ARF INDUCES AUTOPHAGY BY VIRTUE OF INTERACTION WITH BCL-XL Julia Pimkina1,2 ...

19 Pages·2008·5.25 MB·English

JBC Papers in Press. Published on December 2, 2008 as Manuscript M804705200
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M804705200
ARF INDUCES AUTOPHAGY BY VIRTUE OF INTERACTION WITH BCL-XL
Julia Pimkina1,2,#, Olivier Humbey1,#, Jack T. Zilfou3, Michal Jarnik4, and Maureen E. Murphy1
Division of Medical Sciences1, Smolensk State Medical Academy Graduate Program2, and Division
of Basic Sciences4, Fox Chase Cancer Center, Philadelphia PA, 19111; Zilfou Therapeutics, Inc.
Allentown PA 181043
Running title: ARF inhibits Bcl-xl
# These authors contributed equally to this work
Address correspondence to: Maureen E. Murphy, Ph.D. Fox Chase Cancer Center, 333 Cottman
Avenue, Philadelphia PA 19111. FAX [phone]. E-mail: [email protected]
The ARF tumor suppressor controls a shown that full-length ARF, in addition to the
well-described p53/Mdm2-dependent small molecular weight variant, can likewise
oncogenic stress checkpoint. In addition, induce autophagy (3). However, neither of these
ARF has recently been shown to localize to studies revealed a mechanism whereby ARF
mitochondria, and to induce autophagy; induces autophagy.
however, this has never before been
demonstrated for endogenous ARF, and the Autophagy is an evolutionarily-
molecular basis for this activity of ARF has conserved homeostatic process whereby
not been elucidated. Using an unbiased mass cytosolic components are targeted for removal or
spectrometry-based approach, we show that turnover in membrane-bound compartments
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mitochondrial ARF interacts with the Bcl2 (autophagosomes) that fuse with the lysosome w
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family member Bcl-xl, which normally (for review see 4). This process regulates the lo
a
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protects cells from autophagy by inhibiting turnover of damaged organelles and long-lived ed
the Beclin-1/Vps34 complex, which is essential proteins that are too large to be delivered to the fro
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for autophagy. We find that increased proteasome. Autophagy occurs constitutively at h
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expression of ARF decreases Beclin-1/Bcl-xl low levels, and is greatly induced during period ://w
complexes in cells, thereby providing a basis of metabolic stress, where lysosome-mediated w
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for ARF-induced autophagy. Our data also digestion of sequestered molecules serves to .jb
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indicate that silencing p53 leads to high levels release free amino acids and ATP to fuel the .o
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of ARF and increased autophagy, thereby continued survival of the cell. b/
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providing a possible basis for the finding by g
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others that p53 inhibits autophagy. The Several genes are implicated in the st o
combined data support the premise that ARF control of autophagy. Perhaps most notable of n M
induces autophagy in a p53-independent these is Beclin-1, which is an evolutionarily- ay
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manner in part by virtue of its interaction conserved mediator of autophagy, with structural , 2
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with Bcl-xl. similarity to the yeast autophagy gene 19
Apg6/Vps30. Beclin-1 is a component of the
The ARF tumor suppressor, p14ARF in class III PI3 kinase complex that includes
humans and p19ARF in mouse, is a critical growth Vps34; this complex regulates the formation and
suppressor that is upregulated by chronic nucleation of autophagosomes, and the
mitogenic signals and localizes predominantly to regulation of the activity of this complex is
the nucleolus. At the nucleolus and in the tightly regulated. For example, Beclin-1
nucleoplasm, ARF can exert both p53 dependent possesses a BH3 domain that interacts with the
and independent growth suppressive function, by BH3-binding groove of certain members of the
virtue of interaction with and inhibition of Bcl-2 family, including Bcl-2, Bcl-xl, Bcl-w and
MDM2, nucleophosmin, E2F-1, CtBP, c-myc, as to a lesser extent, Mcl-1 (5-9). Binding of Bcl-2
well as others (see 1 for review). Recently, a family members to Beclin-1 inhibits autophagy,
small molecular weight variant of ARF, possibly by decreasing the kinase activity of the
generated by translation from an internal Beclin/Bcl-2/Vps34 complex (5) or by
methionine, has been discovered to localize negatively regulating Beclin-1 oligomerization
primarily to mitochondria and to induce (10). The interaction between Beclin-1 and Bcl-
autophagy (2). More recently, another group has 2 family members is also regulated; for example,
1
Copyright 2008 by The American Society for Biochemistry and Molecular Biology, Inc.
BH3-only proteins can bind directly to Bcl-2 blotting was performed on 100 ug whole cell
family members and disrupt complex formation lysate and 20 ug mitochondrial lysate; antisera
with Beclin-1 (11). Additionally, used were anti-p19ARF (GeneTex), anti-p14ARF
phosphorylation of Bcl-2 by Jun-N-terminal (Ab-1, Ab-2, Calbiochem), anti-Bcl-xl (Cell
kinase (JNK) can interfere with its ability to bind Signaling), anti-p53 (Ab-6, Calbiochem), anti-
to Beclin-1. In all cases, dissociation of the Mdm2 (Ab-1; Ab-2, Calbiochem) anti-actin
Beclin-1/Bcl-2 complex is associated with (AC15, Sigma), anti-GRP75 (C19, Santa Cruz),
induction of autophagy. anti-PCNA (PC10, Santa Cruz), anti-Bak (NT,
Upstate). For IP-westerns equivalent amounts of
In this report we confirm the findings of protein (500 µg total cellular or 100 µg
others that a fraction of ARF protein localizes to mitochondrial fraction) in NP-40 buffer was
mitochondria and can induce autophagy. We immunoprecipitated with 1 µg of anti-p19ARF
show for the first time that endogenous ARF, (GeneTex, Inc), anti-p14ARF (Ab2, Calbiochem)
upregulated in non-transformed cells by or anti-Bcl-xl (Cell Signaling), as described (16).
oncogenes, is capable of inducing autophagy, For GST binding assays, recombinant GST and
and further that silencing of p53 is sufficient to GST-tagged 19ARF proteins were induced in
de-repress ARF and induce autophagy. We BL21 cells for 5-6 h with 0.1 mM IPTG at 300C.
report the identification of Bcl-xl as a The GST-fusion proteins were isolated by batch
mitochondrial ARF-binding protein, and show purification using Glutathione–Sepharose 4B
that ARF-mediated autophagy is enhanced in beads (GE Health Care). Binding reactions were D
cells with Bcl-xl silenced. Finally, we show that performed as described (16) using 1 ug of GST ow
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ARF can reduce complex formation between fusion protein along with 25 uL of a 50 uL in lo
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Bcl-xl and Beclin-1. These data offer the first vitro transcription/translation reaction (TNT T7 ed
mechanistic insights into ARF-mediated Quick Coupled Transcription/Translation fro
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autophagy. They also point to ARF as a novel System, Promega) for full-length human Bcl-xl, h
regulator of Beclin/Bcl-xl complex formation. MDM2 and BAK labeled with [35]S-methionine ttp://w
(Amersham Biosciences), as described (16). w
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Experimental Procedures .jb
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Immunofluorescence for GFP-LC3; electron .o
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Cell culture, transfections, retroviral infections. microscopy. For GFP imaging, cells were b/
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The U2OS/Tet-On/p19ARF-inducible cell line transfected for 24 hours with 0.1 ug pEGFP-C1- g
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(U2OS-ARF) was generously provided by LC3, followed by siRNA for Bcl-xl st o
Pradip Raychaudhuri (University of Illinois, (Dharmacon, 471), p53 (SmartPool, Dharmacon) n M
Chicago), and was cultured in presence of 0.1 and non-targeting control (Dharmacon) for 48 ay
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µg/ml doxycycline (Sigma) to induce ARF, as hours with Dharmafect1 as per the manufacturer. , 2
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described (13). Transfections were carried out The averaged data from two independent 19
using Fugene 6 as per the manufacturer (Roche experiments in which 50 GFP-positive cells
Diagnostic, Indianapolis, US). Plasmids used were counted, plus standard deviations, were
were pcDNA3.1, pcDNA3.1-Mdm2, pcDNA3.1- calculated and analyzed by the student’s t test
p14ARF, pcDNA3.1-p19ARF, pcDNA3.1-Bcl-XL, (GraphPad InStat). Images were taken using a
pcDNA3.1-BAK, and pEGFP-C1-LC3. p53 Nikon E800 upright microscope with BioRad
short hairpin and control vector (shControl) are Radiance 2000 confocal scanhead (60x – 100x
described (14). For retroviral infection, Phoenix magnification).
cells were co-transfected with retroviral vectors
and helper plasmid using Fugene 6; supernatants For electron microscopy, cells were
were filtered and used subsequently for fixed with 2% glutaraldehyde/2% formaldehyde
retroviral infections as described (14). in cacodylate buffer followed by 1% osmium
tetroxide fixation. The samples were embedded
Mitochondria isolation, western analysis, in epoxy resin and cell sections were stained
immunoprecipitation, GST-pull down assay. with uranyl acetate and lead citrate. The area of
Mitochondria were purified using mannitol autophagosomes was calculated using the NIH
gradients as previously described (15). Western ImageJ program. For immunostaining, the cells
2
were trypsinized and prepared for cryosectioning for these studies we initially chose to analyze a
(17). In brief, cell pellets were fixed in 6% U2OS cell line (wt p53) containing a
formaldehyde, washed, cryoprotected with 2.1 tetracycline-regulated ARF transgene (13). We
M sucrose and frozen in liquid nitrogen. Thin induced ARF in these cells using doxycycline,
sections were cut at -120°C, collected on EM and mitochondria were purified using our
grids, and labeled with primary antibody. The previously published protocols (15); the purity
labeling was visualized by secondary labeling of these mitochondria was verified by the
with colloidal gold conjugated to Protein A. All enrichment for the mitochondrial proteins BAK,
samples were viewed in a FEI Tecnai 12 TEM cytochrome c and GRP75, relative to cytosol or
operated at 80 kV, the images were recorded whole cell lysate (Fig. 1A). Additionally, we
using AMT digital camera. confirmed these mitochondria were free from
contamination by the abundant protein PCNA,
Long-lived Protein Degradation Assay. which is nuclear in the DNA synthesis phase of
Measurement of long-lived protein degradation the cell cycle and cytosolic in G1 phase.
was performed as described previously (18). Western analysis for p53 in these purified
The percentage of long-lived protein degraded mitochondria revealed that ARF led to increased
was obtained by the formula % degradation = co-fractionation of p53 with mitochondria.
(14C counts at timepoint / sum of 14C counts at Importantly, however, this analysis revealed a
each time point+ total cell-associated considerable fraction of ARF co-purifying with
radioactivity) x 100. mitochondria (Fig. 1A).
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2D-DIGE, cell death/viability assays. Two To confirm the localization of ARF to lo
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dimensional differential in-gel electrophoresis mitochondria, we used immuno-electron e
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was performed by Applied Biomics (Hayward microscopy, which has not been used previously fro
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CA); briefly, 1000 ug of purified mitochondria to visualize this protein at this organelle. This h
was lysed by freeze-thaw in NP-40 buffer and analysis revealed that ARF was readily ttp://w
immunoprecipitated with 1 ug anti-p19ARF detectable at nucleoli, but was also clearly w
w
antibody (Ab-1, GeneTex) from induced and detectable at mitochondria; each mitochondrion .jb
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uninduced U2OS-ARF cells. These samples contained between 1 to 4 gold particles of ARF .o
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were covalently linked to CyDye and run with immuno-staining, and the overwhelming b/
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100 ug total mitochondrial lysate from induced majority of cytosolic ARF signal co-localized g
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cells on first dimension isoelectric focusing, and with mitochondria (Fig. 1B). Counting of the st o
second dimension SDS-PAGE. Image analysis particles localized to mitochondria, along with n M
was performed using DeCyder software, and quantitative western analysis using a light-based ay
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spots were picked and analyzed by mass western blot detection system (data not shown) , 2
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spectrometry (MALDI/TOF/TOF). Annexin V, revealed that between five and ten percent of 1
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cell viability and mitochondrial depolarization ARF was localized to mitochondria in this cell
assays were performed on a Guava Personal Cell line. The mitochondrial localization of ARF was
Analysis machine, using the Annexin V, supported by confocal microscopy, which
ViaCount and Guava EasyCyte Mitopotential kit indicated that in addition to the nucleolus, ARF
(Guava Technologies). localizes to perinuclear regions that co-stain with
Mito-Tracker dye (Supp. Fig. 1A). The small
Results molecular weight variant of ARF (smARF)
found at mitochondria has been reported to be
ARF localizes to mitochondria, induces resistant to proteinase K treatment (2), and
autophagy therefore likely in the inner organelle. In
We initiated these studies to determine contrast, we found that ARF localized to
whether the ARF tumor suppressor could mitochondria was most consistent with full-
enhance the localization of p53 to mitochondria. length ARF, and further was sensitive to
Because ARF is potently transcriptionally protease digestion. As depicted in Figure 1C,
repressed by p53, it is negligibly expressed in we found that the inner-mitochondrial proteins
cells containing wild type (wt) p53. Therefore, Hsp60 and cytochrome c were protected from
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treatment with increasing concentration of approximately 30% increase in the degradation
trypsin, while the majority of mitochondrial of long-lived proteins; the combined data are
ARF is sensitive to trypsin digestion, and most consistent ARF being associated with the
therefore most likely associated with the outer induction of autophagy.
mitochondrial membrane.
To determine whether mitochondrial or
We could find no indication that nucleolar ARF was inducing autophagy, we
mitochondrial ARF induces programmed cell engineered ARF to localize directly to
death in U2OS-ARF cells, although we were mitochondria, by fusing the coding region of this
consistently able to detect mitochondrial protein to the mitochondrial leader peptide of
membrane depolarization in ARF-induced cells ornithine transcarbamylase (OTC); our group
(Supp. Fig. 1B). These data, coupled with the and others have shown that this leader peptide
report that smARF can induce autophagy (2) directs nearly 100% of fusion proteins to
prompted us to examine this pathway. mitochondria (16). We find that OTC-ARF is as
Autophagy is a pro-survival pathway where-in capable as wild type ARF of inducing
cytosolic organelles and proteins are catabolized autophagy, as assessed by the appearance of
to release necessary nutrients; during this LC3 II (Supp. Fig 1C) and autophagosome
process, the protein LC3 is proteolytically formation (data not shown).
processed and covalently linked to
phosphatidylethanolamine, resulting in a species To date, all of the studies implicating
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with increased mobility in SDS-PAGE. Western ARF in autophagy have relied on transient ow
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analysis indicated that a significant fraction of transfection and overexpression of this protein. lo
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LC3 accumulated in the faster mobility form We therefore attempted to determine whether e
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(LC3 II) following ARF induction, but not in physiological induction of ARF led to fro
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doxycycline-treated parental cells (Fig. 2A). autophagy. ARF is negligibly expressed in h
Next, we transfected U2OS-ARF cells with a normal cells, but it becomes transcriptionally ttp://w
GFP-tagged LC3 construct and then induced upregulated by inappropriate expression of w
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ARF; this analysis revealed that this reporter oncogenes, including c-myc and oncogenic Ha- .jb
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protein stained in a manner consistent with ras. This gene is also transcriptionally repressed .o
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autophagic vesicles in cells following ARF by p53, so silencing or deletion of p53 results in b/
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induction (Fig. 2B). In contrast, doxycycline high ARF levels (19, 20). To determine whether g
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alone did not induce autophagy or cause GFP- endogenous ARF could induce autophagy we st o
LC3 vacuole localization (see for example Fig. infected early passage wild type mouse embryo n M
2A and Fig. 5A, respectively). Electron fibroblasts with E1A and Ras; this led to a ay
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microscopy revealed a significant number of significant upregulation of ARF, but no evidence , 2
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vesicles following ARF induction containing the for autophagy, as assayed by LC3 II abundance 1
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characteristic smooth double membrane of (Fig. 3A) and autophagosome formation (Fig.
autophagosomes (Fig. 2C). Quantification of the 3B). Interestingly however, we found that
area of autophagosomes from EM pictures infection with E1a and Ras combined with a
indicated that the area covered by short hairpin to silence p53 resulted in high ARF
autophagosomes increased from 1% to 37+/-9% levels, along with accumulation of LC3 II (Fig.
following ARF induction. 3A lane 3) and autophagosomes (Fig. 3B).
These data suggested that p53 might inhibit
Because autophagy occurs constitutively ARF-induced autophagy, or that the threshold
at low levels in all cells, the accumulation of level of ARF required for autophagy was only
LC3 II, GFP-LC3 and autophagosomes can efficiently achieved following p53 silencing. In
occur following either the induction of, or order to distinguish between these possibilities,
inhibition of, autophagy. In contrast, the we measured autophagy in U2OS-ARF cells
assessment of the degradation of long-lived transfected with siRNA for p53 or non-targeting
proteins is regarded to be a more reliable control siRNA. We found that silencing of p53
indicator of autophagy induction. As depicted in led to a significant increase in ARF-induced
Figure 2D, ARF induction resulted in an autophagy, as determined by cells containing
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GFP-LC3 vacuoles (p< 0.01, Fig. 3C) and LC3 but not control IgG (Fig. 4C). Similarly, in p53-
II abundance (data not shown). These data null MEFs, which express high levels of ARF
suggest that p53 inhibits ARF-induced and moderate levels of Bcl-xl, we were
autophagy. In support of this contention, we consistently able to detect ARF in Bcl-xl
found that silencing p53 alone, even in the complexes, and Bcl-xl in ARF
absence of E1a or Ras, is sufficient to induce immunocomplexes, in both asynchronous cells
ARF and autophagy in early passage MEFs (Fig. as well as in cells induced to undergo autophagy
3D). The combined data are most consistent by nutrient deprivation (Supp. Fig. 1D).
with the premise that p53 inhibits autophagy in
unstressed cells. We next chose to perform in vitro
binding assays in order to detect a Bcl-xl/ARF
ARF binds to Bcl-xl, and inhibits Beclin/Bcl-xl complex. In these binding assays we were able
complex formation to consistently detect 35S-labeled Bcl-xl
In order to elucidate the molecular basis precipitated with GST-ARF but not GST alone
for ARF-induced autophagy, we chose to purify (Fig. 4D). Additionally, while GST-ARF
mitochondria and immunoprecipitate ARF- precipitated Bcl-xl to levels comparable to the
interacting proteins. For this analysis we used positive control MDM2, the Bcl2 family
the technique of 2 Dimensional Differential In- member BAK failed to precipitate with GST-
gel Electrophoresis (2D-DIGE), in which ARF ARF. These data indicate that Bcl-xl and ARF
antisera was used to precipitate mitochondria can interact both in vivo and in vitro.
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from uninduced and induced U2OS-ARF cells; ow
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these precipitates were separately covalently To assess the relevance of the ARF-Bcl- lo
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labeled with fluorochromes, and run together on xl complex to autophagy induction, we silenced e
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a 2D gel. As depicted in Figure 4A, this analysis Bcl-xl using siRNA in U2OS-ARF cells and fro
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revealed about a dozen ARF-interacting proteins assessed the impact on ARF-mediated h
in mitochondria (red spots); mass spectrometry autophagy, by analyzing the percent of cells ttp://w
revealed their identity with significant coverage. containing GFP-LC3 puncta (Fig. 5A). This w
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One of these was the mitochondrial protein p32, analysis revealed that siRNA-mediated silencing .jb
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which was recently identified as an ARF- of Bcl-xl in U2OS-ARF cells led to a significant .o
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interacting mitochondrial protein (21, 22); increase in the percent of cells undergoing ARF- b/
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another was the mitochondrial chaperone protein induced autophagy (18% to 45%, Fig. 5B). g
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GRP75. At least one ARF-binding protein These data indicate that ARF induces autophagy st o
played a known role in autophagy: Bcl-xl. We in a manner that is inhibited by Bcl-xl. n M
opted to focus on Bcl-xl because of its known ay
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role in autophagy (7), and because it localizes Mechanistically, Bcl-xl is believed to , 2
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primarily to the outer mitochondrial membrane, inhibit autophagy via its direct interaction and 1
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where our data indicate the majority of ARF is interference with the protein Beclin-1, which
localized. Immunoprecipitation-western binds and allosterically activates a key enzyme
analysis confirmed the existence of an ARF-Bcl- in this process, Vps34 (class III PI3 kinase). We
xl complex in doxycycline-treated U20S cells therefore tested the hypothesis that ARF might
(Fig. 4B); we were also able to consistently interfere with the ability of Bcl-xl to complex
detect a complex between GRP75 and ARF (Fig. with Beclin-1. Because the level of Beclin-1 in
4B, bottom panel). U2OS cells is extremely low, we transfected
Beclin-1 and Bcl-xl and analyzed Beclin-1/Bcl-
In order to detect a complex between xl complex formation. As shown in Figure 6,
endogenous ARF and Bcl-xl, we infected induction of ARF led to markedly decreased
primary MEFs with E1A/Ras and a p53 short complex formation between transfected Bcl-xl
hairpin (shp53) and immunoprecipitated Bcl-xl and Beclin-1 (Fig. 6A). In addition, there was a
from whole cell lysate or purified mitochondria. slight but consistent decrease in the steady state
For both whole cell lysate and purified levels of Bcl-xl and Beclin-1 following ARF
mitochondria, we were consistently able to induction, suggesting that ARF might also
detect ARF co-precipitating with Bcl-xl antisera, regulate the stability of Beclin-1/Bcl-xl
5
complexes (Fig. 6A right panel). To determine particular role for inhibiting cytosolic p53 in
whether ARF influenced endogenous Beclin- autophagy induction, our data argue that p53’s
1/Bcl-xl complex formation, took advantage of a ability to transcriptionally repress ARF may also
short hairpin to silence ARF in p53-null MEFs, play a role in its ability to inhibit autophagy.
and measured Beclin-1/Bcl-xl complex
formation. Significantly, p53-null MEFs The data here-in and those of Kroemer
infected with a short hairpin control vector had (24) need to be reconciled with the findings of
high levels of ARF co-precipitating with Bcl-xl, others indicating that genotoxic stress-induced
but no detectable Beclin-1 (Fig. 6B). Notably, p53 has a positive role in autophagy, and that
however, silencing ARF with the short hairpin p53 transactivates the pro-autophagy gene
(shARF) led to readily detectable Beclin-1 in DRAM (25, 26). Such reconciliation likely lies
Bcl-xl immunoprecipitates. These data support with differences in the stresses used to induce
the premise that the ability of Beclin-1 and Bcl- autophagy and the timing of events.
xl to form complexes is influenced by ARF Specifically, these authors used genotoxic stress
level. to induce autophagy, and apoptosis was the
dominant effect of p53 induction; autophagy was
We next purified mitochondria from induced late in this process. In contrast, our data
U2OS-ARF cells in the absence and presence of and those of Kroemer (24) fit best with a model
doxycycline, and assessed Bcl-xl/Beclin-1 in which p53 inhibits autophagy in unstressed
complex formation. Whereas the steady state cells. How p53 inhibits this process remains an
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levels of Beclin-1 and Bcl-xl co-purifying with interesting question to be resolved; our data ow
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mitochondria were unaltered by ARF induction, suggest that at least part of this inhibition is lo
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low levels of a Bcl-xl/Beclin-1 complex were mediated by transcriptional repression of ARF. e
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detectable in untreated cells, and this was fro
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abolished following ARF induction (Fig 6C). These studies identify ARF as a novel h
The combined data suggest that Beclin-1/Bcl-xl regulator of the Beclin-1/Bcl-xl interaction. ttp://w
complexes are inhibited by ARF. This premise Whereas our findings are most consistent with w
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is supported by in vitro binding assays, which the interaction of ARF and Bcl-xl at .jb
indicate that increasing amounts of 35S-labeled mitochondria, it should be noted that only Bcl-xl c.o
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Beclin-1 titrated into a GST-ARF/Bcl-xl binding and Bcl-2 targeted to the endoplasmic reticulum b/
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reaction can diminish the ability of ARF to (ER), not the mitochondria, have been found to g
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interact with 35S-labeled Bcl-xl in vitro (Fig. suppress autophagy (5, 7, 27). Because est o
6C). mitochondria and ER are often intricately linked, n M
we cannot exclude the possibility that our ay
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Discussion mitochondria preparations are contaminated with , 2
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ER, and that the ARF-Bcl-xl interaction also 1
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In this manuscript we show that both occurs at the ER. However, our EM data
endogenous and exogenous ARF can induce support a localization of ARF at mitochondria;
autophagy. In our system of early passage further we find that enforced localization of ARF
MEFs infected with retroviruses, we find that to mitochondria (OTC-ARF) is sufficient to
oncogene-mediated induction of ARF can lead induce autophagy. Therefore, these data support
to autophagy only in cells with silenced p53. the intriguing possibility that ARF may be
Additionally, we find that silencing of p53 in selectively inducing mitophagy (autophagy of
ARF-inducible cells increases the timing and mitochondria) as opposed to reticulophagy
appearance of autophagosomes, implying at least (autophagy of the ER).
in these cell types that p53 inhibits autophagy.
These findings are consistent with a recent report It has been known for some time that
indicating that p53 inhibits autophagy (24). ARF possesses a p53-independent function in
Specifically, Kroemer and colleagues recently tumor suppression. For example, the incidence
showed that inactivation of p53 by deletion, and aggressiveness of tumors from p53/ARF
depletion or chemical inhibition can trigger double knockout mice are more severe than in
autophagy. Whereas these authors a find p53 knockout mice (23). Additionally,
6
overexpression of ARF is known to suppress the play important roles in the induction of
proliferation of p53-null cells (28-30). There are autophagy are UVRAG and Bif-1; heterozygous
compelling data to suggest that autophagy knockout mice for these proteins are predisposed
normally plays a role in tumor suppression. For to multiple spontaneous cancers (33, 34), and
example, the Beclin-1 heterozygous knockout like Beclin-1, UVRAG is mono-allelically
mouse is predisposed to multiple tumor types, mutated in cancer (34). We have recently
including lymphoma and liver cancer; in tumors reported that ARF plays a critical role in
from these mice, the wild type allele of Beclin-1 autophagy, and that p53-null tumor cells with
is not lost, so Beclin-1 is a haplo-insufficient silenced ARF have reduced autophagy, and
tumor suppressor gene (6, 31). Additionally, reduced survival, under conditions of nutrient
Beclin-1 is mono-allelically deleted in a subset depletion (35). It remains to be determined
of tumors of the breast, ovary and prostate (6, whether ARF’s autophagy function plays a role
32). Two Beclin-1 binding proteins that both in tumor suppression by this protein.
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Acknowledgments
The authors thank Steve Hann (Vanderbilt University School of Medicine) for GST-ARF, Scott Lowe
(Cold Spring Harbor Laboratories) for E1A and Ras retroviral constructs, Shengkan Jin (University of
Medicine and Dentistry of New Jersey) for Beclin-1 short hairpin, Tamotsu Yoshimori (Osaka
University, Japan) for GFP-LC3 and Pradip Raychaudhuri (University of Illinois at Chicago) for
U2OS-ARF cells. We thank members of the Murphy lab, Donna George, and Steven McMahon for
critical reading of the manuscript. We thank and acknowledge the support of Harvey Hensley (FCCC
Small Animal Imaging Facility), Tony Lerro (Laboratory Animal Facility) and Sandy Jablonski
(Confocal Facility). This work was supported by NIH R01 CA080854 (M.M.) and R01 CA150002
(M.M.) and by the Philippe Foundation (O.H.).
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Figure Legends
Figure 1. The ARF tumor suppressor protein localizes mitochondria
(A) Western analysis using antisera for the proteins indicated of whole cell lysate (WCL) and
mitochondrial (Mito) extracts isolated from U2OS-ARF cells, treated or untreated for 24 hours with
doxycycline (100 ng/ml) and/or Adriamycin (0.5 µg/ml). The boxed lanes depict mitochondrial
fractions containing ARF. In the panel on the right, 20 ug of mitochondrial and cytosolic extract were
probed for GRP75, BAK and cytochrome c (Cyt c), which are mitochondrial proteins, and PCNA,
which is cytosolic and nuclear, to assess the integrity of mitochondrial purification.
(B) Immuno-electron microscopy using ARF antisera followed by Protein G-Gold in U2OS-ARF
cells following 6 hours treatment with doxycycline. The boxes depict gold particles in mitochondria
(box 1-3) and nucleoli (box 4). Scale markers are shown. Uninduced cells were used as a control and
were clear of ARF immunostaining (not shown).
(C) Mitochondrial ARF is trypsin-sensitive. Mitochondria were purified from U20S-ARF cells
treated with doxycycline for 8 hours (prior to autophagy induction). 20 ug of mitochondria were
probed for ARF, Hsp60 and cytochrome c following 20-minute incubation with increasing
concentration of trypsin (0, 75, 100, 125 and 150 ug/mL, lanes 1-5 respectively).
Figure 2. ARF induces autophagy
(A) Western analysis of parental U2OS or U2OS-ARF cells treated with doxycycline for 24 hours.
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The bottom LC3 band (LC3 II) represents the form that accumulates during autophagy. w
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(B) U2OS-ARF cells were transiently transfected with GFP-LC3 for 24 hours and treated with lo
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doxycycline for 24 and 48 hours, followed by confocal microscopy to detect GFP. The cells depicted ed
are representative of over 100 cells counted for each timepoint; the average number of GFP-LC3 fro
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puncta per GFP-positive cell are indicated. h
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(C) Electron microscopy of U2OS-ARF cells untreated or treated with doxycycline for 24 hours. The ://w
scale bar is 2 um. In the right panel autophagosomes with double membranes are depicted; scale bar w
w
is 200 nm. The average area of autophagosomes calculated with ImageJ software per cell is indicated. .jb
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APG: autophagosomes. .o
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(D) Increased degradation of long-lived proteins in U2OS-ARF cells treated with doxycycline for 24 b/
y
hours (black line), compared to untreated cells (gray line) following radiolabeling of total cellular g
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protein with 14C-valine for twenty four hours, and twenty-four hour chase with cold valine. Data are est o
reported in percent of protein degraded at each time point, and are representative of two independent n M
experiments. ay
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Figure 3. Silencing p53 enhances ARF-induced autophagy 19
(A) Western analysis of primary mouse embryo fibroblasts (MEFs) 48 hours following infection with
parental retrovirus (vector) or retrovirus encoding E1A and Ras (E1A/Ras) and short hairpin to p53
(shp53). Only when p53 is absent or silenced is autophagy induced (LC3 II). The slight increase in
LC3 II in vector-infected cells was not consistent.
(B) Electron microscopy analysis of autophagosomes in wild type MEFs (passage 3) infected with the
retroviruses indicated. Scale bars are 5 um (upper panels) and 500 nm (lower panels). The far right
panel depicts western analysis of these samples. The average area of autophagosomes per cell
calculated with ImageJ software is indicated. APG: autophagosomes.
(C) Confocal microscopy of U2OS-ARF cells transfected with GFP-LC3 vector for 24 hours, then
with siRNA for p53 or non-targeting control for 24 hours, followed by treatment for 6 hours with
doxycycline. Cells with four or more GFP-LC3 vacuoles were considered positive for autophagy.
The silencing of p53 is assessed in the western blot depicted in the top right panel. In the bottom right
panel the data from three independent experiments in which 50 or more GFP-positive cells were
counted are presented as the mean % GFP-LC3-positive cells plus standard deviations. The p value
depicted reflects a comparison between columns 3 and 4.
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