N4BP1 is a newly identified nucleolar protein that undergoes SUMO-regulated polyubiquitylation and proteasomal turnover at promyelocytic leukemia nuclear bodies.
ABSTRACT A number of proteins can be conjugated with both ubiquitin and the small ubiquitin-related modifier (SUMO), with crosstalk between these two post-translational modifications serving to regulate protein function and stability. We previously identified N4BP1 as a substrate for monoubiquitylation by the E3 ubiquitin ligase Nedd4. Here, we describe Nedd4-mediated polyubiquitylation and proteasomal degradation of N4BP1. In addition, we show that N4BP1 can be conjugated with SUMO1 and that this abrogates N4BP1 ubiquitylation. Consistent with this, endogenous N4BP1 is stabilized in primary embryonic fibroblasts from mutants of the desumoylating enzyme SENP1, which show increased steady-state sumoylation levels. We have localized endogenous N4BP1 predominantly to the nucleolus in primary cells. However, a small fraction is found at promyelocytic leukemia (PML) nuclear bodies (NBs). In cells deficient for SENP1 or in wild-type cells treated with the proteasome inhibitor MG132, there is considerable accumulation of N4BP1 at PML NBs. These findings suggest a dynamic interaction between subnuclear compartments, and a role for post-translational modification by ubiquitin and SUMO in the regulation of nucleolar protein turnover.
The eukaryotic nucleus is organized into discrete domains that are
physically and functionally distinct (Dundr and Misteli, 2001;
Spector, 2001; Zimber et al., 2004; Handwerger and Gall, 2006;
Zaidi et al., 2007). The nucleolus, the most prominent nuclear
compartment, has a well-established function in the formation of
ribosomes. In addition, the nucleolus is the site of maturation for
a number of different small RNAs, including components of the
spliceosome and signal recognition particle. It might also play a
role in the cell cycle, on the basis of the observed sequestration of
several important cellular regulatory proteins (Pederson, 1998;
Olson et al., 2000; Raska et al., 2006; Boisvert et al., 2007; Sirri
et al., 2008; Pederson and Tsai, 2009). Another much-studied
nuclear compartment is the promyelocytic leukemia (PML) nuclear
body (NB), also known as the PML oncogenic domain (POD) or
nuclear domain 10 (ND10). Although its function is still not
completely understood, proteins crucial for apoptosis, transcription
and cell-cycle regulation, among other cellular functions, localize
to PML NBs, and a number of human diseases are associated with
their loss or alteration (Rego et al., 2001; Strudwick and Borden,
2002; Bernardi and Pandolfi, 2007; Bernardi et al., 2008; Borden,
2008; Torok et al., 2009).
Covalent post-translational modification of PML protein by the
small ubiquitin-related modifier (SUMO), as well as noncovalent
SUMO binding mediated by SUMO interaction motif (SIM) domains,
is crucial for the proper formation of PML NBs and the recruitment
of other sumoylated proteins to these structures (Ishov et al., 1999;
Zhong et al., 2000; Lin et al., 2006; Shen et al., 2006). A number of
studies have shown colocalization of PML NBs with proteasomes
(reviewed in Wojcik and DeMartino, 2003). The ubiquitin proteasome
pathway plays a fundamental role in a number of nuclear processes,
including maintenance of chromatin structure and DNA repair,
control of cell proliferation and programmed cell death (Hershko and
Ciechanover, 1998). It was recently shown that proteasome inhibition
leads to the accumulation of both sumoylated and ubiquitylated
proteins at PML NBs, providing evidence that PML NBs might
function to regulate proteasomal degradation of polyubiquitylated
proteins and suggesting an intimate connection with the SUMO
pathway in this function (Bailey and O’Hare, 2005). It has also been
noted that proteasome inhibition can lead to PML NB component
proteins being found in the nucleolus (Mattsson et al., 2001; Matafora
et al., 2009). Other recent findings have illustrated a dynamic
relationship between the nucleolus and PML NBs (Condemine et al.,
2007; Janderova-Rossmeislova et al., 2007; Rokaeus et al., 2007).
We originally identified N4BP1 in a yeast two-hybrid screen for
developmentally expressed proteins that interact with the WW and
HECT domains of the E3 ubiquitin ligase Nedd4, as part of an effort
to discover targets of ubiquitin-mediated protein degradation that
might play key roles in normal development (Murillas et al., 2002).
We showed that transiently transfected N4BP1 localizes to discrete
subnuclear domains and is a substrate for Nedd4-mediated
monoubiquitylation. Recent work has shown that N4BP1 also
interacts with the related E3 ligase ITCH, but is not a substrate for
ITCH-mediated ubiquitylation (Oberst et al., 2007). Rather, N4BP1
N4BP1 is a newly identified nucleolar protein that
undergoes SUMO-regulated polyubiquitylation and
proteasomal turnover at promyelocytic leukemia
Prashant Sharma1, Rodolfo Murillas2, Huafeng Zhang1,* and Michael R. Kuehn1,‡
1Laboratory of Protein Dynamics and Signaling, Center for Cancer Research, National Cancer Institute, NCI-Frederick, Frederick, MD 21702, USA
2Epithelial Biomedicine Division, Centro de Investigaciones Energeticas, Medioambientales y Tecnologicas-CIBERER U714, Madrid, Spain
*Present address: Institute for Cell Engineering, Department of Genetic Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
‡Author for correspondence (email@example.com)
Accepted 19 January 2010
Journal of Cell Science 123, 1227-1234
© 2010. Published by The Company of Biologists Ltd
A number of proteins can be conjugated with both ubiquitin and the small ubiquitin-related modifier (SUMO), with crosstalk between
these two post-translational modifications serving to regulate protein function and stability. We previously identified N4BP1 as a substrate
for monoubiquitylation by the E3 ubiquitin ligase Nedd4. Here, we describe Nedd4-mediated polyubiquitylation and proteasomal
degradation of N4BP1. In addition, we show that N4BP1 can be conjugated with SUMO1 and that this abrogates N4BP1 ubiquitylation.
Consistent with this, endogenous N4BP1 is stabilized in primary embryonic fibroblasts from mutants of the desumoylating enzyme
SENP1, which show increased steady-state sumoylation levels. We have localized endogenous N4BP1 predominantly to the nucleolus
in primary cells. However, a small fraction is found at promyelocytic leukemia (PML) nuclear bodies (NBs). In cells deficient for
SENP1 or in wild-type cells treated with the proteasome inhibitor MG132, there is considerable accumulation of N4BP1 at PML NBs.
These findings suggest a dynamic interaction between subnuclear compartments, and a role for post-translational modification by
ubiquitin and SUMO in the regulation of nucleolar protein turnover.
Key words: N4BP1, PML nuclear bodies, SUMO, Nucleolus, Ubiquitin
Journal of Cell Science
JCS ePress online publication date 16 March 2010
binding to ITCH negatively regulates ITCH E3 activity directed
toward its substrates, which include the p53-related tumor-
suppressor proteins p73 and p63, as well as c-Jun. Whether this is
the primary function of N4BP1 is not known, but a recent report
predicted that N4BP1 could have ribonuclease activity, on the basis
of a newly identified protein fold termed the N4BP1, YacP-like
nuclease (NYN) domain, which is shared with previously
characterized nucleases (Anantharaman and Aravind, 2006). Indeed,
two other NYN-domain-containing proteins, ZC3H12A and
KIAA0391, have been shown to have ribonuclease activity
(Holzmann et al., 2008; Matsushita et al., 2009).
Here we localize endogenous N4BP1 to the nucleolus in primary
cells, suggesting a role in nucleolar RNA processing. We also find
that N4BP1 can undergo polyubiquitylation and proteasome-
dependent degradation. Several lines of evidence point to N4BP1
turnover not in the nucleolus, but in PML NBs, with this process
being regulated by N4BP1 sumoylation. Together our findings
provide further support for SUMO and ubiquitin post-translational
modification pathways localized specifically at PML NBs regulating
nuclear protein levels and extend this paradigm to nucleolar proteins.
Endogenous N4BP1 localizes to the nucleolus in mouse
We showed previously that N4BP1 transfected into HEK293 cells
localizes to the nucleus within discrete circular domains (Murillas
et al., 2002). To determine whether N4BP1 colocalizes with any of
the well-characterized nuclear bodies, we co-stained with antisera
against PML, coilin, SC35 and PSP1, which are signature proteins
of PML NBs, Cajal bodies, GEMs and paraspeckles, respectively
(Handwerger and Gall, 2006; Spector, 2006). This analysis revealed
colocalization only with PML (supplementary material Fig. S1A-
C), indicating that overexpressed N4BP1 accumulates at PML NBs.
A number of proteins have been found to localize to PML NBs only
when expressed above physiological levels (Borden, 2002). To
determine the biological significance of the presence of N4BP1 at
PML NBs, we asked whether the endogenous protein also localizes
To investigate endogenous N4BP1 localization, we carried out
indirect immunofluorescence using a rabbit polyclonal anti-N4BP1
antiserum raised against the mouse protein. We were unable to detect
endogenous N4BP1 in HEK293 cells (supplementary material Fig.
S2A). However, examination of primary mouse embryonic
fibroblasts (MEFs) revealed strong anti-N4BP1 immunostaining in
multiple subnuclear domains (Fig. 1A). Some cytoplasmic staining
was seen, but this was also found in MEFs derived from N4BP1-
knockout mice, indicating non-specific reactivity (supplementary
material Fig. S3). The nuclear anti-N4BP1 staining had a distinct
clumpy and irregular appearance that was variable in size and
number from cell to cell, suggestive of the nucleolus rather than
PML NBs. Indeed, we found extensive overlap with upstream
binding factor (UBF), which localizes to the fibrillar center of the
Journal of Cell Science 123 (8)
Fig. 1. N4BP1 localizes predominantly to the nucleolus. MEFs
were examined by indirect immunofluorescence using anti-N4BP1
(A,C,D,F,G,I,J,L,M,O), anti-UBF (B,C), anti-fibrillarin (E,F,M-O),
anti-B23 (H,I) and anti-PML (K,L), as labeled in each panel. In
A,D,G,J, DAPI staining of nuclei is shown. The anti-N4BP1
cytoplasmic staining is non-specific (see supplementary material
Fig.S3). N4BP1 is found predominantly in discrete subnuclear
domains of varying size that colocalize extensively with UBF (A-C),
less so with fibrillarin (D-F) and to only a limited extent with B23
(G-I). In a fraction of cells, N4BP1 domains that do not colocalize
with nucleolar markers are seen (arrows). A similar fraction of cells
show N4BP1 colocalization with PML (J-L; arrow). (M-O)PML-
mutant MEFs reconstituted with GFP-PML fusion. In (M), a merge
of anti-N4BP1 (red) and anti-fibrillarin (green) is shown. In (N), a
merge of anti-fibrillarin (green) and GFP-PML (blue) is shown. In
(O), a triple merge of anti-N4BP1 (red), anti-fibrillarin (green) and
GFP-PML (blue) is shown. Non-nucleolar N4BP1 (arrows)
colocalizes with GFP-PML (magenta).
Journal of Cell Science
Nucleolar N4BP1 turnover at PML nuclear bodies
nucleolus (Fig. 1B,C). Analysis using antibodies against fibrillarin,
which localizes to the dense fibrillar component of the nucleolus,
also showed overlap with N4BP1 (Fig. 1D-F). B23 (also known as
nucleophosmin), found in the granular component of the nucleolus,
showed only limited overlap at the edges of the N4BP1-containing
domains (Fig. 1G-I). Similar results were found with other mouse
primary cells, including tail-tip fibroblasts, trophoblast stem cells
and giant cells. These results point to endogenous N4BP1 being a
nucleolar protein predominantly resident in the fibrillar regions.
We also detected small discrete sites of nuclear anti-N4BP1
staining that did not overlap with nucleolar markers (Fig. 1, white
arrows). These had the size and appearance of PML NBs, but were
found infrequently, on average less than one per cell (Table 1),
whereas there are multiple PML NBs per cell. However, analysis
with anti-PML revealed co-localization of N4BP1 with PML NBs
at a similar low frequency, with on average less than one PML NB
per cell showing co-staining with N4BP1 (Fig. 1J-L, arrow). This
suggested that the small fraction of N4BP1 that is non-nucleolar is
present at a subset of PML NBs. To confirm this, we examined
PML, fibrillarin and N4BP1 in the same cell. We derived MEFs
from PML mutant mice and stably expressed a GFP-PML fusion
protein. These cells were then co-immunostained for N4BP1 and
fibrillarin. This allowed us to determine that non-nucleolar N4BP1
always overlapped with GFP-PML (Fig. 1M-O). Thus, although
the majority of N4BP1 is nucleolar, at least in MEFs, there is a
small amount of endogenous protein present at PML NBs. This
suggests that the localization of transfected N4BP1 at this site might
be physiologically relevant.
Proteasome inhibition leads to increased N4BP1
localization to PML NBs
The inability to detect endogenous N4BP1 in HEK293 cells
(supplementary material Fig. S2A) could be due to either low
expression levels or limited crossreactivity with the human protein.
As an approach to increase endogenous N4BP1 to detectable levels,
we treated HEK293 cells with the proteasome inhibitor MG132.
This treatment resulted in the appearance of nuclear anti-N4BP1
immunostaining (supplementary material Fig. S2D,G). Interestingly,
endogenous N4BP1 did not accumulate in the nucleolus of MG132-
treated HEK293 cells (supplementary material Fig. S2E,F), but
rather in PML NBs (supplementary material Fig. S2H,I). This
suggests that N4BP1 localizes to PML NBs normally for
proteasomal turnover. The accumulation of overexpressed
exogenous N4BP1 at PML NBs could reflect the same phenomenon.
To determine whether the same mechanism is at work in primary
cells, in which N4BP1 is predominantly found at the nucleolus, we
carried out immunofluorescence on MEFs treated with MG132. We
found a significant increase in non-nucleolar N4BP1-containing
nuclear bodies (Fig. 2A-C), the frequency of which correlated
perfectly with the increase in PML NBs that co-stained with anti-
N4BP1 (Fig. 2D-F). There was an approximately tenfold increase
from on average 0.6 per wild-type MEF cell to 6 per MG132-treated
cell (Table 1). Thus, similar to HEK293 cells, MG132 treatment
leads to accumulation of N4BP1 at PML NBs. We repeated the
above experiment using PML-mutant MEFs, which lack PML NBs.
Although there were non-nucleolar N4BP1-containing nuclear
bodies in these cells (not shown), the frequency was significantly
lower than in wild- type MEFs and, upon MG132 treatment, there
was only a small increase (Table 1). This suggests that N4BP1 can
re-localize outside the nucleolus for turnover in the absence of PML,
but this is very inefficient compared to the process occurring at
PML NBs. In PML-mutant MEFs reconstituted with GFP-PML,
we found the usual level of non-nucleolar N4BP1 (Table 1), even
though there appeared to be a higher number of PML NBs than in
wild-type MEFs. Upon MG132 treatment, there was again an
approximately tenfold increase in non-nucleolar N4BP1 co-
localizing with PML NBs.
The above results are consistent with N4BP1 turnover being
mediated by the proteasome and occurring predominantly at PML
NBs, which are tightly associated with the nuclear insoluble fraction.
To gain additional insight into N4BP1 turnover with respect to cell
compartment, we examined N4BP1 levels in the detergent-soluble
and -insoluble fractions of untreated MEFs and following proteasomal
inhibition. Either whole-cell extracts or separate Triton X-100 soluble
Table 1. Number of non-nucleolar N4BP1-containing nuclear
bodies per cell
Mean ± s.e.m.
*P<0.0001 compared to untreated or MG132-treated wild-type MEFs. The
difference between wild-type MEFs and PML-mutant MEFs reconstituted
with GFP-PML is not statistically significant. †P<0.0001 compared to
littermate wild-type MEFs.
Fig. 2. Proteasome inhibition leads to accumulation of N4BP1 at PML
NBs. (A-F)MEFs treated with MG132 were examined by indirect
immunofluorescence using anti-N4BP1 (A,C,D,F), anti-fibrillarin (B,C) and
anti-PML (E,F), as labeled in each panel. (A-C)A significant increase in
N4BP1 outside the nucleolus was found (arrows point to non-nucleolar
N4BP1). (D-F)There is a comparable increase in the colocalization of N4BP1
with PML (arrows point to colocalized N4BP1 and PML). (G)Total protein
extracts and detergent-soluble and -insoluble fractions were prepared from
either untreated or MG132-treated MEFs, followed by immunoblotting (IB)
with anti-N4BP1 to detect N4BP1 levels. MG132 treatment leads to
accumulation in the detergent-insoluble fraction. Lower panels show reblotting
with anti-b-actin to confirm equivalent sample loading.
Journal of Cell Science
and insoluble fractions were prepared. In total protein extracts and
in the detergent-soluble fraction, there was no obvious change upon
MG132 treatment (Fig. 2G, lanes 1-4). However, there was significant
accumulation of N4BP1 in the detergent-insoluble fraction of MG132-
treated MEFs (Fig. 2G, lanes 5 and 6). These biochemical experiments
show that the majority of endogenous N4BP1 exists as a stable protein
and suggest that only a small fraction undergoes proteasomal turnover.
Turnover is restricted to the detergent-insoluble fraction, which
includes the nuclear-matrix-bound PML NBs.
N4BP1 can be polyubiquitylated
Proteasomal degradation is almost invariably associated with
polyubiquitylation of the target protein. However, we previously
reported that N4BP1 transfected into HEK293 cells only undergoes
monoubiquitylation mediated by the Nedd4 E3 ubiquitin ligase
(Murillas et al., 2002), which is not a signal for proteasomal
degradation. One possible explanation for our failure to detect
polyubiquitylated N4BP1 is that we had examined only the non-
ionic detergent-soluble fraction; it is clear from the above results
for endogenous N4BP1 that turnover is restricted to the detergent-
insoluble fraction. Therefore, we reinvestigated N4BP1
polyubiquitylation by examining total protein extracts from HEK293
cells transfected with N4BP1 and Myc-epitope-tagged ubiquitin,
with or without hemagglutinin (HA)-tagged Nedd4. Cells were lysed
in buffer containing 3% SDS followed by a tenfold dilution into
0.5% Triton X-100 buffer. This enabled complete extraction of
proteins, but still allowed immunoprecipitation using anti-N4BP1
antiserum. Ubiquitylation was then evaluated by immunoblotting
with anti-Myc. Under these conditions we detected not only a strong
immunoreactive band of 120 kDa, representing monoubiquitylated
N4BP1, but also a high molecular mass smear (Fig. 3, lane 2),
indicating that N4BP1 can indeed undergo polyubiquitylation.
Coexpression with wild-type Nedd4 stimulated a marked increase
in N4BP1 polyubiquitylation levels (Fig. 3, lane 3), indicating
Nedd4 may mediate both mono- and poly-ubiquitylation of N4BP1.
N4BP1 undergoes SUMO1 modification and is
desumoylated by SENP1
The above results suggest that N4BP1 localizes to PML NBs to
undergo ubiquitin-mediated proteasomal degradation. Many of the
proteins localizing to PML NBs, including PML, are sumoylated
(reviewed in Bernardi and Pandolfi, 2007). To determine whether
endogenous N4BP1 is also a SUMO substrate, we first carried out
N4BP1 immunoprecipitation from MEFs, followed by anti-SUMO1
or anti-SUMO2 and 3 immunoblotting, but we were unable to detect
any sumoylated forms of the protein. It is possible that the anti-
N4BP1 antibody does not efficiently immunoprecipitate SUMO-
modified N4BP1. As an alternative approach to provide evidence
of sumoylation, we took advantage of our previous finding that
MEFs derived from embryos deficient for the desumoylating
enzyme SENP1 accumulate higher levels of SUMO1-conjugated
proteins (Yamaguchi et al., 2005). We carried out direct anti-N4BP1
immunoblotting on total protein extracts from wild-type and
SENP1-mutant MEFs, and looked for higher molecular mass
species accumulating in the absence of the desumoylating enzyme.
Indeed, we were able to detect an approximately 130 kDa form of
N4BP1 that was present at a significantly higher level in whole-
cell extracts from SENP1-mutant MEFs compared with the wild
type (Fig. 4A, compare lanes 1 and 2). This suggests not only that
endogenous N4BP1 can be sumoylated, but also that the sumoylated
form is normally a substrate for SENP1.
To confirm N4BP1 sumoylation, we carried out overexpression
studies in HEK293 cells. We transfected expression vectors for
N4BP1 and polyoma (Pyo)-epitope-tagged SUMO1, and
immunoprecipitated Pyo–SUMO1-conjugated proteins using anti-
Pyo antibody followed by immunoblotting with anti-N4BP1. This
analysis revealed two higher molecular mass species of
approximately 130 kDa and 150 kDa (Fig. 4B, lane 2, upper panel),
consistent with N4BP1 having two SUMO conjugation sites.
Recently, an active site cysteine to serine mutant form of human
SENP1 was shown to block endogenous desumoylation, resulting
in the accumulation of high molecular mass SUMO1 conjugates
(Bailey and O’Hare, 2004). We generated a similar mutant of the
mouse enzyme, SENP1 C599S, and found that it also behaves as
a dominant negative. Coexpression of SENP1 C599S with N4BP1
led to enhanced sumoylation levels, providing additional evidence
that N4BP1 is normally a substrate for SENP1. The higher molecular
mass forms of N4BP1 were more easily detected in the
immunoprecipitation and were found even in whole-cell extracts
(Fig. 4B, top and middle panels, lane 3). This analysis also revealed
a third higher molecular mass species of approximately 170 kDa
(Fig. 4B, upper panel, lane 3). Thus, N4BP1 might well have three
potential SUMO1 conjugation sites. Inspection of the N4BP1 amino
acid sequence revealed three lysine residues (K157, K183 and K607)
lying within the KXE sumoylation consensus motif (Rodriguez
et al., 2001; Sampson et al., 2001). However, lysine to arginine
mutations for each, either singly or in combination, failed to abrogate
N4BP1 sumoylation (not shown). Either these three lysines do not
serve as SUMO1 acceptor sites or they are used interchangeably
for conjugation with other non-consensus sites, as has been reported
for Daxx (Lin et al., 2006).
SENP1 regulates endogenous N4BP1 stability
Detection of endogenous sumoylated N4BP1 in SENP1-mutant
MEFs required immunoblotting of a minimum of 100 g of total
protein extract. When we analyzed lower amounts (10-20 g), we
could not detect the higher molecular mass form. However,
immunoblotting lower amounts of protein did reveal that the
Journal of Cell Science 123 (8)
Fig. 3. N4BP1 can undergo polyubiquitylation. N4BP1, Myc-tagged
ubiquitin (Ub) and HA-tagged Nedd4 were overexpressed in HEK293 cells.
Immunoprecipitations (IP) were performed with anti-N4BP1, followed by
immunoblotting (IB) either with anti-Myc (upper panel) to detect ubiquitylated
forms of N4BP1 (arrow indicates position of monoubiquitylated N4BP1) or
with anti-N4BP1. Lower panels show immunoblotting of whole-cell extracts
(WCE) with anti-Myc (to show overall polyubiquitylation levels), anti-N4BP1,
anti-HA (to detect Nedd4 levels) and anti-b-actin.
Journal of Cell Science
Nucleolar N4BP1 turnover at PML nuclear bodies
steady-state level of N4BP1 protein in SENP1-mutant MEFs was
significantly higher than in wild-type cells (Fig. 4A, compare lanes
3 and 4). This was seen consistently over a number of experiments
and in independently derived SENP1-mutant MEFs. Further analysis
revealed no difference in N4BP1 mRNA levels between wild-type
and SENP1-mutant cells (not shown), suggesting that decreased
turnover is responsible for the increased protein levels. Sumoylation
has been shown to stabilize protein levels by competing with
ubiquitylation (reviewed in Ulrich, 2005). Thus, the increase in
sumoylation of N4BP1 in SENP1-mutant cells might cause a
compensatory decrease in ubiquitylation and proteasomal turnover.
The increased level of N4BP1 in SENP1-mutant MEFs was
accompanied by a noticeable increase in the number of N4BP1-
containing NBs outside of the nucleolus, on the basis of
immunofluorescence with anti-fibrillarin and anti-N4BP1 (Fig. 5A-
C). There were, on average, two such bodies in every SENP1-mutant
MEF cell examined, compared with approximately one in every
other MEF cell derived from wild-type littermates (Table 1). Given
our above findings indicating that non-nucleolar N4BP1 localizes
to PML NBs in wild-type MEFs, we asked whether there was
increased colocalization of N4BP1 with PML in SENP1-mutant
MEFs. Indeed, we found that every SENP1-mutant MEF had, on
average, two PML NBs that colocalized with N4BP1 (Fig. 5D-F).
These numbers are consistent with the increased non-nucleolar
N4BP1 staining in SENP1-mutant MEFs representing increased
localization to PML NBs. Accumulation at PML NBs might either
be due to increased steady-state sumoylation levels driving
additional N4BP1 to the PML NBs or indicate that N4BP1
degradation at PML NBs is blocked in the absence of desumoylation,
SUMO modification negatively regulates N4BP1
To address our hypothesis that sumoylation of N4BP1 competes
with its ubiquitylation, we examined N4BP1 polyubiquitylation in
the presence of overexpressed components of the sumoylation
pathway. Expression vectors for Pyo-SUMO1 and Ubc9 were
cotransfected along with HA-tagged Nedd4 and Myc-tagged
ubiquitin. Under these conditions, there was a significant decrease
in the level of N4BP1 polyubiquitylation (Fig. 6, compare lanes 1
and 2). This decrease was not due to any general reduction in overall
ubiquitylation levels in transfected cells (Fig. 6, WCE, anti-Myc
panel). By contrast, immunoprecipitation with anti-Pyo showed that
sumoylation levels of N4BP1 were similar in the presence or
absence of overexpressed components of the ubiquitylation
machinery (Fig. 6, compare lanes 2 and 3). Thus, whereas N4BP1
sumoylation clearly can negatively
polyubiquitylation, the converse does not appear to be true, at least
under these conditions. These results are consistent with sumoylated
N4BP1 not being a substrate for polyubiquitylation and suggest that
Fig. 4. N4BP1 is SUMO1 conjugated and desumoylated by SENP1.
(A)Immunoblot (IB) analysis of whole-cell extracts (WCE) of wild-type (WT,
lanes 1 and 3) and SENP1-mutant (SENP1–/–, lanes 2 and 4) MEFs with anti-
N4BP1 to detect endogenous N4BP1. Filters were reblotted with anti-b-actin
to ensure equal loading. Arrow indicates position of endogenous N4BP1.
Arrowhead indicates position of a higher molecular mass species (obvious in
lane 2), presumed to be sumoylated N4BP1, seen in long exposures of
immunoblots with at least 100g total protein (lanes 1 and 2). Immunoblotting
of less total protein (10-20g) revealed significantly greater N4BP1 levels in
SENP1-mutant cells (lanes 3 and 4). (B)N4BP1 was overexpressed in
HEK293 cells with or without Pyo-SUMO1, and with or without SENP1
C599S active-site mutant. Immunoprecipitation (IP) was performed with anti-
Pyo to pull down Pyo–SUMO1-conjugated proteins, followed by
immunoblotting (IB) with anti-N4BP1 to detect sumoylated N4BP1 isoforms
(upper panel). The arrows indicate three N4BP1 isoforms with sizes consistent
with conjugation of one, two and three Pyo-SUMO1 moieties, the levels of
which were enhanced in the presence of the SENP1 active-site mutant (lane 3).
The middle panel shows immunoblotting of whole-cell extracts (WCE) with
anti-N4BP1, revealing sumoylated N4BP1 in the presence of SENP1 C599S
(lane 3, arrows). The lower panel shows immunoblotting of whole-cell extracts
with anti-Pyo to detect high molecular mass (HMM) Pyo-SUMO1 conjugates.
The open arrowhead points to the position of SUMO1-conjugated RanGAP1.
Fig. 5. N4BP1 accumulates at PML NBs in SENP1-mutant MEFs. SENP1-
mutant MEFs were examined by indirect immunofluorescence using anti-
N4BP1 (A,C,D,F), anti-fibrillarin (B,C) and anti-PML (E,F), as labeled in each
panel. (A-C)There is an increase in the level of N4BP1 outside of the
nucleolus compared to wild-type MEFs (arrows point to non-nucleolar
N4BP1). (D-F)There is a comparable increase in the colocalization of N4BP1
with PML (arrows point to co-localized N4BP1 and PML).
Journal of Cell Science
desumoylation of N4BP1 is an obligate step for its ubiquitin-
mediated proteasomal turnover.
We originally identified N4BP1 as a developmentally expressed
target of the HECT domain ubiquitin ligase Nedd4 and previously
showed, in overexpression studies, that it can undergo Nedd4-
mediated monoubiquitylation (Murillas et al., 2002). Here, we have
found that N4BP1 can be polyubiquitylated and SUMO conjugated
as well. Whereas overexpressed N4BP1 localizes almost exclusively
to PML NBs in HEK293 cells, the endogenous protein in MEFs
and other primary mouse cells localizes predominantly to the
nucleolus. N4BP1 was recently shown to share a domain with
known RNases (Anantharaman and Aravind, 2006). Localization
in the nucleolus suggests a potential role in nucleolar RNA
processing. An important future avenue of research will be to
confirm RNase activity and identify substrates.
Although the majority of endogenous N4BP1 is nucleolar, a small
fraction localizes to PML NBs. Several lines of evidence point to
this fraction specifically undergoing polyubiquitylation and
proteasomal degradation, with sumoylation playing a role in the
regulation of this process. It was recently shown that proteasome
inhibition leads to the accumulation of both sumoylated and
ubiquitylated species at discrete foci in the nucleus, in proximity
to PML NBs. Thus, one function of PML NBs might be to regulate
proteasomal degradation of polyubiquitylated proteins, in concert
with the SUMO pathway (Bailey and O’Hare, 2005). A number of
other studies have shown that PML NBs partially colocalize with
proteasomes (reviewed in Wojcik and DeMartino, 2003), including
the 20S proteolytic core complex (Lafarga et al., 2002; Rockel and
von Mikecz, 2002), the 19S regulatory complex (Lafarga et al.,
2002) and PA28 (the 11S regulatory complex) of the
immunoproteasome (Fabunmi et al., 2001; Lallemand-Breitenbach
et al., 2001), and might serve as sites of proteolytic degradation for
cellular and viral proteins (Anton et al., 1999; Rockel et al., 2005).
For N4BP1, proteasome inhibition results in a significant increase
in colocalization with PML NBs, suggesting that this is where
turnover normally occurs.
It has been known for some time that proteasomes are not found
in nucleoli (Wojcik and DeMartino, 2003). Thus, proteasomal
turnover of a nucleolar protein requires redistribution to sites outside
of nucleoli, as has been shown for fibrillarin (Chen et al., 2002).
Although the non-nucleolar sites where fibrillarin undergoes
degradation were not examined for colocalization with PML, a
similar process to that demonstrated here for N4BP1 might be
occurring. Recent findings on the relationship between PML NBs
and the nucleolus (Condemine et al., 2007; Janderova-Rossmeislova
et al., 2007; Rokaeus et al., 2007) might also be relevant to N4BP1
turnover in MEFs. These reports document a dynamic association
between PML and nucleoli, providing a direct conduit for N4BP1
relocalization to PML NBs from the nucleolus. This might also be
facilitated or stabilized by sumoylation of N4BP1, similar to other
proteins that transiently associate with PML NBs (Weidtkamp-Peters
et al., 2008). The recent findings on the intimate relationship of
PML and nucleoli have also documented a tight physical linkage
with the ubiquitin proteasome pathway, adding to previous work
documenting proximity of the proteasomal machinery and PML
Journal of Cell Science 123 (8)
Fig. 6. N4BP1 ubiquitylation is inhibited by sumoylation. N4BP1 was
overexpressed in HEK293 cells with Myc-tagged ubiquitin (Ub) and HA-
tagged Nedd4 (lane 1), or additionally with Pyo-SUMO1 and Ubc9 (lane 2), or
with Pyo-SUMO1 and Ubc9 only (lane 3). Immunoprecipitations (IP) were
performed with anti-N4BP1, followed by immunoblotting (IB) with anti-Myc
(upper panel) to detect ubiquitylated N4BP1 species. There is a significant
reduction in ubiquitylated N4BP1 in the presence of SUMO1 and Ubc9.
Immunoblotting of whole-cell extracts (WCE) with anti-Myc (third panel
down) shows global ubiquitylation is not affected. Immunoprecipitations
performed with anti-Pyo, followed by immunoblotting with anti-N4BP1
(second panel down), show no change in sumoylated N4BP1 levels in the
presence or absence of ubiquitylation machinery. Lower panels show
immunoblotting of whole-cell extracts with anti-N4BP1, anti-HA (to detect
Nedd4 levels), anti-Ubc9 and anti-b-actin.
Fig. 7. Model of N4BP1 post-translational modification and turnover.
N4BP1 is predominantly resident in the nucleolus. Sumoylation might
promote association with PML NBs (i), where N4BP1 might undergo
desumoylation by SENP1. It will then either traffic back to the nucleolus (ii)
or be retained (iii) and undergo polyubiquitylation (iv) and degradation at
proteasomes physically associated with PML NBs (v). The accumulation of
N4BP1 at PML NBs in SENP1-mutant MEFs might be due to reduced
relocalization to the nucleolus or because polyubiquitylation is blocked, or
both. Treatment with MG132 leads to N4BP1 accumulation as a result of the
blocking of degradation at PML-NB-associated proteasomes.
Journal of Cell Science
Nucleolar N4BP1 turnover at PML nuclear bodies
SENP1-mutant cells, which have a significantly higher steady-
state level of N4BP1, also show a marked increase in the number
of PML NBs containing N4BP1. This result provided the first
indication that sumoylation plays a regulatory role in N4BP1
turnover. We subsequently demonstrated using transfected N4BP1
that sumoylation can occur at three sites and that this modification
dramatically decreases the potential for polyubiquitylation. The
marked inhibitory effect of sumoylation might be due to conjugation
to the same lysine residue(s) used for polyubiquitylation, a
competition mechanism first proposed for IkBa (Desterro et al.,
1998). Ubiquitylation and sumoylation have opposing effects on
the stability of other proteins, but apparently through mechanisms
that are more complex than simply blocking the acceptor lysine,
such as inducing localization or conformational changes that
prevent the protein from undergoing the other modification
(reviewed in Ulrich, 2005). We favor a model derived from a recent
proposal that sumoylated proteins dynamically traffic through PML
NBs and must be desumoylated to undergo proteasomal degradation
in a process tightly coordinated with the ubiquitylation pathway
(Bailey and O’Hare, 2005). This is schematically represented in
Fig. 7. Sumoylation might allow stable association of N4BP1 with
PML NBs (Fig. 7, i). SENP1-mediated desumoylation might lead
to trafficking back to the nucleolus (Fig. 7, ii). Alternatively,
desumoylated N4BP1 might remain associated with the PML NB
(Fig. 7, iii), as is the case for the PML protein itself (Nefkens et
al., 2003). This could explain why only very low levels of
endogenous sumoylated N4BP1 can be detected biochemically,
whereas the protein is easily seen in PML NBs by
immunofluorescence. Desumoylation catalyzed by SENP1 might
be a prerequisite, and perhaps a signal, for N4BP1 to undergo
polyubiquitylation (Fig. 7, iv), followed by degradation by PML-
NB-associated proteasomes (Fig. 7, v). Polyubiquitylation is
possibly mediated by Nedd4, which has been shown to function in
the nucleus (Hamilton et al., 2001). In the absence of desumoylation,
as in the SENP1 mutant, N4BP1 cannot be polyubiquitylated or
degraded and thus accumulates at PML NBs. In the absence of
proteasomal degradation, as in MG132-treated cells, N4BP1 also
accumulates at PML NBs. Thus, the apparent antagonism between
sumoylation and ubiquitylation might reflect tightly coordinated
sequential action to first direct N4BP1 to the NB, where subsequent
desumoylation and ubiquitylation can occur. A similar dynamic for
the bulk of nuclear sumoylated proteins has been proposed (Bailey
and O’Hare, 2005).
In conclusion, the detailed characterization of N4BP1 presented
here indicates that localization to PML NBs and turnover are tightly
linked and regulated by the dynamic interplay of post-translational
modification by both ubiquitin and SUMO. Our findings provide
additional support for PML NBs serving as specialized sites for
integrating these two post-translational modifications with
proteasomal degradation. N4BP1 is the first nucleolar protein
identified whose regulation fits this newly emerging function for
Materials and Methods
Reagents and antibodies
Stock solution of MG132 (carbobenzoxy-L-leucyl-L-leucyl-L-leucinal; Sigma) was
prepared in DMSO and applied to cells at 50 M final concentration for 5 hours. An
equivalent concentration of DMSO (0.1%) was used for control cells. Primary
antibodies used were: anti-b-actin (clone AC-15; Sigma), anti-Ubc9 (clone 50; BD),
anti-HA (clone 3F10; Roche), anti-GMP-1 (anti-SUMO1; clone 21C7; Zymed), anti-
Myc (clone 9E10; American Tissue Culture Collection), anti-PML (clone 36.1-104;
Millipore), anti-UBF (clone F-9; Santa Cruz Biotechnology) and anti-B23 (Invitrogen).
Rabbit polyclonal anti-N4BP1 generated in our laboratory was described previously
(Murillas et al., 2002). Rabbit polyclonal anti-SUMO1, mouse monoclonal antibody
against the polyoma epitope (anti-Pyo) and mouse monoclonal anti-fibrillarin clone
72B9 were gifts from Mary Dasso (NICHD, Bethesda, MD), Deborah Morrison (NCI,
Frederick, MD) and Patrick DiMario (Louisiana State University, LA), respectively.
Expression vectors and in vitro mutagenesis
The mouse N4BP1 cDNA has been described previously (Murillas et al., 2002). The
HA-epitope-tagged Nedd4 and N-terminal Myc-epitope-tagged ubiquitin expression
constructs were described previously (Magnifico et al., 2003). The expression vector
for mouse Ubc9 was provided by Mary Dasso. Double polyoma (Pyo)-epitope-tagged
SUMO1 and SENP1 constructs were generated in pcDNA3.1 by PCR (details
available upon request). Amino acid changes in SENP1 and N4BP1 were made using
the QuikChange site-directed mutagenesis kit (Stratagene). All constructs were
confirmed by DNA sequencing.
Cell culture and transfection
HEK293 cells and MEFs were maintained in DMEM medium (Invitrogen)
supplemented with 10% heat-inactivated FBS and antibiotics. MEFs were maintained
in 3% O2(Parrinello et al., 2003). All experiments were performed using exponentially
growing cells and repeated at least twice. Transfections were performed with
Lipofectamine Plus reagent (Invitrogen) according to the manufacturer’s protocol.
Cells were harvested from 24 to 48 hours after transfection. PML-mutant MEFs were
infected with pBabe-GFP-PML virus-containing supernatants generated by transient
transfection of Phoenix-Ampho packaging cells. After 24 hours, infected cultures
were subjected to selection in the presence of 2 g/ml puromycin.
Whole-cell lysates were prepared by suspending cells in 62.5 mM Tris-HCl (pH 6.8)
and then adding an equal amount of 2?SDS sample buffer (10% glycerol, 2% 2-
mercaptoethanol, 6% SDS, 62.5 mM Tris-HCl pH 6.8) and boiling for 5 minutes, as
described (Ogiso et al., 2000). For preparation of non-ionic detergent-soluble and -
insoluble fractions, cells were suspended in ice-cold Triton X-100 lysis buffer
[150 mM NaCl, 50 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.5% Triton X-100, 1 mM
phenylmethylsulphonylfluoride (PMSF), 10 mM iodoacetamide, protease inhibitor
mixture (Roche)] and kept on ice for 45 minutes with occasional mixing, followed
by centrifugation. The supernatant represented the detergent-soluble fraction. The
pellet, representing the detergent-insoluble fraction, was resuspended in 62.5 mM
Tris-HCl (pH 6.8) with an equal volume of 2?SDS sample buffer added, followed
by sonication and boiling for 5 minutes. Protein concentrations were determined using
a modified Bradford assay (Bio-Rad). For immunoblot analysis, equal amounts of
protein (depending on experiment from 10 to 100 g) were resolved on SDS-
polyacrylamide gels, electroblotted onto nitrocellulose membrane (Novex), and probed
with specific primary antibodies and appropriate secondary antibodies.
Chemiluminescent detection was performed using SuperSignal West Pico
chemiluminescent substrate (Pierce).
For in vivo ubiquitylation and sumoylation experiments, cell lysates were prepared
using 2?SDS sample buffer as above. After sonication and boiling for 5 minutes,
equal amounts of protein were diluted ten times with the above-described Triton X-
100 lysis buffer in the presence of protease inhibitor mixture (Roche), precleared
with protein G agarose and immunoprecipitated with the indicated antibodies. Triton
X-100 lysis buffer was used for co-immunoprecipitation. Antibodies used for
immunoprecipitation were anti-N4BP1 (1:200 dilution) and anti-Pyo (1:10 dilution).
After incubation at 4°C overnight, immunoprecipitates were washed five times with
1 ml of the Triton X-100 lysis buffer and subsequently resuspended in 2 ? SDS
sample buffer. After boiling for 5 minutes, the complexes were evaluated by
Immunofluorescence analysis of MEFs was done as described previously (Evdokimov
et al., 2008). Samples were visualized on a Zeiss LSM510 confocal microscope with
all images acquired using identical parameters. N4BP1 antiserum was purified using
a Melon gel IgG spin purification kit (Pierce) and used at 1:100 dilution. Other primary
antibodies used were anti-UBF (1:50), anti-PML (1:100), anti-fibrillarin (1:75,
hybridoma supernatant) and anti-B23 (2.5 g/ml).
We thank Margaret Lualdi for expert technical assistance, Mary Dasso
and Deborah Morrison for antibodies and reagents, Stephen Lockett
for assistance with imaging, and Øyvind Dahle, Alessandra Mazzoni
and Allan Weissman for critically reading the manuscript. This research
was supported by the Intramural Research Program of the National
Institutes of Health, National Cancer Institute, Center for Cancer
Research. The content of this publication does not necessarily reflect
the views or policies of the Department of Health and Human Services
and nor does mention of trade names, commercial products or
Journal of Cell Science
organizations imply endorsement by the US Government. Deposited
in PMC for release after 12 months.
Supplementary material available online at
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Journal of Cell Science 123 (8)
Journal of Cell Science
Fig. S1. Transfected N4BP1 colocalizes with PML in HEK293 cells
Fig. S2. Endogenous N4BP1 accumulates at PML NBs in MG132 treated HEK293 cells
Fig. S3. Non-specific anti-N4BP1 cytoplasmic staining in MEFs