1 Natural Sciences Section, Södertörns University College, S-141 89
Huddinge, Sweden
2 Department of Biosciences, Karolinska Institute, S-141 57 Huddinge,
Sweden
3 Department of Neurochemistry and Neurotoxicology, Stockholms University, S-106
91 Stockholm, Sweden
Author for correspondence (e-mail:
azadeh.arabi{at}sh.se)
Accepted 15 January 2003
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Summary |
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Key words: Transcription factor, Proteasome inhibitor, Degradation, Stability, Photobleaching
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Introduction |
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In addition to gene rearrangements and amplifications that cause elevated
levels of c-Myc, mutations in the coding sequence are frequently found in
human cancers (Axelson et al.,
1995; Murphy et al.,
1986
). These mutations cluster in the N-terminal transactivation
domain within or close to the evolutionarily conserved Myc-box I region with
Thr58, a phosphorylation site, being a major hotspot. Characterisation of the
N-terminal sequences, including the Myc box I and II region as elements
responsible for c-Myc rapid proteolysis
(Flinn et al., 1998
;
Salghetti et al., 1999
),
suggested that increased protein stability might be a consequence of these
alterations. It was subsequently shown that these cancer-related mutations
impair the rapid turnover of c-Myc by proteasomes and cause a substantially
prolonged half life of the protein (Bahram
et al., 2000
; Gregory and
Hann, 2000
; Salghetti et al.,
1999
). Aberrant ubiquitin/proteasome-mediated degradation of c-Myc
is therefore likely to play an important role in the pathogenesis of cancer.
However, the mechanisms that target the regulated degradation of c-Myc via the
proteasome remain unknown.
26S Proteasomes are multi-subunit proteases consisting of the 20S catalytic
core and the 19S regulatory particle
(DeMartino and Slaughter,
1999; Myung et al.,
2001
). Proteasomes have been detected both in the nucleus and the
cytoplasm of mammalian cells (Brooks et
al., 2000
; Palmer et al.,
1996
), and photobleaching analysis of GFP-tagged proteasomes has
revealed that they are highly mobile
(Reits et al., 1997
).
Different models have emerged to explain how these proteases and their
substrates come into contact. One is the `recruitment model', according to
which the function and distribution of proteasomes are regulated by the
distribution of their substrates (Anton et
al., 1999
; Baumann et al.,
2001
). In contrast, other current models propose that the
substrates of the proteasomes are translocated to cellular compartments
containing appropriate proteasomes. For example, nuclear proteins such as the
transcriptional regulators p53 and the Arylhydrocarbon receptor are presumed
to be substrates for cytoplasmic proteasomes
(Davarinos and Pollenz, 1999
;
Freedman and Levine, 1998
)
since nuclear export via the Leptomycin-B-sensitive CRM1 system has been shown
to be a necessary step in regulating the degradation of these proteins. In
addition, there is evidence for proteasome-mediated protein degradation within
the nucleus (Floyd et al.,
2001
). One of the nuclear structures associated with proteasomal
degradation is the ND10 bodies (also called POD or PML-bodies)
(Anton et al., 1999
).
Interestingly, the list of the proteins interacting and/or co-localising with
these nuclear foci is growing and includes transcription regulators as well as
proteasomes (Matera, 1999
;
Reyes, 2001
).
In spite of the previous identification of regions within the c-Myc protein that contribute to its rapid degradation as well as their demonstrated importance for the oncogenic potential of c-Myc, we still have no knowledge about the mechanisms that target regulated c-Myc degradation. In this work we show that even though c-Myc is predominantly nuclear, it is potentially available for degradation in both the nucleus and the cytoplasm since the protein shuttles between these compartments. Furthermore, increased c-Myc levels, resulting form ectopic expression or proteasome inhibition, are associated with accumulation of the protein at the nucleoli. Nucleolus-associated c-Myc is relatively stably associated with regions distinct from the fibrillarin containing structures in nucleoli that also accumulate proteasomes concurrent with c-Myc accumulation. These results are consistent with a model in which excess c-Myc is sequestered and subsequently degraded in the nucleoli of mammalian cells.
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Materials and Methods |
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Transfections
c-myc-GFP encoding the full-length human c-Myc protein fused to GFP at its
C-terminal, has been used previously for functional studies of c-Myc
(Satou et al., 2001;
Yin et al., 2001
). We are
grateful to H. Ariga, Hokkaido University, for providing us this plasmid. In
experiments with c-Myc-GFP the cells were transiently transfected with 1 µg
(or an otherwise indicated amount) of the c-Myc-GFP expression vector using
Effectene transfection kit (Qiagen, Kebo Lab, Stockholm) according to the
manufacturer's protocol. Proteasome inhibitors were added to the culture
medium 24 hours post transfection.
Cells were transfected with 1, 2 or 4 µg of the c-Myc-GFP expression vector. The total amount of DNA in each transfection was kept constant at a total of 4 µg by adding complementary amounts of the empty vector. The efficiency of the transfection, with the expression vector was determined by scoring the number of c-Myc-GFP-expressing cells. The values were 20%, 35% and 40% in cells transfected with 1, 2 and 4 µg, respectively. A total of 5000 cells in three independent transfections were analysed.
Western blotting
Whole-cell extracts were made as described previously
(Flinn et al., 1998). Lysates
were subjected to polyacrylamide SDS-PAGE. Proteins were elecrtophoretically
transferred to Hybond C-super membrane (Amersham), and detected by
immunostaining and chemoluminescence.
Immunofluorescence staining and microscopy
Cells grown on glass cover slips were fixed in 3.7% paraformaldehyde in PBS
for 20 minutes, permeabilised in 0.5% Triton X-100 in PBS for 5 minutes. All
steps were performed on ice. The cells were blocked with 2% BSA in PBS
containing 0.1% Tween-20 for 1 hour, at room temperature or over night at
4°C. Cells were then incubated at room temperature with primary antibodies
for 1 hour, washed four times in blocking buffer and then incubated at room
temperature with fluorophore-conjugated secondary antibody for 1 hour. After
incubating with the secondary antibody the cells were washed twice in 0.1%
Tween-20 in PBS, before mounting.
Anti-c-Myc C33 (Santa Cruz Biotechnology, Santa Barbara, CA), anti-20S proteasome mAb PW 8265 (Affiniti Research Products), anti-20S proteasome `core' rabbit pAb PW8155 (Affiniti Research Products), and Anit-Fibrillarin mAb (Cytoskeleton Inc., Denver) were used. Purified 20S proteasomes (mammalian), PW8720, was purchased from Affiniti Research Products.
Images were acquired using a Leica DM IRBE fluorescence microscope or a Leica TCS-SP confocal laser scanning microscope (Leica, Heidelberg, Germany). For the Leica DM IRBE fluorescence microscope a C4742-95 Hamamatsu CCD camera (Hamamatsu Photonics, Sweden) was used. The FITC/GFP fluorescence was analysed using a BP 470/40 nm excitation and a BP 525/50 nm (Dic 500 nm) emission filter combination. The TRITC fluorescence was analysed using a BP 546/14 nm excitation and a LP 590 nm (Dic 580 nm) emission filter combination. For the Leica TCS-SP laser scanning confocal microscope a 488 nm laser line from an Argon laser (20 mW) and a 568 nm laser line from a Krypton laser (20 mW) were used. The images were acquired by using a wavelength interval between 500-550 nm and 600-650 nm, respectively.
Live cell imaging and photobleaching experiments
A Leica TCS-SP laser scanning confocal microscope with a 488 nm laser line
from an Argon laser (20 mW) was used to perform all live cell and
photobleaching experiments. The images were acquired by using a wavelength
interval between 500-580 nm. In the time-lapse experiment, cells were followed
for 3 hours after proteasome inhibitor addition. Images were acquired every
120 seconds.
In fluorescence recovery after photobleaching (FRAP) experiments
(Lippincott-Schwartz et al.,
2001; Pederson,
2001
), a defined region of cells was exposed to 100% laser
intensity for 0.5-1 seconds. The fluorescence recovery was calculated from
images acquired. For quantitative analysis the post-bleach intensities were
normalised to correct for total loss of fluorescence due to photobleaching.
The relative fluorescence intensity (RFI) was calculated as
RFI=(IB,t/IN,t)x100), where IB,t is the
fluorescence intensity of the bleached area at each time point and
IN,t is the intensity of the unbleached area at the corresponding
time points.
The mobile fraction (MF) from the FRAP experiment was calculated as MF=(Ii-I0)/(Ii-I0), where Ii is the fluorescence intensity in the bleached region after full recovery, Ii is the fluorescence intensity before bleaching and I0 is the fluorescence intensity just after bleaching.
In fluorescence loss in photobleaching (FLIP) experiments, cells expressing c-Myc-GFP were repeatedly bleached in a defined region with 100% laser intensity for 1 second. The cell was then imaged between the bleach pulses. The relative fluorescence intensity (RFI) was calculated as RFI=IB,t/IB,0 x100, where IB,t is the fluorescence intensity of region of interest (ROI), at each time point and IB,0 is the intensity of the ROI before bleaching.
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Results |
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To study the mobility of c-Myc-GFP in the nucleoplasm we performed FRAP (fluorescence recovery after photobleaching) analysis. A defined area in the nucleus was bleached for 1 second. The cell was then imaged immediately after bleaching and then sequentially every second, to measure the recovery rate of the fluorescence signal. Fluorescence in the bleached spot recovered too quickly for determination of fluorescence recovery using our instrumentation (data not shown), demonstrating that c-Myc diffuses very rapidly within the nucleoplasm.
To determine whether the mobility of the nuclear c-Myc protein is restricted to the nuclear compartment we used FLIP (fluorescence loss in photobleaching). Cells expressing c-Myc-GFP were repeatedly bleached in a defined area of the cytoplasm. A series of time-lapse images of the cells were obtained showing the fluorescence signal before bleaching and after every bleach pulse. Repeated bleaching in the cytoplasm caused fading of the nuclear signal, indicating that nuclear c-Myc-GFP shuttles to the cytoplasm (Fig. 1B). The reduction in nuclear fluorescence was specific because no reduction was seen in the nucleus of an adjacent cell. Fig. 1C shows that substantial quenching of the fluorescence was observed within the order of minutes, indicating a relatively rapid rate of nuclear cytoplasmic exchange. Thus we conclude that the observed, steady state distribution of c-Myc results from a dynamic equilibrium in which c-Myc molecules move rapidly within the nucleoplasm and continuously shuttle between the nucleus and the cytoplasm.
Nucleolar association of c-Myc correlates with elevated levels of its
expression
c-Myc was not observed in the nucleoli of most cells under normal growth
conditions (Fig. 1A). However,
careful inspection of endogenous c-Myc in COS-7 cells revealed that a very
small portion of cells (<1%) accumulated c-Myc at the nucleoli. Similarly,
we detected nucleolus-associated c-Myc-GFP in less than 1% of the transfected
cells in our initial experiments (Fig.
1B).
To assess whether c-Myc expression levels could influence its nucleolar localisation, we progressively increased the levels of c-Myc-GFP expression by transfecting cells with increasing amounts of the expression plasmid. As expected, observation of the transfected cells showed that both the proportion of cells transfected, and the amount of expressed c-Myc-GFP in individual cells increase by increasing the amount of transfected vector. Interestingly, the proportion of cells containing c-Myc-GFP at the nucleoli (Fig. 2A, lower panels, yellow arrows), compared with cells containing nucleoplasmic c-Myc-GFP (Fig. 2A, upper panels), increased from <1% in cells transfected with 1 µg, to 4% and 8% in cells transfected with 2 and 4 µg, respectively (Fig. 2B).
|
Thus, although c-Myc is normally not detectable at the nucleoli, a small fraction of cells accumulate the protein in the nucleoli under physiological conditions of growth. The fraction of cells with nucleolar c-Myc-GFP increases progressively, as c-Myc expression levels increase. This suggests that the nucleolus might serve as a sequestration site for c-Myc when protein expression levels are high.
c-Myc accumulates at the nucleoli of cells treated with proteasome
inhibitors
Since c-Myc levels increase in response to proteasome inhibitors
(Salghetti et al., 1999), we
investigated the cellular location of c-Myc in cells treated with proteasome
inhibitor. COS-7 cells were cultured in the presence of the proteasome
inhibitor, ALLN (100 µM), for 4 hours prior to analysis by indirect
immunofluorescence microscopy. Strikingly, after proteasome inhibition c-Myc
accumulated at the nucleoli of essentially all cells
(Fig. 3A), which suggests that
nucleolar-association becomes a bottle-neck in c-Myc degradation. Similarly to
the endogenous protein, transiently transfected c-Myc-GFP was localised to the
nucleoli in proteasome-inhibitor-treated cells. Similar results were obtained
using other proteasome inhibitors such as MG132 (50 µM) as shown in
Fig. 3B, and Epoxomicin (10
µM) (data not shown).
|
Using time-lapse analysis of c-Myc-GFP in living cells, we followed the translocation of c-Myc-GFP to the nucleoli as a result of proteasome inhibition in live cells. Within 3 hours of proteasome inhibitor addition c-Myc-GFP started to accumulate at the nucleolar periphery (Fig. 3B). The western blot in Fig. 3C shows that c-Myc accumulation occurs in the same time frame as its relocation to nucleoli during proteasome inhibition. These results are consistent with the existence of a cellular mechanism that targets c-Myc to the nucleolus of cells containing elevated levels of the protein.
Using fibrillarin as a marker for the dense fibrillar region of the nucleolus, and 3D reconstitution of the confocal images we show at higher resolution that c-Myc is not detected in the nucleoli (Fig. 4A). In proteasome-inhibitor-treated cells c-Myc accumulates in regions distinct from the fibrillarin-containing regions of the nucleolus (Fig. 4B).
|
c-Myc is relatively stably associated with the nucleolus in cells
treated with proteasome inhibitors
The data presented so far suggest that nucleoli might be a sequestration
site for c-Myc. In order to determine whether nucleolus-associated c-Myc in
these cells is still in equilibrium with other cellular compartments we
performed FLIP analysis. As shown in Fig.
5A, in cells treated with 50 µM MG 132 for 3 hours, the
fluorescence intensity of c-Myc-GFP in both the nucleoplasm and the nucleolus
fades as a result of repeated bleaching in the cytoplasm, indicating that
nucleolar c-Myc exchanges with the cytoplasmic pool of the protein. The
residual mobility of nucleolar c-Myc further suggests that the accumulation of
c-Myc cannot be attributed to the formation of a protein aggregate in the
nucleoli.
|
To quantify the relative mobility of c-Myc accumulated at the nucleoli we used FRAP. c-Myc-GFP-expressing COS-7 cells were cultured in the presence of ALLN (100 µM) for 3 hours and then subjected to FRAP analysis. Cells with nucleolus-associated c-Myc-GFP were bleached within the nucleoli (Fig. 5B) or the nucleoplasm (data not shown), and then sequentially imaged to assess the recovery of the fluorescence signal. The fluorescence in the bleached nucleoli recovered within 30 minutes, with an average half-life of 6 minutes (Fig. 5C). The mobile fraction was calculated to 84%. The relatively slow fluorescence recovery kinetics of nucleolus-associated c-Myc-GFP compared with the recovery of the nucleoplasmic c-Myc-GFP is indicative of a relatively stable biological interaction between c-Myc-GFP and the nucleoli. As shown for untreated cells, the repopulation of c-Myc-GFP after bleaching in the nucleoplasm was too fast to measure in this experiment, indicating that the slower mobility of nucleolus-associated c-Myc is not just a general consequence of proteasome inhibition. Thus, although nucleolus-associated c-Myc is less mobile than nucleoplasmic c-Myc it is still dynamic and continues to shuttle to the cytoplasm. The reduced mobility of c-Myc associated with nucleoli compared with its mobility in the nucleoplasm shows that nucleoli provide sequestration sites for c-Myc when the protein is in excess.
Proteasomes co-localise with nucleolus-associated c-Myc
c-Myc accumulates and is sequestered at nucleoli when its cellular level is
increased. A potential consequence of this sequestration is subsequent
proteasomal degradation. Degradation of c-Myc at the nucleoli would, however,
require the presence of proteasomes at this location. To address this issue we
investigated the localisation of proteasomes in the absence and the presence
of proteasome inhibitors in COS-7 cells by immunofluorescence, using one
monoclonal and one polyclonal antibody, both of which react with the core
subunits of the proteasomes. In cells growing under normal conditions,
proteasomes were localised in the nucleus and cytoplasm but could not be
detected in nucleoli (Fig. 6A,
upper panel). However, within 5 hours of proteasome inhibition a fraction of
the proteasomes accumulated in the nucleoli, as shown by both antibodies
(Fig. 6A, lower panel).
Accumulation of the proteasomes in the nucleoli of
proteasome-inhibitor-treated cells is shown at higher magnification in
Fig. 6B (left panels). Confocal
images of proteasome-inhibitor-treated cells stained for fibrillarin and
proteasomes show that, like c-Myc, proteasomes accumulate in regions adjacent
to but distinct from the fibrillarin-containing structures of the nucleolus
(Fig. 6B, right panels).
|
Further investigation of the localisation of proteasomes in relation to c-Myc shows that proteasomes co-localise with c-Myc in the nucleoli of proteasome-inhibitor-treated cells (Fig. 7A). We detected proteasomes in the nucleoli of cells expressing elevated levels of c-Myc, in the absence of proteasome inhibitors (Fig. 7B). Interestingly, in these cells proteasomes accumulate specifically in the subset of nucleoli, which accumulate c-Myc. This strongly suggests a specific coupling of proteasome translocation to the nucleoli and accumulation of nucleolus-associated c-Myc.
|
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Discussion |
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Accumulation of c-Myc in nuclear bodies or inclusions has been observed
previously in transiently transfected cells overexpressing c-Myc
(Henriksson et al., 1992;
Koskinen et al., 1991
). These
inclusions are similar to the inclusions we also observe in cells, which
appear to express the highest amounts of c-Myc-GFP
(Fig. 2A, class 3 cells).
Co-localisation of c-Myc with Hsp70 in such inclusions has led to speculation
that they might be artefacts of the transfection procedure and formed by
aggregates of unfolded c-Myc. However, as suggested by the same authors, it is
equally possible that formation of these c-Myc/Hsp70-containing bodies may be
a mechanism for regulating c-Myc activity
(Henriksson et al., 1992
;
Koskinen et al., 1991
). In
support of this view it should be noted that c-Myc inclusions are also
observed in non-transfected, cytokine-activated lymphocytes.
(Heikkila et al., 1987
;
Larsson et al., 1991
).
Importantly, the nucleolar accumulation sites in our experiments are distinct
from the nuclear bodies that have been observed previously since cells
containing nucleolar c-Myc do not contain other sites of c-Myc accumulation
(Figs
2,3,4,5).
A recent proteomic analysis of human nucleoli has shown that a surprisingly
large number of proteins, many with no previously known nucleolar functions,
are found in nucleoli (Andersen et al.,
2002). This study reveals that the nucleolar protein composition
is not static and can alter significantly in response to the metabolic state
of the cell. Although conditions analogous to ours were not investigated in
that study, the findings are in general agreement with the idea that c-Myc and
additional proteins translocate to the nucleoli under particular conditions
such as proteasome inhibition or elevated expression. Interestingly, a number
of proteins, including POD-associated proteins PML and sp100, SUMO-1
(Mattsson et al., 2001
),
EBNA-5 (Pokrovskaja et al.,
2001
) and MDM2 (Xirodimas et
al., 2001
) have been shown to localise to the nucleolus in
response to proteasome inhibition. However, it has also been shown that some
other proteins such as p27, p21 and cyclin D do not localise to the nucleolus
under such conditions (Mattsson et al.,
2001
). Thus, it appears that the nucleolar association in response
to proteasome inhibitors applies to only a subset of nuclear proteins and, to
our knowledge, c-Myc is the first example of a transcription factor belonging
to this class.
Regulation of protein activity by nucleolar sequestration has been reported
previously (Tao and Levine,
1999; Visintin et al.,
1999
). It is thus likely that c-Myc activity can also be regulated
in this way. Such a model would predict a relatively stable interaction of
c-Myc with the nucleolus in order to remove it from the functional pool of
c-Myc in the nucleoplasm. We used FRAP to measure the mobility of c-Myc in
alternative nuclear locations. The results show that c-Myc diffuses very
rapidly within the nucleoplasm. This is consistent with other studies of
transcriptional regulators including HP1
(Kourmouli et al., 2000
), GR
(Hager et al., 2000
) and ER
(Stenoien et al., 2001
). Based
on these findings a `hit and run' model for gene regulation has been proposed
according to which transcriptional regulators are in continual flux and move
rapidly in and out of the chromatin. Consistent with sequestration in the
nucleolus, the mobility of c-Myc-GFP associated with the nucleolus is
strikingly reduced compared with the rapid dynamics seen in the nucleoplasm.
Although re-population of bleached nucleoli with actively fluorescent
c-Myc-GFP molecules from the nucleoplasm or other nucleolar sources is slow,
nucleolar c-Myc is still mobile. The rates of re-population are much faster
than those observed for components of well-characterised stable structures,
such as the nuclear pore complexes (Daigle
et al., 2001
). The relatively stable but still mobile nucleolar
c-Myc pool shows that the nucleolus fulfils the requirements for a nuclear
sequestration site, from which the protein has the opportunity to re-enter the
active c-Myc population in the nucleoplasm.
Our observation that proteasome inhibition causes c-Myc accumulation in the
nucleoli of essentially all cells suggested that nucleolar sequestration of
accumulating c-Myc might be augmented by inhibition of its degradation at
nucleolar sites. Accordingly, our results demonstrate changes in proteasome
localisation. We show that under normal growth conditions proteasomes are not
detectable in nucleoli. In contrast, in response to proteasome inhibitors a
fraction of the proteasomes accumulates in the nucleoli concurrent with the
re-localisation of c-Myc. This is consistent with a recent report where
proteasomes were shown to accumulate in the nucleoli of other mammalian cells
following proteasome inhibition (Mattsson
et al., 2001). Notably, redistribution of proteasomes to the
nucleoli is not restricted to the conditions of proteasome inhibitor treatment
since we show that, in cells expressing high levels of c-Myc in the absence of
proteasome inhibitors, proteasomes are also associated with the subset of
nucleoli that contain c-Myc.
There is independent evidence for alterations in proteasome distribution
under experimental conditions similar to ours. Proteasome inhibitor treatment
has been shown to promote accumulation of the proteasomes at the mitotic
organising centre (MTOC) (Wigley et al.,
1999; Wojcik et al.,
1996
). Moreover, studies with the mutant influenza virus
nucleoprotein, which is a target of rapid proteasomal degradation, reveal that
in the presence of proteasome inhibitors nucleoprotein accumulates in MTOC and
PML bodies and recruits the proteasomes to both locations
(Anton et al., 1999
). When
proteasome function is restored proteasomes degrade substrates in MTOC and PML
bodies. One possible explanation to such alterations in proteasome
localisation is that these enzymes might be able to seek out their targets at
their accumulation/sequestration sites. Several lines of evidence support this
view. Proteasome inhibitor treatment has been shown not to induce a general
increase in proteasome levels (Wigley et
al., 1999
), but rather the observed accumulation of proteasomes in
specific regions appears to be caused by redistribution of the existing pools
to the regions of substrate sequestration. Taken together with our data, these
results support a model in which proteasomes are recruited to the nucleoli,
where a subset of their substrates, including c-Myc, is sequestered. In most
normal cells c-Myc would be rapidly degraded and this would be a simple way to
explain the observation that c-Myc and proteasomes are not normally observed
in the nucleoli.
The molecular chaperone protein Hsp70 has been shown to interact with the
nuclear accumulations of c-Myc (Henriksson
et al., 1992; Koskinen et al.,
1991
). In agreement with this we have observed that Hsp70 also
co-localises with excess c-Myc, which is sequestered at the nucleoli (A.A. and
A.P.H.W., unpublished). However, the outcome of Hsp70 interaction with
accumulated c-Myc remains unclear. In addition to its role in regulating
protein folding, Hsp70 interacts directly with the proteasomes via co-factors
such as the CHIP (C-terminus of Hsp70-interacting protein) and BAG1 proteins
(Alberti et al., 2002
;
Demand et al., 2001
), which
have been shown to regulate the proteasomal degradation of chaperone
substrates. It is therefore tempting to speculate that Hsp70 might influence
the fate of c-Myc sequestered at the nucleolus either by inducing c-Myc
release or by targeting it for proteasomal degradation.
In summery, our results suggest that c-Myc activity is negatively regulated by both sequestration and proteasome-mediated degradation at the nucleoli. Our observation that c-Myc and proteasomes concurrently translocate to the nucleolus suggests that these mechanisms are coordinated and at least partly overlapping. Further studies will be required to determine the extent to which c-Myc sequestration is directly coupled to its degradation. It is possible that the extent of coupling may vary under different conditions of cell growth and proliferation.
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Acknowledgments |
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Footnotes |
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