPORS to glutathione-Sepharose beads as
described.14 HeLa nuclear extracts were prepared as described
by Dignam, et al.15 Extract was pre-cleared on GST beads before
affinity purification using GST-TOPORS-loaded beads as described previously.14
In vitro Sumoylation Assays. A protease-deficient bacterial
strain (BLR(DE3); Invitrogen)) was used for expression of
recombinant GST-SAE2/SAE1, GST- and His-tagged TOPORS
proteins. Purification of these proteins from bacterial lysates
was performed as described previously.16,17 Sumoylation reactions were performed in sumoylation buffer, containing 50 mM
HEPES, pH 8.0, 5 mM MgCl2, 15 µM ZnCl2, and 4 mM ATP.
Unless stated otherwise, reactions contained 150 nM SAE2/
10.1021/pr0703674 CCC: $37.00
 2007 American Chemical Society
research articles
Proteomic Screen for TOPORS Substrates
Figure 1. TOPORS-dependent sumoylation of HeLa nuclear proteins in vitro. (A) HeLa nuclear extract was subjected to affinity
chromatography using GST-TOPORS covalently linked to glutathione beads. Bound proteins were eluted and concentrated. One milligram
of the eluate was used in an in vitro sumoylation assay containing 100 ng of SAE1/2 and 20 ng of Ubc9. As indicated, certain reactions
also contained 300 ng of SUMO-1 and 400 ng of His-TOPORS. Reaction products were analyzed by SDS-PAGE and immunoblotting
with a SUMO-1 antibody. Migration of free SUMO-1 and SUMO-1-conjugated Ubc9 are indicated. Asterisks indicate SUMO-1 conjugates
detected in the presence of His-TOPORS. (B) Purification of His-SUMO-1-p53 conjugates induced by GST-TOPORS. Reaction products
from reconstituted in vitro sumoylation reactions containing the indicated components were analyzed by SDS-PAGE and p53
immunoblotting, either without purification (lanes 1-3) or after purification of His-SUMO-1 conjugates using nickel beads (lane 4). (C)
p53 is detected as a sumoylation substrate for TOPORS in HeLa nuclear extract. One-hundred micrograms of HeLa nuclear extract was
affinity purified using GST-TOPORS beads, followed by the addition of SAE1/2, Ubc9, and His-tagged SUMO-1. As indicated, sumoylation
reactions were performed in the presence or absence of ATP, followed by affinity chromatography using nickel beads under denaturing
conditions. Eluates were analyzed by SDS-PAGE and p53 immunoblotting. The lane marked input HeLa represents 10 µg of nonpurified
HeLa nuclear lysate. (D) Sumoylation of HeLa nuclear proteins by TOPORS. One milligram of HeLa nuclear extract was subjected to
affinity chromatography followed by an in vitro sumoylation in the presence or absence of ATP, with subsequent purification of HisSUMO-1 conjugates using nickel beads. Eluates were analyzed by Coomassie staining.
SAE1, 30 nM UbcH9 (Sigma), 30 nM of purified recombinant
p53 (BD Pharmingen), and 1 µM of His-SUMO-1 (Boston
Biochem). After incubation at 30 °C for 2 h, reactions were
terminated by the addition of SDS sample buffer.
For the substrate screen, HeLa nuclear proteins bound to
GST-TOPORS beads were washed with sumoylation buffer.
Duplicate sumoylation assays were performed in the presence
and absence of 4 mM ATP, with the addition of His-SUMO-1
(Boston Biochem), SAE1/2 (Boston Biochem), and Ubc9 (Sigma).
Reactions were allowed to proceed at 30 °C for 2 h and then
were stopped by the addition of buffer A (6 M guanidine-HCl,
with 0.10% NP-40, 10 mM β-mercaptoethanol, and 5% glycerol
in phosphate-buffered saline, pH 8.0). Reaction products were
subjected to affinity chromatography using nickel-NTA beads
(Qiagen) and buffer A containing 5 mM imidazole. Proteins
were eluted by boiling in SDS sample buffer (60 mM Tris-Cl,
pH 6.8, 2% SDS, 10% glycerol, and 0.1% phenol red) containing
1 mM DTT and then analyzed by immunoblotting and Coomassie staining.
Mass Spectrometry. In-gel tryptic digest samples were
analyzed by LC-MS/MS as described.18 Peak list files for MS/
MS spectra were generated by Bioworks software (ThermoFinnigan, San Jose, CA) and searched against a human
database (ENSEMBL 28.35a.1 NCBI35, May 2005) using a local
implementation of X! Tandem.19 Identified proteins of interest
were confirmed manually.
Sumoylation of Transfected and Endogenous mSin3A.
H1299 human lung cancer cells were grown in Dulbecco’s
modified Eagle’s medium supplemented with 10% fetal bovine
serum and antibiotics at 37 °C and 5% CO2. Plasmid transfections were performed using the Lipofectamine 2000 transfection
reagent according to manufacturer’s protocol (Invitrogen). Cells
were cotransfected with various combinations of 1 µg pCS2MT-mSin3A, 2.5 µg pcDNA-His-TOPORS, 500 ng pGFP-SUMO1, and 500 ng pHA-SENP2. Transfected DNA was adjusted to
4.5 µg by addition of pRC-CMV (Invitrogen). Thirty hours after
transfection, cells were lysed in SDS sample buffer containing
1 mM DTT and 5 mM N-ethyl maleimide (Sigma). For immu-
noprecipitations, cells were lysed in PBS containing 1% igepal,
0.5% sodium deoxycholate, 0.1% SDS, 2 mM PMSF, 5 µg/mL
leupeptin, and 1 µg/mL pepstatin. Immunoprecipitations were
performed at 4 °C overnight using 1 µg of MYC or mSin3A
antibodies (Sigma). Immune complexes were recovered using
50 µL of pre-equilibrated 50% Protein G- or A-sepharose beads
(Sigma). Whole cell lysates and immunoprecipitates were
analyzed by SDS-PAGE and immunoblotting with MYC and
GFP (Roche) antibodies.
Results
Identification of Sumoylation Substrates for TOPORS.
Initial experiments were designed to determine whether TOPORS-binding proteins present in HeLa nuclear extract could
be sumoylated by TOPORS using a reconstituted in vitro
sumoylation assay. TOPORS-bound HeLa nuclear proteins that
were eluted from covalently linked GST-TOPORS beads were
subjected to an in vitro sumoylation assay in the presence or
absence of His-TOPORS as a SUMO-1 E3 ligase. In the presence
of His-TOPORS, SUMO-1 conjugates of various gel mobilities
were detected after immunoblotting with a SUMO-1 antibody
(Figure 1A). Importantly, the results indicated that in the
absence of TOPORS, only free SUMO-1 and a probable Ubc9SUMO-1 conjugate were evident after immunoblotting of
reaction products (Figure 1A). Therefore, under these conditions, there was little background SUMO-1 E3 ligase activity in
HeLa nuclear extracts that might confound identification of
sumoylation substrates for TOPORS.
To enrich for identification of sumoylation substrates for
TOPORS rather than nonsumoylated TOPORS-binding proteins,
we employed polyhistidine-tagged SUMO-1 and nickel-based
affinity chromatography under denaturing conditions to purify
SUMO-1 conjugates from the in vitro sumoylation reaction
mixture. In a validation experiment using purified p53 as a
substrate, immunoblotting with a p53 antibody indicated that
under these conditions only sumoylated p53 (and not unmodified p53) was detected in affinity-purified reaction products
(Figure 1B).
Journal of Proteome Research • Vol. 6, No. 10, 2007 3919
research articles
Pungaliya et al.
Table 1. Candidate Sumoylation Substrates for TOPORS
log(e)a
SUMOb
protein name/function
UniProt/
Swiss-Prot
Chromatin modification/transcriptional regulation
-145.7
X
TFII-I
P78347
-142.9
X
p54nrbc
Q15233
-104.7
X
PSF
P23246
-65.7
Sin3a
Q96ST3
-59.2
X
Poly (ADP-ribose) polymerase 1
P09874
-57.6
X
Scaffold attachment factor B2
Q14151
-50.6
DNA damage binding protein 2
Q92466
-49.6
RbAp46
Q16576
-48.7
X
Transcription intermediary
Q13263
factor 1-beta
-38.6
RbAp48
Q09028
-33.1
BRG1-associated factor 170
Q8TAQ2
Nuclear mRNA Processing and Transport
-112.4
X
DEAH box protein 15
O43143
-110
X
hnRNP L
P14866
-97.5
X
Ran GTPase-activating protein 1
P46060
-88.5
UPF3A
Q9H1J1
-75.1
X
hnRNP M
P52272
-39.4
X
hnRNP A1
P09651
-33.4
Splicing factor 3B subunit 1
O75533
Translational Regulation
-71.1
Elongation factor 1-alpha 1
P68104
Other
-32.7
Calgranulin B
P06702
a
Expectation value (e) is defined as the number of proteins expected to
have at least the score of the indicated protein.50 b “x” indicates that the
protein was identified in a screen for cellular sumoylated proteins.12,20-22
c
Italic font indicates a member of an mSin3A complex.
As a further validation experiment for the screen, we evaluated TOPORS-induced sumoylation of p53 present in HeLa
nuclear extracts. HeLa nuclear extract was affinity purified using
GST-TOPORS beads, followed by addition of SAE1/2, Ubc9, and
His-tagged SUMO-1. In this case, the bead-bound GST-TOPORS protein served as the SUMO-1 E3 ligase. Despite the
relatively low levels of p53 present in HeLa nuclear extracts,
monosumoylation of p53 was detected in affinity-purified
reaction products (Figure 1C).
Next, the assay was scaled up to include 1 mg of HeLa
nuclear lysate. Duplicate reactions were performed in the
presence and absence of ATP. Analysis of affinity-purified
reaction products by SDS-PAGE and Coomassie staining indicated that proteins of various molecular weights were generated
from reactions containing ATP (Figure 1D). From the lane
including reactions containing ATP, 34 2-mm gel slices were
excised and analyzed by liquid chromatography-tandem mass
spectrometry (LC-MS/MS). Table 1 lists the 20 highest-scoring
protein matches, according to cellular function and expectation
value (see Supplemental Table 1 for a list of all matches with
a log(expectation value) e -9). Most of the candidate substrates
are involved in chromatin modification or nuclear mRNA
processing, and many were identified previously in efforts to
detect SUMO-1-conjugates in cells.12,20-22 p53 was not identified
among the highest scoring matches (Supplemental Table 1).
This indicates that our screening method may not be able to
detect low-abundance proteins that are substrates of TOPORS.
Verification of mSin3A as a Cellular TOPORS Substrate.
In addition to mSin3A, several mSin3A-binding proteins were
identified as potential sumoylation substrates for TOPORS
(Table 1). mSin3A was also identified as a TOPORS-binding
protein in an independent analysis of proteins that co-purified
with polyhistidine-tagged TOPORS expressed in H1299 lung
cancer cells (data not shown). mSin3A functions in transcrip3920
Journal of Proteome Research • Vol. 6, No. 10, 2007
tional repression and chromatin modification by association
with histone deacetylases (HDACs) 1 and 2 in multi-protein
complexes.23 RbAp46 and RbAp48, which were identified as
putative TOPORS substrates (Table 1), are part of a core
mSin3A-HDAC complex.24,25 Additional candidate TOPORS
substrates listed in Table 1 that associate with mSin3A include
BRG1-associated factor 170,26 polypyrimidine tract-binding
protein-associated splicing factor (PSF), and p54nrb.27 Given
that alterations in mSin3A function are implicated in carcinogenesis,23,28 we chose to further investigate mSin3A as a
sumoylation substrate for TOPORS.
Transient cotransfection studies were performed using TOPORS, MYC-mSin3A, and GFP-SUMO-1 expression plasmids.
No sumoylation of MYC-mSin3A was detected in H1299 cells
expressing MYC-mSin3A alone or in cells expressing MYCSin3A and GFP-SUMO-1 (Figure 2A). By contrast, in cells
coexpressing mSin3A, GFP-SUMO-1, and TOPORS, a low
mobility mSin3A band was detected, consistent with the
addition of a GFP-SUMO-1 conjugate on mSin3A (Figure 2A,
lane 3). This band was not detected in cotransfectants expressing the SUMO-1 isopeptidase SENP212 (Figure 2A, lane 5),
providing additional evidence that this band represents a GFPSUMO-1-conjugate on mSin3A. Notably, the finding that coexpression of GFP-SUMO-1 was required for detection of
sumoylation of mSin3A by TOPORS (Figure 2A) is similar to
studies of other SUMO E3 ligases and may be due to a limiting
amount of endogenous SUMO-1.29,30
GFP immunoblotting of cotransfectant lysates yielded similar
results, indicating that in cells cotransfected with GFP-SUMO1, TOPORS induced multiple low-mobility GFP-SUMO-1 conjugates, with these conjugates sensitive to expression of SENP2
(Figure 2B). Immunoprecipitation studies confirmed that sumoylation of MYC-mSin3A occurred in cells coexpressing TOPORS
and GFP-SUMO-1 but not in cells coexpressing GFP-SUMO-1
alone (Figure 2C).
Additional studies were performed to determine whether
TOPORS was capable of sumoylating endogenous mSin3A.
Using an antibody recognizing GFP, a low mobility band
corresponding to a GFP-SUMO-1-mSin3A conjugate was detected in mSin3A immunoprecipitates obtained from H1299
cells cotransfected with GFP-SUMO-1 and TOPORS plasmids.
This conjugate was not detected in mSin3A immunoprecipitates from cells expressing GFP-SUMO-1 alone, or in control
immunoprecipitates obtained from cells cotransfected with
GFP-SUMO-1 and TOPORS plasmids (Figure 2D). The lack of
detection of the GFP-SUMO-1-mSin3A conjugate upon immunoblotting with an mSin3A antibody may reflect the relatively
small amount of endogenous mSin3A obtained upon immunoprecipitation, relative to transfected mSin3A (Figure 2).
Together, these results indicate that TOPORS is capable of
stimulating sumoylation of both transfected and endogenous
mSin3A.
Discussion
Although proteomic analyses of ubiquitinated or sumoylated
cellular proteins have been reported previously,21,31-34 our
results are the first report of a proteomic screen for substrates
of a specific SUMO-1 E3 ligase. The results implicate TOPORS
in the sumoylation of proteins involved in chromatin modification and transcription, which is consistent with several lines
of evidence implicating sumoylation in the regulation of
chromatin structure and transcription.30 In addition, our results
Proteomic Screen for TOPORS Substrates
research articles
Figure 2. TOPORS induces sumoylation of mSin3A in H1299 cells. A plasmid expressing MYC-mSin3A was cotransfected with the
indicated plasmids in H1299 cells. Thirty hours after transfection, cell lysates were analyzed by SDS-PAGE and immunoblotting. (A)
Results of anti-MYC immunoblotting. Migration of unmodified MYC-mSin3A and a GFP-SUMO-1-MYC-mSin3A conjugate are indicated
by an asterisk and arrow, respectively. (B) Results of GFP immunoblotting. TOPORS-induced GFP-SUMO-1 conjugates are indicated
by a bracket. (C) Lysates were subjected to immunoprecipitation with a MYC antibody before immunoblotting with a GFP antibody.
The arrow indicates a GFP-SUMO-1-mSin3A conjugate. (D) H1299 cells were transfected with the indicated plasmids. As indicated,
lysates were subjected to immunoblotting with mSin3A and GFP antibodies either before or after immunoprecipitation with control
rabbit serum or an mSin3A antibody. Migration of unmodified mSin3A and a GFP-SUMO-1-mSin3A conjugate are indicated by an
asterisk and arrow, respectively. The arrow indicates migration of endogenous mSin3A conjugated with GFP-SUMO-1.
Figure 3. Identification of a conserved consensus sumoylation site in the histone interaction domain of Sin3A. H. sapiens, M. musculus,
and S. cerevisiae Sin3A orthologs were aligned using a ClustalW algorithm. The conserved consensus sumoylation site (underlined,
residues 701-704) resides within the histone deacetylase interaction domain (residues 524-899 of the H. sapiens ortholog).
are consistent with the observation that a Drosophila TOPORS
ortholog is involved in the functioning of a chromatin insulator.35,36
Little is known regarding substrate specificity for SUMO-1
E3 ligases, and it is not known whether TOPORS serves as a
unique SUMO-1 ligase for the proteins identified in our screen.
Currently, at least three other SUMO-1 E3 ligases have been
described: the transcription factor Siz/PIAS family of proteins,
the nucleoporin RanBP2, and the polycomb protein Pc2. The
Siz/PIAS family were the first described SUMO-1 E3 ligases,
and sumoylate a variety of substrates, including septins, LEF1,
c-Jun, and p53.37 RanBP2 sumoylates SP100,38 PML,39 and
HDAC4.40 Notably, RanBP2 may be a unique SUMO-1 E3 ligase
for HDAC4, because neither PIAS1 nor PIAS3 were able to
stimulate sumoylation of HDAC4 in cells.40 Pc2 sumoylates the
C-terminal binding protein transcriptional corepressor (CtBP)41
and homeodomain interacting protein kinase 2 (HIPK2).42 With
the exception of p53, none of these proteins were identified as
potential substrates for TOPORS. However, Gocke et al. reported that PIASxβ enhanced sumoylation of TFII-I and
Ku80,12 which were both identified as potential substrates for
TOPORS in our screen. Therefore, our results support the idea
that SUMO-1 E3 ligases exhibit only partial substrate specificity.
Our results are also the first report of sumoylation of mSin3A.
Sin3 (switch independent 3) was originally identified in yeast
genetic screens for proteins involved in mating-type switching.43 Subsequent studies revealed that Sin3 orthologs, including mSin3A, function as scaffolds in multi-protein transcriptional repressor complexes, which include HDACs.44 mSin3A
has four paired amphipathic R-helix (PAH) domains that
function in binding transcription factors or adapter proteins
such as SAP30.44 In addition, a conserved histone deacetylase
interaction domain (HID; residues 524-899) is located between
PAH3 and PAH4.13 mSin3A contains four ΨKxE consensus
sumoylation sites (122-124, 191-193, 702-704, and 11811183), where Ψ represents a nonhydrophilic residue, K is the
lysine to which SUMO-1 is attached, and x is any amino
acid.45,46 Interestingly, among these sites, the only one that is
conserved between yeast and humans (residues 702-704) is
within the HID, suggesting that sumoylation of Sin3 proteins
may regulate HDAC binding (Figure 3). Additional studies are
necessary to identify the sumoylation sites on mSin3A and to
determine the physiologic role of mSin3A sumoylation.
Notably, because TOPORS is a dual function E3 ligase, the
identified substrates may also be targeted for ubiquitination
by TOPORS. Alterations in protein ubiquitination and sumoylation have been implicated in a variety of human diseases
including cancer.47-49 In addition, alterations in mSin3A function lead to chromosomal and genomic instability and accelerate tumorigenesis.23,28 The results of our proteomic screen
suggest that loss of TOPORS may predispose to malignancy as
a result of dysregulation of the function of mSin3A and other
chromatin-modifying proteins.
Acknowledgment. We thank Peter Lobel for assistance
with mass spectrometry. This work was supported by United
States Public Health Service grants CA99951 (E.H.R.) and
RR017992 (Dr. Lobel).
Supporting Information Available: Supplemental
Table 1 listing all protein matches with a log(expectation value)
e -9. This material is available free at http://pubs.acs.org.
Journal of Proteome Research • Vol. 6, No. 10, 2007 3921
research articles
References
(1) Rajendra, R.; Malegaonkar, D.; Pungaliya, P.; Marshall, H.;
Rasheed, Z.; Brownell, J.; Liu, L. F.; Lutzker, S.; Saleem, A.; Rubin,
E. H. Topors Functions as an E3 Ubiquitin Ligase with Specific
E2 Enzymes and Ubiquitinates p53. J. Biol. Chem. 2004, 279(35),
36440-36444.
(2) Saleem, A.; Dutta, J.; Malegaonkar, D.; Rasheed, F.; Rasheed, Z.;
Rajendra, R.; Marshall, H.; Luo, M.; Li, H.; Rubin, E. H. The
topoisomerase I- and p53-binding protein topors is differentially
expressed in normal and malignant human tissues and may
function as a tumor suppressor. Oncogene 2004, 23(31), 52935300.
(3) Bredel, M.; Bredel, C.; Juric, D.; Harsh, G. R.; Vogel, H.; Recht, L.
D.; Sikic, B. I. High-resolution genome-wide mapping of genetic
alterations in human glial brain tumors. Cancer Res. 2005, 65(10),
4088-4096.
(4) Oyanagi, H.; Takenaka, K.; Ishikawa, S.; Kawano, Y.; Adachi, Y.;
Ueda, K.; Wada, H.; Tanaka, F. Expression of LUN gene that
encodes a novel RING finger protein is correlated with development and progression of non-small cell lung cancer. Lung Cancer
2004, 46(1), 21-28.
(5) Shinbo, Y.; Taira, T.; Niki, T.; Iguchi-Ariga, S. M.; Ariga, H. DJ-1
restores p53 transcription activity inhibited by Topors/p53BP3.
Int. J. Oncol. 2005, 26(3), 641-648.
(6) Weger, S.; Hammer, E.; Heilbronn, R. Topors acts as a SUMO-1
E3 ligase for p53 in vitro and in vivo. FEBS Lett. 2005, 579(22),
5007-5012.
(7) Zhao, X.; Blobel, G. A SUMO ligase is part of a nuclear multiprotein complex that affects DNA repair and chromosomal
organization. Proc. Natl. Acad. Sci. U.S.A. 2005, 102(13), 47774782.
(8) Matunis, M. J.; Coutavas, E.; Blobel, G. A novel ubiquitin-like
modification modulates the partitioning of the Ran-GTPaseactivating protein RanGAP1 between the cytosol and the nuclear
pore complex. J. Cell Biol. 1996, 135(6 Pt 1), 1457-1470.
(9) Girdwood, D. W.; Tatham, M. H.; Hay, R. T. SUMO and transcriptional regulation. Semin. Cell Dev. Biol. 2004, 15(2), 201210.
(10) Desterro, J. M.; Rodriguez, M. S.; Hay, R. T. SUMO-1 modification
of IkappaBalpha inhibits NF-kappaB activation. Mol. Cell 1998,
2(2), 233-239.
(11) Alarcon-Vargas, D.; Ronai, Z. SUMO in cancer-wrestlers wanted.
Cancer Biol. Ther. 2002, 1(3), 237-242.
(12) Gocke, C. B.; Yu, H.; Kang, J. Systematic identification and analysis
of mammalian small ubiquitin-like modifier substrates. J. Biol.
Chem. 2005, 280(6), 5004-5012.
(13) Laherty, C. D.; Yang, W. M.; Sun, J. M.; Davie, J. R.; Seto, E.;
Eisenman, R. N. Histone deacetylases associated with the mSin3
corepressor mediate mad transcriptional repression. Cell 1997,
89(3), 349-356.
(14) Edwards, T. K.; Saleem, A.; Shaman, J. A.; Dennis, T.; Gerigk, C.;
Oliveros, E.; Gartenberg, M. R.; Rubin, E. H. Role for Nucleolin/
Nsr1 in the Cellular Localization of Topoisomerase I. J. Biol.
Chem. 2000, 275(46), 36181-36188.
(15) Dignam, J. D.; Lebovitz, R. M.; Roeder, R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from
isolated mammalian nuclei. Nucleic Acids Res. 1983, 11, 14751489.
(16) Rajendra, R.; Malegaonkar, D.; Pungaliya, P.; Marshall, H.;
Rasheed, Z.; Brownell, J.; Liu, L. F.; Lutzker, S.; Saleem, A.; Rubin,
E. H. Topors functions as an E3 ubiquitin ligase with specific E2
enzymes and ubiquitinates p53. J. Biol. Chem. 2004.
(17) Tatham, M. H.; Jaffray, E.; Vaughan, O. A.; Desterro, J. M.; Botting,
C. H.; Naismith, J. H.; Hay, R. T. Polymeric chains of SUMO-2
and SUMO-3 are conjugated to protein substrates by SAE1/SAE2
and Ubc9. J. Biol. Chem. 2001, 276(38), 35368-35374.
(18) Perez, E.; Zheng, H.; Stock, A. M. Identification of methylation
sites in Thermotoga maritima chemotaxis receptors. J. Bacteriol.
2006, 188(11), 4093-4100.
(19) Craig, R.; Cortens, J. P.; Beavis, R. C. Open source system for
analyzing, validating, and storing protein identification data. J.
Proteome Res. 2004, 3(6), 1234-1242.
(20) Vertegaal, A. C.; Ogg, S. C.; Jaffray, E.; Rodriguez, M. S.; Hay, R.
T.; Andersen, J. S.; Mann, M.; Lamond, A. I. A proteomic study
of SUMO-2 target proteins. J. Biol. Chem. 2004, 279(32), 3379133798.
3922
Journal of Proteome Research • Vol. 6, No. 10, 2007
Pungaliya et al.
(21) Li, T.; Evdokimov, E.; Shen, R. F.; Chao, C. C.; Tekle, E.; Wang,
T.; Stadtman, E. R.; Yang, D. C.; Chock, P. B. Sumoylation of
heterogeneous nuclear ribonucleoproteins, zinc finger proteins,
and nuclear pore complex proteins: a proteomic analysis. Proc.
Natl. Acad. Sci. U.S.A. 2004, 101(23), 8551-8556.
(22) Rosas-Acosta, G.; Russell, W. K.; Deyrieux, A.; Russell, D. H.;
Wilson, V. G. A Universal Strategy for Proteomic Studies of SUMO
and Other Ubiquitin-like Modifiers. Mol. Cell. Proteomics 2005,
4(1), 56-72.
(23) Dannenberg, J. H.; David, G.; Zhong, S.; van der Torre, J.; Wong,
W. H.; Depinho, R. A. mSin3A corepressor regulates diverse
transcriptional networks governing normal and neoplastic growth
and survival. Genes Dev. 2005, 19(13), 1581-1595.
(24) Hassig, C. A.; Fleischer, T. C.; Billin, A. N.; Schreiber, S. L.; Ayer,
D. E. Histone deacetylase activity is required for full transcriptional repression by mSin3A. Cell 1997, 89(3), 341-347.
(25) Zhang, Y.; Iratni, R.; Erdjument-Bromage, H.; Tempst, P.; Reinberg, D. Histone deacetylases and SAP18, a novel polypeptide,
are components of a human Sin3 complex. Cell 1997, 89(3), 357364.
(26) Sif, S.; Saurin, A. J.; Imbalzano, A. N.; Kingston, R. E. Purification
and characterization of mSin3A-containing Brg1 and hBrm
chromatin remodeling complexes. Genes Dev. 2001, 15(5), 603618.
(27) Mathur, M.; Tucker, P. W.; Samuels, H. H. PSF is a novel
corepressor that mediates its effect through Sin3A and the DNA
binding domain of nuclear hormone receptors. Mol. Cell. Biol.
2001, 21(7), 2298-2311.
(28) Silverstein, R. A.; Ekwall, K. Sin3: a flexible regulator of global
gene expression and genome stability. Curr. Genet. 2005, 47(1),
1-17.
(29) Johnson, E. S. Protein modification by SUMO. Annu. Rev.
Biochem. 2004, 73, 355-382.
(30) Hay, R. T. SUMO: a history of modification. Mol. Cell 2005, 18(1),
1-12.
(31) Peng, J.; Schwartz, D.; Elias, J. E.; Thoreen, C. C.; Cheng, D.;
Marsischky, G.; Roelofs, J.; Finley, D.; Gygi, S. P. A proteomics
approach to understanding protein ubiquitination. Nat. Biotechnol. 2003, 21(8), 921-926.
(32) Wohlschlegel, J. A.; Johnson, E. S.; Reed, S. I.; Yates, J. R., 3rd.
Global analysis of protein sumoylation in Saccharomyces cerevisiae. J. Biol. Chem. 2004, 279(44), 45662-45668.
(33) Vertegaal, A. C.; Ogg, S. C.; Jaffray, E.; Rodriguez, M. S.; Hay, R.
T.; Andersen, J. S.; Mann, M.; Lamond, A. I. A proteomic study
of SUMO-2 target proteins. J. Biol. Chem. 2004.
(34) Sato, K.; Hayami, R.; Wu, W.; Nishikawa, T.; Nishikawa, H.; Okuda,
Y.; Ogata, H.; Fukuda, M.; Ohta, T. Nucleophosmin/B23 is a
candidate substrate for the BRCA1-BARD1 ubiquitin ligase. J. Biol.
Chem. 2004, 279(30), 30919-30922.
(35) Capelson, M.; Corces, V. G. The ubiquitin ligase dTopors directs
the nuclear organization of a chromatin insulator. Mol. Cell 2005,
20(1), 105-116.
(36) Capelson, M.; Corces, V. G. SUMO conjugation attenuates the
activity of the gypsy chromatin insulator. EMBO J. 2006, 25(9),
1906-1914.
(37) Schmidt, D.; Muller, S. PIAS/SUMO: new partners in transcriptional regulation. Cell. Mol. Life Sci. 2003, 60(12), 2561-2574.
(38) Pichler, A.; Gast, A.; Seeler, J. S.; Dejean, A.; Melchior, F. The
nucleoporin RanBP2 has SUMO1 E3 ligase activity. Cell 2002,
108(1), 109-120.
(39) Tatham, M. H.; Kim, S.; Jaffray, E.; Song, J.; Chen, Y.; Hay, R. T.
Unique binding interactions among Ubc9, SUMO and RanBP2
reveal a mechanism for SUMO paralog selection. Nat. Struct. Mol.
Biol. 2005, 12(1), 67-74.
(40) Kirsh, O.; Seeler, J. S.; Pichler, A.; Gast, A.; Muller, S.; Miska, E.;
Mathieu, M.; Harel-Bellan, A.; Kouzarides, T.; Melchior, F.;
Dejean, A. The SUMO E3 ligase RanBP2 promotes modification
of the HDAC4 deacetylase. EMBO J. 2002, 21(11), 2682-2691.
(41) Kagey, M. H.; Melhuish, T. A.; Wotton, D. The polycomb protein
Pc2 is a SUMO E3. Cell 2003, 113(1), 127-137.
(42) Roscic, A.; Moller, A.; Calzado, M. A.; Renner, F.; Wimmer, V. C.;
Gresko, E.; Ludi, K. S.; Schmitz, M. L. Phosphorylation-dependent
control of Pc2 SUMO E3 ligase activity by its substrate protein
HIPK2. Mol. Cell 2006, 24(1), 77-89.
(43) Nasmyth, K.; Stillman, D.; Kipling, D. Both positive and negative
regulators of HO transcription are required for mother-cellspecific mating-type switching in yeast. Cell 1987, 48(4), 579587.
(44) Ayer, D. E. Histone deacetylases: transcriptional repression with
SINers and NuRDs. Trends Cell Biol. 1999, 9(5), 193-198.
research articles
Proteomic Screen for TOPORS Substrates
(45) Melchior, F. SUMO-nonclassical ubiquitin. Annu. Rev. Cell Dev.
Biol. 2000, 16, 591-626.
(46) Rodriguez, M. S.; Dargemont, C.; Hay, R. T. SUMO-1 conjugation
in vivo requires both a consensus modification motif and nuclear
targeting. J. Biol. Chem. 2001, 276(16), 12654-12659.
(47) Alkuraya, F. S.; Saadi, I.; Lund, J. J.; Turbe-Doan, A.; Morton, C.
C.; Maas, R. L. SUMO1 haploinsufficiency leads to cleft lip and
palate. Science 2006, 313(5794), 1751.
(48) 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.; Marsh, J. L. SUMO modification
of Huntingtin and Huntington’s disease pathology. Science 2004,
304(5667), 100-104.
(49) Seeler, J. S.; Bischof, O.; Nacerddine, K.; Dejean, A. SUMO, the
three Rs and cancer. Curr. Top. Microbiol. Immunol. 2007, 313,
49-71.
(50) Fenyo, D.; Beavis, R. C. A method for assessing the statistical
significance of mass spectrometry-based protein identifications
using general scoring schemes. Anal. Chem. 2003, 75(4), 768-774.
PR0703674
Journal of Proteome Research • Vol. 6, No. 10, 2007 3923