Viral oncoproteins E1A and E7 and cellular LxCxE proteins repress
SUMO modification of the retinoblastoma tumor suppressor
Andreas Ledl1, Darja Schmidt1and Stefan Mu ¨ ller*,1
1Department of Molecular Cell Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany
The retinoblastoma tumor suppressor protein (pRB) is a
major regulator of cell-cycle progression and cellular
differentiation. Central to pRB function is the pocket
domain, which serves as the main binding region for
cellular regulators. In tumors pRB is frequently inacti-
vated by mutations in the pocket domain or by binding of
viral oncoproteins to this region. A characteristic feature
of these viral oncoproteins and many cellular pRB-binding
partners is an LxCxE sequence motif, which interacts with
pRB’s pocket domain. Here, we show that the ubiquitin-
like modifier SUMO is covalently attached to a distinct
residue (K720) of pRB within the B-box of the pocket
region that binds LxCxE-motif proteins. We provide
evidence that SUMO preferentially targets the active,
hypophosphorylated form of pRB and show that tumori-
genic mutations of pRB in the pocket domain lead to a loss
of SUMOylation. Notably, the level of pRB SUMOyla-
tion is controlled by the interaction of pRB with viral and
cellular LxCxE-motif proteins. Inhibitors of pRB func-
tion, including the viral oncoproteins E1A and E7 and the
cellular E1A-like inhibitor of differentiation EID-1,
completely abolish SUMO modification of pRB. Con-
versely, pRB mutants deficient in binding of LxCxE-motif
proteins exhibit a drastically enhanced modification by
SUMO. Finally, we provide evidence that SUMOylation
can influence pRB function, as the SUMO-deficient
pRBK720Rmutant exerts a slightly higher repressive
potential on an E2F-responsive reporter gene than wild-
type pRB. Taken together, these data identify SUMO
modification as a novel post-translational modification of
pRB that may control pRB activity by modulating
Oncogene (2005) 24, 3810–3818. doi:10.1038/sj.onc.1208539
Published online 4 April 2005
Keywords: SUMO; pRB; viral oncoproteins; LxCxE
The retinoblastoma-susceptibility gene product pRB is a
prototypical tumor suppressor (Classon and Harlow,
2002; Sherr, 2004). pRB plays a critical role in the
control of cell proliferation and differentiation. The
inhibitory role of pRB on cell-cycle progression is
mainly linked to the repression of E2F-dependent
transcription of genes that are required for entry into
S phase. Binding of pRB masks the transactivation
domain of E2F, thereby directly preventing E2F-
dependent gene activation. Additionally, pRB recruits
chromatin-remodelling factors of the SWI/SNF family
and co-repressor complexes, containing histone deace-
tylases (HDACs) and methyltransferases, to promoters
to silence E2F target genes (Harbour and Dean, 2000).
The pRB-mediated repression of E2F is relieved upon
sequential phosphorylation of pRB by the cyclin-
dependent kinase complexes cyclinD-CDK4/6 and
In addition to its role in cell-cycle control, pRB is
crucial for terminal differentiation and controls adipo-
genic, myogenic and osteogenic differentiation programs.
In these processes pRB appears to function indepen-
dently of E2F-repression, but likely acts as a coactivator
of transcription factors that govern cell differentiation
(Liu et al., 2004).
In neoplastic cells, the pRB pathway is inactivated by
mutation/deletion of pRB or by alterations in upstream
components of the pathway, such as loss of the CDK
inhibitor p16 or overexpression of cyclinD1 or CDK4
(Sherr and McCormick, 2002). Importantly, pRB is also
a direct target of viral oncoproteins, such as the
papillomavirus E7 protein, the adenovirus E1A protein
and the SV40 largeT antigen. Viral oncoproteins and
most cellular interactors of pRB bind to a highly
conserved region termed pocket domain. This domain
is central for pRB function and defines the ‘pocket
protein’ family, which includes the pRB-related mem-
bers p107 and p130. Most of the pRB alterations in
tumors affect the pocket domain. The pocket can be
structurally subdivided in an A- and a B-box. While E2F
binds at the interface of the A- and B-Box (Lee et al.,
2002; Xiao et al., 2003), viral proteins and most
corepressors bind to pocket B (Brehm et al., 1998;
Magnaghi-Jaulin et al., 1998; Nielsen et al., 2001). A
characteristic feature of viral and many cellular proteins
that target pocket B is the presence of a short LxCxE
sequence motif, which binds to a hydrophobic groove in
the B domain (Lee et al., 1998; Kim et al., 2001).
Post-translational modification of lysine residues with
ubiquitin and ubiquitin-like proteins controls the
activity of many cellular key factors. The ubiquitin-like
Received 21 October 2004; revised 15 December 2004; accepted 19
January 2005; published online 4 April 2005
*Correspondence: S Mu ¨ ller; E-mail: firstname.lastname@example.org
Oncogene (2005) 24, 3810–3818
& 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00
SUMO modifier is conjugated to proteins that are
involved in central processes, such as nuclear transport,
transcription, signal transduction, genome stability and
cell-cycle progression (Melchior, 2000; Muller et al.,
2001; Seeler and Dejean, 2003). The currently available
data suggest that SUMO regulates protein–protein
interactions and in some particular cases is implicated
in the regulation of subcellular localization. In humans
at least three SUMO forms are expressed from distinct
genes. Conjugation of SUMO generally involves the
ATP-dependent dimeric SUMO activating E1 enzyme
(AOS1/UBA2) and the E2 conjugating enzyme Ubc9.
At least in some cases, E3-like factors stimulate
conjugation and act as specificity factors in the
modification pathway (Johnson and Gupta, 2001;
Pichler et al., 2002; Kagey et al., 2003; Schmidt and
Transcription factors and transcriptional co-regula-
tors represent a particular large subgroup of cellular
SUMO target proteins indicating that SUMO is critical
in the control of transcriptional processes (Verger et al.,
2003; Muller et al., 2004). This led us to investigate
whether pRB, which predominantly acts as a regulator
of transcription, may undergo modification by SUMO.
We identified SUMOylation as a novel post-transla-
tional modification of pRB, which targets a specific
residue in the lysine cluster of pocket B region and
demonstrate that SUMOylation is controlled by binding
of LxCxE-containing proteins to the pocket region.
pRB is covalently modified by SUMO
To investigate whether pRB is targeted by SUMO, an
HA-tagged version of pRB was expressed together with
either untagged or His-tagged SUMO-1 in the pRB-
negative C33a cell-line. Western blotting performed with
an anti-HA antibody on whole-cell extracts detected the
characteristic doublet band corresponding to the hypo-
and hyperphosphorylated pRB species migrating at
about 110kDa (Figure 1a). Coexpression of SUMO
led to the formation of a slower migrating anti-HA-
reactive pRB species with an apparent molecular weight
of about 130kDa. In extracts from His-SUMO trans-
fected cells, the His-SUMO-pRB form is slightly up-
shifted and could be specifically recovered on Ni-NTA
beads, indicating that it corresponds to a covalent
SUMO-pRB conjugate. Importantly, a SUMO modified
form of endogenous pRB was also specifically enriched
on Ni-NTA beads upon expression of His-SUMO in
U2OS cells, which express wild-type pRB (Figure 1b).
To further validate these findings, we studied SUMO
modification of pRB in a reconstituted in vitro system,
where in vitro translated
with the recombinant SUMOylation machinery, con-
sisting of E1, Ubc9 and SUMO-1. Consistent with the in
vivo data, addition of the assay components efficiently
triggered the formation of a pRB-SUMO conjugate
35S-labelled pRB is incubated
His-SUMO conjugates recovered on Ni2þ-NTA agarose beads were separated by SDS–PAGE and probed with anti-HA antibody. (b)
U2OS cells were transfected as indicated. Lysates and Ni2þ-NTA precipitates were probed with anti-pRB antibody (c) pRB is
SUMOylated in vitro. In vitro translated35S-labelled pRB was incubated either in the absence (?) or presence (þ) of the assay mix
containing recombinant E1 (AOS1/UBA2), Ubc9 and SUMO-1. SUMO modification of p53 served as the positive control. Proteins
were resolved by SDS–PAGE and visualized by autoradiography. Asterisk indicates a translational truncation product. (d) pRB is
SUMOylated in a fully recombinant system. Purified GST-pRB (379-928) was incubated with the assay mix, containing no SUMO or
either SUMO-1 or SUMO-2. Proteins were resolved on SDS–PAGE and stained with Coomassie-blue
pRB undergoes SUMO modification. (a) pRB is SUMOylated in vivo. C33a cells were transfected as indicated. Lysates and
SUMO modification of pRB
A Ledl et al
We thank Jochen Rech for excellent technical assistance,
Alexander Buchberger and Olaf Stemmann for critical
reading of the manuscript and Stefan Jentsch for helpful
discussions and continuous support. Our work is suppor-
tedby the Deutsche Forschungsgemeinschaft
German Israeli Foundation for Development and Scientific
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SUMO modification of pRB
A Ledl et al