Author Correction
Romo1 is a negative-feedback regulator of Myc
Seung Baek Lee, Jung Jin Kim, Jin Sil Chung, Myeong-Sok Lee, Kee-Ho Lee, Byung Soo Kim and
Young Do Yoo
Journal of Cell Science 124, 2512
© 2011. Published by The Company of Biologists Ltd
doi:10.1242/jcs.095042
There was an error published in J. Cell Sci. 124, 1911-1924.
William P. Tansey should not have been included as an author on this paper. The author list should read: Seung Baek Lee, Jung Jin Kim,
Jin Sil Chung, Myeong-Sok Lee, Kee-Ho Lee, Byung Soo Kim and Young Do Yoo.
The authors apologise for this mistake.
1911
Research Article
Romo1 is a negative-feedback regulator of Myc
Seung Baek Lee1, Jung Jin Kim1, Jin Sil Chung1, Myeong-Sok Lee2, Kee-Ho Lee3, Byung Soo Kim4,
William P. Tansey5 and Young Do Yoo1,*
1
Laboratory of Molecular Cell Biology, Graduate School of Medicine, Korea University College of Medicine, Korea University, Seoul 136-705,
Republic of Korea
2
Department of Biological Sciences, Sookmyung Women’s University, Seoul 140-742, Republic of Korea
3
Department of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul 139-706, Republic of Korea
4
Department of Internal Medicine, Korea University College of Medicine, Korea University, Seoul 136-705, Republic of Korea
5
Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA
*Author for correspondence ([email protected])
Journal of Cell Science
Accepted 18 January 2011
Journal of Cell Science 124, 1911-1924
© 2011. Published by The Company of Biologists Ltd
doi:10.1242/jcs.079996
Summary
Degradation of Myc protein is mediated by E3 ubiquitin ligases, including SCFFbw7 and SCFSkp2, but much remains unknown about
the mechanism of S-phase kinase-associated protein (Skp2)-mediated Myc degradation. In the present study, we show that upregulated
Myc protein, which triggers the G1–S phase progression in response to growth-stimulatory signals, induces reactive oxygen species
modulator 1 (Romo1) expression. Romo1 subsequently triggers Skp2-mediated ubiquitylation and degradation of Myc by a mechanism
not previously reported in normal lung fibroblasts. We also show that reactive oxygen species (ROS) derived from steady-state Romo1
expression are necessary for cell cycle entry of quiescent cells. From this study, we suggest that the generation of ROS mediated by
pre-existing Romo1 protein is required for Myc induction. Meanwhile, Romo1 expression induced by Myc during G1 phase stimulates
Skp2-mediated Myc degradation in a negative-feedback mechanism.
Key words: Myc, Romo1, ROS, Skp2
Introduction
Myc protein levels are increased in response to mitogenic stimuli
to stimulate G1–S phase progression of the cells, and the expression
level of Myc is tightly controlled through transcriptional,
translational and post-translational mechanisms (Arnold and Sears,
2008). Although Myc transcription is induced during G0–G1
transition, fine modulation of the Myc protein level occurs at the
post-translational level through regulation of its stability (Bhatia et
al., 1993). The main mechanism for Myc degradation involves
ubiquitin-mediated proteolysis (Gross-Mesilaty et al., 1998). Myc
is polyubiquitylated by E3 ubiquitin ligases, including the F-box
proteins Fbw7 and Skp2, and a series of sequential phosphorylation
events is required for Fbw7-mediated proteasomal degradation.
Phosphorylation sites of Myc include Thr58 and Ser62.
Phosphorylation at Ser62 occurs via the Ras–Raf–MEK–ERK
pathway (Alvarez et al., 1991; Seth et al., 1991). Ras activation
also inhibits the phosphatidylinositol-3-OH-kinase (PI3K)/Akt
pathway to inhibit GSK-3, resulting in stabilization of the Myc
protein (Cross et al., 1995; Sears et al., 2000). During the later
stages of G1, Ras activity decreases, and GSK-3 is reactivated to
phosphorylate Myc at Thr58 (Henriksson et al., 1993; Saksela et
al., 1992). Myc phosphorylated at Thr58 is ubiquitylated by the
SCFFbw7 ubiquitin machinery for degradation by the 26S
proteasome (Welcker et al., 2004; Yada et al., 2004).
Another important mechanism for ubiquitin-mediated
proteasomal degradation of Myc is the Skp2 pathway (Kim et al.,
2003; von der Lehr et al., 2003). Skp2 is also reported to
ubiquitylate Cdk inhibitors and tumor suppressor proteins such as
p27Kip1 (Carrano et al., 1999), p57Kip2 (Kamura et al., 2003), p130
(Tedesco et al., 2002) and Tob1 (Hiramatsu et al., 2006). Skp2 is
overexpressed in cancer (Bashir and Pagano, 2003). The
expressions of Skp2 and Myc are induced by mitogenic stimulation;
however, Skp2 expression continues into S phase (Lisztwan et al.,
1998). Skp2 interacts with two domains of Myc (residues 129–
147: the N-terminal Myc box II domain and residues 379–418: the
C-terminal bHLHZip domain) at the G1 to S phase transition to
induce Myc degradation and turnover (Kim et al., 2003; von der
Lehr et al., 2003). Skp2-mediated ubiquitylation does not correlate
with Myc phosphorylation because Myc mutated at Thr58 is well
ubiquitylated (Kim et al., 2003).
Romo1 (reactive oxygen species modulator 1) was first identified
in 2006, and forced expression of Romo1 increases the level of
cellular ROS that originate from mitochondria (Chung et al., 2006).
Romo1 is localized to the mitochondria and releases mitochondrial
ROS through complex III of the mitochondrial electron transport
chain (Chung et al., 2008). Although the ROS produced by cytosolic
enzymes such as NADPH oxidase have a role in cell proliferation,
mitochondrial ROS are not known to be involved in cell
proliferation. Recently, we reported that ROS originating from the
endogenous Romo1 protein are necessary for both normal and
cancer cell proliferation (Chung et al., 2009; Na et al., 2008).
Suppression of Romo1 expression inhibits cell growth through
inhibition of ERK activation and p27Kip1 expression, demonstrating
that ROS derived from Romo1 are required for cell proliferation.
Romo1 expression is enhanced in senescent cells and in most
cancer cells (Chung et al., 2006; Chung et al., 2008). Romo1 is
also upregulated by serum deprivation and contributes to the serumdeprivation-mediated increase in ROS (Lee et al., 2010).
Furthermore, a recent paper demonstrated that Romo1 modulates
ROS production in the mitochondria (Kim et al., 2010). Romo1
recruits the anti-apoptosis regulator Bcl-XL to decrease the
mitochondrial membrane potential in response to tumor necrosis
factor- (TNF-), resulting in ROS production. Although many
studies have been conducted on Romo1, its physiological function
1912
Journal of Cell Science 124 (11)
is not well elucidated. In the present study, we investigated the role
of Myc-induced Romo1 in Myc turnover after serum stimulation.
Results
Myc expression induced after serum stimulation increases
Romo1 expression
Journal of Cell Science
Myc has been reported to stimulate ROS generation, which in turn
induces DNA damage (Vafa et al., 2002). Romo1 has been also
reported to recruit Bcl-XL to reduce the mitochondrial membrane
potential in response to TNF-, resulting in mitochondrial ROS
generation (Chung et al., 2006; Kim et al., 2010). Therefore, we
investigated the correlation between Myc and Romo1 after serum
stimulation. The expressions of Myc, Romo1 and p27Kip1
(CDKN1B) were examined after addition of serum to cultures of
normal human lung fibroblasts (IMR-90 and WI-38 cells) and
human embryo kidney cells (HEK293). Low basal levels of Myc
and Romo1 and high levels of p27Kip1 were detected in unstimulated
cells by western blot analysis (Fig. 1A). When the cells were
stimulated with serum, Myc expression was induced at 1 hour and
its level peaked at 3–6 hours after serum treatment. Interestingly,
Romo1 expression was enhanced after Myc induction, peaking at
9–24 hours (Fig. 1A). By contrast, p27Kip1 was downregulated at
6 hours after serum stimulation.
To observe whether Myc induced Romo1 expression, HEK293
and HeLa cells were transfected with Myc, and Romo1 expression
was observed by western blot analysis. As shown in Fig. 1B, Myc
Fig. 1. Myc-induced Romo1 expression
after serum stimulation. (A)Myc, Romo1
and p27Kip1 expression levels after serum
stimulation were examined by western blot
analysis in IMR-90, WI-38 and HEK293
cells. The cells were serum-starved for 48
hours and then treated with 30% serum. actin was used as a loading control. (B)Myc
induces Romo1 expression. HEK293 and
HeLa cells were transfected with Myc or
vector alone and western blot analysis of
Romo1 was performed at the indicated
times. (C,D)After the cells were transfected
with MYC siRNA and were serum-starved
for 48 hours, serum was added to the cells
and western blot analyses of Myc (C) and
Romo1 (D) were performed. (E)ROMO1
mRNA induction after serum stimulation.
After HEK293 cells were serum-starved,
semi-quantitative RT-PCR (upper panel) and
real time-PCR (lower panel) analyses were
performed. Results represent the means
(± s.e.m.) of three independent experiments
performed in triplicate. The relative
induction of ROMO1 mRNA was
normalized to ACTB (-actin) or GAPDH.
Myc regulation by Romo1
increased the Romo1 protein level, demonstrating that Romo1 is
downstream of Myc. Next, we examined whether serum-stimulated
Myc expression also increased Romo1 expression. To observe
whether knockdown of Myc blocked serum-induced Romo1
expression, WI-38 cells and IMR-90 cells were transfected with
MYC siRNA. MYC siRNA transfection efficiently inhibited Myc
induction by serum stimulation (Fig. 1C), and Myc knockdown
blocked the serum-induced Romo1 expression (Fig. 1D).
Furthermore, semi-quantitative RT-PCR and real-time PCR analyses
demonstrated that serum stimulation upregulated ROMO1
expression transcriptionally (Fig. 1E). These results demonstrate
that serum stimulation enhances Myc-mediated Romo1 expression.
Journal of Cell Science
Romo1 regulates serum-induced ROS generation and Myc
protein level
To investigate whether knockdown of Romo1 suppressed seruminduced ROS generation, IMR-90 cells were transfected with
ROMO1 siRNA and ROS levels were measured by staining the
cells with MitoSOX, a probe for superoxide in the mitochondria.
First, Romo1 knockdown in cells transfected with ROMO1 siRNA
was examined by western blot analysis (supplementary material
Fig. S1A). To exclude off-target effects of the ROMO1 siRNA
construct, a rescue experiment was performed. The N-terminal
deletion mutant of Romo1 (Romo1-N), which does not include
the ROMO1 siRNA-1 sequence, is known to induce mitochondrial
ROS generation (Kim et al., 2010). ROMO1 siRNA-1 transfection
efficiently caused Romo1 knockdown and decreased ROS levels
in various cell lines (Hwang et al., 2007; Na et al., 2008). However,
Romo1-N was resistant to ROMO1 siRNA-1 (supplementary
material Fig. S1B). In the present study, we showed that both wildtype Romo1 and Romo1-N decreased Myc expression, but
Romo1-C did not (Fig. 4D). Therefore, we examined whether
Romo1-N can downregulate Myc expression in cells transfected
with ROMO1 siRNA-1. As shown in supplementary material Fig.
S1C, FLAG–Romo1 (wt) decreased Myc expression and ROMO1
siRNA-1
transfection
blocked
Romo1-induced
Myc
downregulation. However, ROMO1 siRNA-1 transfection failed to
suppress Romo1-N-induced Myc downregulation. This result
showed the specificity of the ROMO1 siRNA construct. Next, we
examined whether Romo1 knockdown suppressed serum-induced
ROS production. As shown in Fig. 2A, high ROS levels were
observed in serum-deprived cells and Romo1 knockdown inhibited
serum deprivation-induced ROS production. The ROS increases
were suppressed by serum addition. These results are consistent
with a previous report (Lee et al., 2010). In Fig. 1A, we showed
that serum stimulation increased Romo1 expression. This Romo1
induction was also observed by fluorescence microscopy (Fig.
2A). The serum-stimulated ROS increases were completely blocked
by Romo1 knockdown (Fig. 2A). The decreases in Romo1
expression and ROS formation in primary human fibroblasts IMR90 (Fig. 2B,C) or WI-38 (supplementary material Fig. S2A) were
quantified using MetaMorph software. In Fig. 1D, we showed that
knockdown of Myc blocked the serum-induced Romo1 expression.
Therefore, we also measured the ROS formation after serum
stimulation in cells transfected with MYC siRNA. As shown in
supplementary material Fig. S2B, the serum-stimulated ROS
increases were also blocked by Myc knockdown. Next, we
examined Myc expression in IMR-90, WI-38 and HEK293 cells
transfected with ROMO1 siRNA. Interestingly, Romo1 knockdown
blocked the elimination of Myc after 6 hours of serum stimulation
(Fig. 2D,E). Instead, Myc expression was gradually increased until
1913
24 hours. From this result, we suggest that the Romo1 expression
induced by Myc during G1 phase is necessary for elimination of
Myc and we assume that increased Romo1 expression might be
involved in Myc degradation in a negative-feedback mechanism.
Although Myc expression was increased in cells transfected
with ROMO1 siRNA after 6 hours of serum stimulation compared
with control cells, its expression was very low at early times after
mitogenic stimulation (0–3 hours, Fig. 2D). Recently, we reported
that ROS derived from Romo1 expression also regulate cell
proliferation through activation of ERK in various normal and
cancer cell lines (Chung et al., 2009; Na et al., 2008). Therefore,
we examined whether ROS derived from Romo1 expression were
required for induction of Myc in early G1 phase. As shown in Fig.
3A, Myc induction for 2 hours after serum stimulation was
suppressed by Romo1 knockdown. Treatment with antioxidants
also inhibited Myc induction. However, hydrogen peroxide (H2O2)
treatment of cells transfected with ROMO1 siRNA recovered Myc
expression (Fig. 3B). Myc expression was also examined using
various kinase inhibitors. MEK1/2-specific inhibitors, PD98059
and U0126, blocked Myc induction and ERK activation (Fig. 3B).
These results demonstrate that ROS derived from Romo1 in
response to serum stimulation are necessary for Myc induction and
cell cycle progression.
To further investigate the correlation between Romo1 expression
and cell cycle transition triggered by serum stimulation, flow
cytometric analysis was carried out in IMR-90 cells transfected
with ROMO1 siRNA. In this experiment, Romo1 knockdown
delayed cell cycle progression into S phase (Fig. 3C). This finding
was also confirmed in WI-38 cells (supplementary material Fig.
S2C). These results indicate that Romo1 expression has an
important role in cell cycle entry triggered by mitogenic stimulation
via ERK activation and Myc induction. We also suggest that the
basal level of ROS derived from the steady-state level of Romo1
is required for ERK activation and Myc stabilization. By contrast,
enhanced ROS levels generated from Romo1 expression, which is
induced by Myc, trigger the elimination of Myc.
Romo1 expression induces Myc degradation
As shown in Fig. 2D, knockdown of Romo1 blocked the
elimination of Myc. Therefore, we investigated whether increased
Romo1 expression caused Myc downregulation. Romo1 was
transfected into HeLa cells, and Myc expression was measured by
western blot and immunofluorescence analysis. Romo1
overexpression triggered downregulation of Myc (Fig. 4A and
supplementary material Fig. S3A). To confirm this finding, cells
were co-transfected with Myc and Romo1, and Myc expression
was again measured by western blot analysis. As shown in Fig. 4B,
Romo1 also induced the downregulation of Myc that was expressed
exogenously. Expression of Romo1 also decreased the expression
of Myc in Huh-7, HeLa, A549 and H1299 cells (supplementary
material Fig. S3B).
Next, we analyzed which domain of Myc was responsible for its
expression. Romo1 and Myc deletion constructs (Herbst et al., 2004;
Tworkowski et al., 2002) were co-transfected into the cells, and Myc
expression was measured by western blot analysis. We found that
Romo1 promoted the downregulation of wild-type (WT) Myc, a A
construct including Myc box (Mb) I, a B construct, a E construct
and a G construct. However, Romo1 failed to downregulate a C
construct including the Mb II domain, a D construct including the
PEST domain, and a F construct (Fig. 4C). This result demonstrates
that the Mb I domain, which is needed for Fbw7-mediated
1914
Journal of Cell Science 124 (11)
Journal of Cell Science
proteasomal degradation of Myc, is not required for Myc degradation
and that another mechanism exists for ubiquitin-mediated
proteasomal degradation of Myc. Recently, we reported that the Cterminal region of Romo1 is important for TNF--induced ROS
production (Kim et al., 2010). To examine the effects of Romo1
deletion constructs on Myc expression, two deletion constructs of
Romo1, designated FLAG–Romo1-C (deletion of the C-terminal
48–79 residues) and FLAG–Romo1-N (deletion of the N-terminal
1–16 residues), were transfected into HeLa cells and Myc expression
was assessed. As shown in Fig. 4D, both wild-type Romo1 and
Romo1-N decreased Myc expression, but Romo1-C did not.
From this result, we suggest that ROS derived from the C-terminal
domain of Romo1 induced Myc degradation through the Mb II
domain, Mb III domain and a F construct (316–378 residues).
Fig. 2. Blockage of seruminduced ROS production and
Myc elimination by
knockdown of Romo1.
(A)Serum-induced ROS
production is blocked by
ROMO1 siRNA transfection.
After transfection with ROMO1
siRNA, IMR-90 cells were
serum-starved for 48 hours and
then treated with serum. The
cells were stained with
MitoSOX for 30 minutes and
then observed by fluorescence
microscopy. (B)For
quantification purposes, the
images were overlaid, and
Romo1 expression (green) was
analyzed with MetaMorph
software. Results represent the
means (± s.e.m.) of three
independent experiments
performed in triplicate. *P<0.05
versus control siRNA; #P<0.05
and ##P<0.01 versus control
siRNA at 0 hours by two-way
ANOVA. (C)For quantification
purposes, the images were
overlaid by a computer, and
MitoSOX fluorescence (red)
was analyzed with MetaMorph
software. Results represent the
means (± s.e.m.) of three
independent experiments
performed in triplicate.
*P<0.05; **P<0.01 versus
control siRNA; #P<0.05 versus
control siRNA at 1 hour by twoway ANOVA. (D)After
transfection with ROMO1
siRNA, IMR-90, WI-38 and
HEK293 cells were serumstarved for 48 hours and then
treated with serum. Myc
expression was measured by
western blot analysis at the
indicated times. (E)The
intensity of Myc expression in
IMR-90 cells was quantified by
scanning densitometry. Results
represent the means (± s.e.m.)
of three independent
experiments performed in
triplicate. **P<0.01;
***P<0.001 versus control
siRNA by one-way ANOVA.
Myc regulation by Romo1
Romo1 is localized in the mitochondria and induces
mitochondrial ROS production through complex III of the
mitochondrial electron transport chain (Chung et al., 2008). To
determine whether mitochondrial ROS production through complex
III is required for downregulation of Myc, HeLa cells were cultured
in the presence of an antioxidant (trolox), mitochondrial respiratory
chain complex III inhibitors (myxothiazol and stigmatellin),
complex I inhibitor (rotenone), complex II inhibitor (malonate) or
complex IV inhibitor (sodium azide), and Myc downregulation
was assessed by western blot analysis (Fig. 4E). Romo1-triggered
downregulation of Myc was blocked by myxothiazol and
stigmatellin and by trolox. By contrast, the other inhibitors failed
to inhibit Romo1-mediated Myc downregulation. Next, we treated
the cells with increasing amounts of H2O2, and Myc downregulation
was analyzed by western blot analysis. As shown in Fig. 4F,
treatment with a low concentration of H2O2 increased the amount
of Myc protein. By contrast, treatment with higher concentrations
of H2O2 decreased the amount of Myc protein. These findings
indicate that Romo1-derived ROS have an important role in Myc
regulation.
Journal of Cell Science
Romo1 induces Myc degradation through cytoplasmic
translocation of Skp2
Myc degradation is regulated by Fbw7 and Skp2, and Myc
degradation by Fbw7 is dependent on the phosphorylation of Thr58
and Ser62 in the MB1 domain (Welcker et al., 2004; Yada et al.,
2004). To ascertain whether Myc degradation controlled by Romo1
1915
is related to the phosphorylation of Thr58 and Ser62, wild-type
(WT) Myc or Myc mutants (T58A, S62A or T58AS62A) were
transfected into HeLa cells, and Myc expression was examined by
western blot analysis. As shown in Fig. 5A, Myc expression was
decreased in cells expressing Romo1, Skp2 or Fbw7. As expected,
Fbw7 expression failed to degrade the Myc protein in cells
transfected with Myc mutants (Fig. 5B). This result is consistent
with a previous report (Welcker et al., 2004; Yada et al., 2004).
However, Romo1 and Skp2 efficiently degraded the Myc protein
in cells transfected with Myc mutants, demonstrating that Romo1triggered Myc degradation is not mediated by Fbw7 (Fig. 5B,C).
H2O2 treatment also efficiently triggered Myc degradation in cells
transfected with Myc mutants (Fig. 5D). Next, we investigated
whether Romo1 stimulated Myc degradation through Skp2. SKP2
siRNA was transfected into cells to knock down Skp2
(supplementary material Fig. S4) and Myc expression was
examined. Interestingly, Skp2 knockdown suppressed Romo1induced Myc degradation (Fig. 5E).
Recent reports have shown that the phosphorylation of Skp2 at
Ser72 by Akt leads to cytoplasmic translocation of Skp2 (Gao et
al., 2009; Lin et al., 2009). To investigate whether Romo1
expression regulates Skp2 cytoplasmic translocation, we observed
HeLa cells by fluorescence microscopy after Romo1 transfection.
As shown in Fig. 6A, Romo1 expression induced the cytoplasmic
translocation of Skp2. Cytoplasmic Skp2 levels were quantified by
fluorescence microscopy and analysis with MetaMorph software.
This finding was also confirmed in HEK293 cells (supplementary
Fig. 3. ROS derived from Romo1 regulate
Myc induction through Erk activation for
cell cycle entry. (A)After HEK293 cells were
transfected with ROMO1 siRNA and serumstarved for 48 hours, the cells were treated with
serum to induce cell cycle entry. Western blot
analysis was performed using antibodies
against the indicated proteins. (B)After
HEK293 cells were treated with MEK1/2specific inhibitors (25M PD98059 and 1 mM
U0126), PI3K-specific inhibitors (20M
LY294002 and 1M wortmannin), JNK
inhibitor (20M SP600215), p38 kinase
inhibitor (25M SB203580), GSK-3 inhibitor
(25M TWS119), antioxidants (1 mM NAC
and 1M trolox) or H2O2 (10M), western
blot analysis was performed using antibodies
against the indicated proteins. (C)After IMR90 cells were treated with ROMO1 siRNA and
serum-starved for 48 hours, the cells were
treated with serum and analyzed by flow
cytometry.
1916
Journal of Cell Science 124 (11)
hours (Fig. 6E). A similar finding was also observed in other
normal human fibroblasts, WI-38 cells (supplementary material
Fig. S5B).
We showed that downregulation of Myc by Romo1 is required
for mitochondrial ROS production through complex III of the
mitochondrial electron transport chain (Fig. 4E). Therefore, we
explored whether cytoplasmic translocation of Skp2 by Romo1
was blocked by inhibitors of complex III of the mitochondrial
electron transport chain. After HeLa cells were transfected with
FLAG–Romo1, the cells were incubated with various inhibitors of
the mitochondrial electron transport chain. Cytoplasmic
translocation of Skp2 was detected in cells transfected with Romo1
(Fig. 6F). However, the Romo1-induced Skp2 cytoplasmic
translocation was inhibited by myxothiazol and stigmatellin, but
was not affected by other inhibitors. Trolox was used as a positive
control. We also investigated whether Romo1 expression regulated
cytoplasmic translocation of Myc in HeLa cells after Romo1
Journal of Cell Science
material Fig. S5A). To determine whether H2O2 treatment also led
to the cytoplasmic translocation of Skp2, the cells were treated
with H2O2. H2O2 treatment promoted the cytoplasmic translocation
of Skp2 (Fig. 6B), which was confirmed in cells exogenously
transfected with Skp2 (Fig. 6C). We also confirmed the localization
of Skp2 by cellular fractionation of HeLa cells. The cells were
treated with H2O2 or transfected with FLAG–Romo1. Both Romo1
and H2O2 induced cytoplasmic translocation of Skp2 (Fig. 6D).
These results demonstrate that ROS derived from Romo1 promote
the cytoplasmic translocation of Skp2.
Next, we performed an immunofluorescence assay to detect the
subcellular localization of Skp2 in response to serum stimulation
in normal human fibroblasts, IMR-90 cells. The cells were serumstarved for 48 hours and then treated with serum for 15 hours. The
cells were then harvested for immunofluorescence analysis. Skp2
was observed in the nucleus before serum stimulation. However,
Skp2 was translocated into cytoplasm after serum addition for 15
Fig. 4. ROS derived from Romo1 stimulate downregulation of Myc. (A)Romo1 expression induces Myc downregulation. HeLa cells were transfected with
FLAG–Romo1, and Myc expression was examined by western blot analysis. (B)WI-13 VA13 and HEK293 cells were transfected with Myc or FLAG–Romo1, and
Myc expression was examined by western blot analysis. (C)Schematic representation of the structural organization of Myc (top). Myc deletion constructs were
transfected into HeLa cells and Myc expression was examined by western blot analysis (bottom). TAD, transactivation domain; Mb, myc box; NLS, nuclear
localization sequence; BR, basic region; HLH, helix-loop-helix motif; Zip, leucine zipper motif. (D)Schematic representation of the structural organization of
Romo1 (top). Romo1 deletion constructs (C or N) were transfected into HeLa cells and Myc expression was examined by western blot analysis (bottom). TM,
transmembrane domain; Wt, Romo1 wild-type; C, Romo1 C-terminus deletion; N, Romo1 N-terminus deletion. (E)After HeLa cells were transfected with Myc
or FLAG–Romo1, cells were incubated with trolox (1 or 10M), myxothiazol (1 or 10M), stigmatellin (1 or 10M), rotenone (1 or 10M), malonate (10 or
100M) or sodium azide (1 or 10 mM) for 4 hours. Myc expression was examined by western blot analysis. (F)HEK293, WI-38 VA13 and H1299 cells were
treated with increasing amounts of H2O2 (1–1,000M) and Myc expression was examined by western blot analysis.
Myc regulation by Romo1
transfection. Romo1 expression enhanced the cytoplasmic
translocation of Myc (Fig. 6G). We also confirmed the localization
of Myc by cellular fractionation of HeLa cells transfected with
FLAG–Romo1 (Fig. 6H).
To investigate whether Romo1 promotes the cytoplasmic
translocation of Skp2 through the PI3K–Akt pathway, HeLa cells
were transfected with FLAG–Romo1. As shown in Fig. 7A, Romo1
expression triggered Akt activation and Myc degradation. The Akt
activation and Myc degradation were inhibited by the PI3K inhibitor
LY294002. Immunofluorescence experiments revealed that Romo1
expression induced the cytoplasmic translocation of Myc and Skp2,
and their translocations were suppressed by LY294002 (Fig. 7B).
We further investigated whether the H2O2-induced cytoplasmic
translocation of Skp2 was blocked by LY294002.
Immunofluorescence analysis showed that cytoplasmic
translocation of exogenous Skp2 by H2O2 treatment was inhibited
by LY294002 and trolox (Fig. 7C). These results suggest that the
cytoplasmic translocation of Skp2 and Myc induced by Romo1 is
mediated by Akt.
Journal of Cell Science
Romo1 enhances the interaction between Skp2 and Myc
and Myc ubiquitylation
To determine whether Romo1 expression enhances the interaction
between Skp2 and Myc, FLAG–Romo1 was transfected into HeLa
cells and a co-immunoprecipitation experiment was performed. As
shown in Fig. 8A, Romo1 expression increased Skp2 binding to
Myc and trolox treatment inhibited this interaction. To confirm this
interaction, Skp2 was immunoprecipitated with its antibody and
western blot analysis was performed with anti-Myc antibody (Fig.
1917
8B). Next, we examined whether Romo1 enhanced Myc
ubiquitylation. Myc was transfected into HeLa cells, together with
FLAG–Romo1, FLAG–Fbw7 or FLAG–Skp2. To identify the
extent of Myc ubiquitylation, ubiquitylated-Myc was
immunoprecipitated with anti-HA antibody and subjected to western
blot analysis using anti-Myc antibody. As shown in Fig. 8C,D,
Romo1 significantly increased the amount of Myc-ubiquitin
conjugates, and trolox treatment suppressed Myc ubiquitylation.
Fbw7 and Skp2 were used as positive controls. Fig. 8E showed
that Romo1-N significantly increased the amount of Mycubiquitin conjugates, but Romo1-C had no effect on Myc
ubiquitylation. This finding was also examined in HEK 293 cells
(supplementary material Fig. S6). We also explored whether H2O2
enhances ubiquitylation of Myc (Fig. 8F). These results indicate
that ROS increase modulated by Romo1 expression induces an
interaction between Skp2 and Myc and then enhances Myc
ubiquitylation. To identify whether Romo1 regulates the stability
of Myc protein, Romo1 was expressed by transient transfection in
HeLa cells and the cells were treated with cycloheximide (CHX).
The half-life of Myc was decreased in cells transfected with Romo1
(Fig. 8G, upper panel). Romo1 also reduced the stability of
exogenously transfected Myc in the cells (Fig. 8G, lower panel).
These results suggest that Romo1 negatively controls Myc stability
and that it is an important post-translational regulator of Myc
expression.
Discussion
Myc is an unstable protein with a half-life of 20–30 minutes (Hann
and Eisenman, 1984) and Myc degradation during G1-S phase
Fig. 5. ROS derived from Romo1
trigger Myc degradation through
Skp2. (A)HeLa cells were cotransfected with Myc, FLAG–Skp2,
FLAG–Fbw7 or FLAG–Romo1.
Myc expression was examined by
western blot analysis. (B)After
HeLa cells were co-transfected with
FLAG–Myc (Wt, a Thr58 mutant, a
Ser62 mutant or a Thr58,Ser62
mutant of Myc), HA–Fbw7 or
FLAG–Skp2, Myc expression was
examined by western blot analysis.
(C)Romo1 induces the Thr58- or
Ser62-phosphorylation-independent
degradation of Myc. HeLa cells
were co-transfected with FLAG–
Romo1 and FLAG–Myc (Wt, T58A,
S62A or T58AS62A). Myc
expression was examined by western
blot analysis. Asterisk, non-specific
band. (D)ROS trigger Thr58 or
Ser62 phosphorylation-independent
degradation of Myc. After HeLa
cells were transfected with FLAG–
Myc (Wt, a Thr58 mutant, a Ser62
mutant or a Thr58,Ser62 mutant of
Myc), cells were treated with H2O2
(100M) for 2 hours. (E)After
transfection of SKP2 siRNA into
HeLa cells for 24 hours, cells were
transfected with HA–Myc and
FLAG–Romo1.
1918
Journal of Cell Science 124 (11)
Journal of Cell Science
progression has been well identified. Serum stimulation enhances
Ras activation in the PI3K–Akt pathway during early G1, resulting
in GSK-3 inhibition (Gregory and Hann, 2000; Sears et al., 2000).
During late G1 phase, the Ras activity decreases and GSK-3
phosphorylates Myc on Thr 58, resulting in its ubiquitylation and
degradation (Sears et al., 2000). Phosphorylation of Myc on Thr
58 plays a key role in Myc degradation, and a mutation on Thr 58
contributes to tumorigenesis (Bhatia et al., 1993). However, Myc
degradation by GSK-3-mediated Myc phosphorylation on Thr 58
is not sufficient for Myc degradation during late G1 phase. Indeed,
Myc mutated at Thr 58 was reported to have a half-life of 30–40
minutes, compared to the 20–30 minutes half-life of wild-type
Myc (Bader et al., 1986; Sears et al., 1999; Salghetti et al., 1999).
Moreover, Myc mutated at Thr 58 is still targeted for ubiquitylation
and degradation (Hann, 2006). Therefore, an additional pathway
Fig. 6. See next page for legend.
should exist for Myc degradation. In the present study, we showed
that Myc expression reached a peak at 3–6 hours and declined at
9 hours. However, Myc expression levels continuously increased
until 24 hours when Romo1 expression was suppressed (Fig. 2D).
We also showed that Romo1 expression promoted the ubiquitylation
and degradation of Myc through cytoplasmic translocation of Skp2
and Myc (Figs 6 and 8). Therefore, we suggest that the
Romo1/ROS/Skp2 pathway is another pathway for Myc turnover.
The Romo1-mediated pathway appears to be one of the main
pathways for Myc degradation, because the Myc level was
significantly decreased when the cells were transfected with Romo1
to enhance Romo1 expression (Fig. 4).
Two main pathways of ubiquitin-mediated degradation of Myc
exist for Myc turnover. One is mediated by Fbw7. The other
pathway is mediated by Skp2. Myc ubiquitylation through Fbw7
Myc regulation by Romo1
Journal of Cell Science
has been well elucidated. A series of sequential phosphorylation
events occur after mitogenic stimulation and are followed by Fbw7mediated degradation of Myc (Sears et al., 1999; Yeh et al., 2004).
1919
However, Skp2-mediated Myc degradation is not well understood.
In the present study, we demonstrate that Romo1 induces Myc
degradation through a novel mechanism not previously reported.
Fig. 6. Romo1 regulates cytoplasmic
translocation of Skp2.
(A)Immunofluorescence staining of HeLa
cells transfected with FLAG–Romo1. The
data represent the average of three
experiments and 150 cells were monitored
in each experiment. Scale bar: 20m.
(B)After HeLa cells were incubated in the
presence of H2O2 (200M) for 2 hours,
cells were stained as indicated. Cells (150–
200) were scored and a representative
result from three independent experiments
is shown. Scale bar: 20m.
(C)Immunofluorescence staining of HeLa
cells transfected with FLAG–Skp2. The
cells were treated with H2O2 (200M) for
2 hours, then fixed and stained as
indicated. The data represent the average of
three experiments and 150 cells were
monitored in each experiment. Scale bar:
20m. (D)Western blot analysis of nuclear
(N) and cytoplasmic (C) fractions of HeLa
cells treated with H2O2 (200M) for 2
hours or transfected with FLAG–tagged
Romo1 for 48 hours. Cell lysates were
subjected to western blot analysis with
antibodies against Skp2, FLAG (Romo1),
-actin (cytosolic marker) or lamin B1
(nuclear marker). (E)IMR-90 cells were
serum-starved for 48 hours and then treated
with serum for 15 hours. Cells were
harvested for immunofluorescence
analysis. Cells (150–200) were scored and
a representative result from three
independent experiments is shown. Scale
bar: 20m. (F)After HeLa cells were
transfected with FLAG–Romo1, the cells
were cultured in the presence of
myxothiazol (10M), stigmatellin
(10M), rotenone (10M), malonate
(100M), sodium azide (10 mM) or trolox
(10M) for 4 hours, and harvested for
immunofluorescence analysis. The data
represent the average of three experiments;
100 cells were monitored in each
experiment. Scale bar: 20m. (G)HeLa
cells were transfected with the indicated
plasmids, treated with MG132 for 6 hours.
Scale bar: 20m. Arrow indicates cells
expressing FLAG–Romo1; asterisks
indicate cytoplasmic Myc protein.
(H)Western blot analysis of nuclear (N)
and cytoplasmic (C) fractions of HeLa
cells transfected with FLAG–Romo1 for
48 hours. Cell lysates were subjected to
western blot analysis with antibodies
against Myc, Flag (Romo1), -actin
(cytosolic marker) or lamin B1 (nuclear
marker).
Journal of Cell Science
1920
Journal of Cell Science 124 (11)
Fig. 7. Romo1-induced cytoplasmic translocation of Skp2 and Myc through the PI3K–Akt pathway.
(A)HeLa cells were transfected with FLAG–Romo1 and treated with LY294002 (20M) or trolox
(1M) for 6 hours before western blot analysis. (B)After HeLa cells were transfected with FLAG–
Romo1, the cells were treated with MG132 (10M), LY294002 (20M), then immunofluorescence
analysis was performed. Scale bar: 20m. (C)Immunofluorescence staining of HeLa cells transfected
with FLAG–Skp2. The cells were treated with H2O2 (200M) for 2 hours in the presence of LY294002
or trolox.
Romo1 induced by the enhanced Myc level increased the cellular
ROS level to trigger the cytoplasmic translocation of Skp2 (Fig.
6). Skp2 was induced by mitogenic stimulation and reached a peak
in S phase. Skp2 binds to two domains of Myc (Kim et al., 2003;
von der Lehr et al., 2003). It is unlikely that Romo1 induced Skp2
expression because increased Romo1 expression did not increase
the Skp2 level (Fig. 8A). Instead, Romo1 contributed to the
cytoplasmic translocation of Skp2. Skp2 is reportedly located in
the nucleus (Miura et al., 1999). However, a recent report showed
that Skp2 translocates into the cytoplasm after Akt-mediated
phosphorylation of Ser 72 (Gao et al., 2009; Lin et al., 2009).
Romo1 also regulated the cytoplasmic translocation of Myc in the
presence of the proteosomal inhibitor, MG-132 (Fig. 6G,H). In
addition to enhancing the cytoplasmic translocation of Skp2 and
Myc, Romo1 promoted the interaction between Skp2 and Myc,
resulting in Myc ubiquitylation (Fig. 8). It seems that Romo1 does
not directly interact with Skp2 or Myc in Myc degradation because
H2O2 treatment increased the cytoplasmic translocation of Skp2
and Myc (Fig. 6B). Antioxidant treatment also suppressed the
cytoplasmic translocation of Skp2 (Fig. 6F). Previously, we reported
that ROS originate from complex III of the mitochondrial
respiratory chain (Chung et al., 2008). Therefore, we examined
whether cytoplasmic translocation of Skp2 was blocked by
mitochondrial complex III inhibitors, and we showed that treatment
of complex III inhibitors efficiently suppressed the cytoplasmic
translocation of Skp2 (Fig. 6F). From these results, we suggest that
ROS derived from Romo1 play an important role in Myc turnover.
Although we showed that Myc degradation occurs via Romo1mediated cytoplasmic translocation of Skp2, the exact mechanism
by which Romo1 regulates the cytoplasmic translocation of Skp2
remains to be studied in the future.
Appropriate ROS levels maintained inside the cells play an
important role in cell growth and survival, and the physiological
range of H2O2 concentrations is 0.001 to 0.7 M (Burdon and
Rice-Evans, 1989). Although excessive ROS production reportedly
contributes to many pathological disorders, including cancer, aging,
and neurological diseases, ROS are required for redox signaling
and their main source is NADPH oxidase (Finkel, 2003; Turrens,
2003). This enzyme responds to growth or cell survival signals to
induce ROS production and is subsequently eliminated by
antioxidant enzymes. Although the increase in ROS triggered by
Romo1, which is induced by Myc, contributes to Myc degradation,
appropriate levels of ROS are critical for Myc stabilization. The
results presented in this study are consistent with a previous report
implicating ROS produced by hematopoietic cytokines in G1 to S
progression in Myc stabilization (Iiyama et al., 2006). This previous
study also showed that NAC treatment reduced the stability of
Myc protein, while H2O2 treatment of the cells enhanced its
stability. H2O2 treatment induced ERK-dependent Myc
phosphorylation on Ser 62 (Benassi et al., 2006). Recently, we
reported that Romo1 is necessary for cell growth and that Romo1
knockdown induces cell cycle arrest at G1 through inhibition of
Journal of Cell Science
Myc regulation by Romo1
1921
Fig. 8. Romo1-induced
interaction of Myc with Skp2
and Myc ubiquitylation.
(A)After HeLa cells were
transfected with FLAG–Romo1
and treated with MG132 (10M)
or trolox (1M) for 6 hours,
Myc was immunoprecipitated
with anti-Myc antibody for
western blot analysis. WB,
western blot analysis; WCL,
whole cell lysates. (B)After
HeLa cells were transfected with
FLAG–Romo1, Skp2 was
immunoprecipitated with antiSkp2 antibody for western blot
analysis. IgG HC,
immunoglobulin heavy chain.
(C)After HeLa cells were
transfected with Myc, UbiquitinHA, FLAG–Romo1, FLAG–
Fbw7 or FLAG–Skp2, the cells
were treated with MG132.
Ubiquitylated proteins were
immunoprecipitated with antiHA antibody for western blot
analysis with anti-Myc antibody.
(D)After HeLa cells were
transfected with Myc, Ubiquitin–
HA, or FLAG–Romo1, the cells
were treated with MG132 or
trolox. Ubiquitylated proteins
were immunoprecipitated with
anti-HA antibody for western
blot analysis with anti-Myc
antibody. (E)After HeLa cells
were transfected with Myc,
Ubiquitin-HA, FLAG–Romo1
(Wt), FLAG–Romo1 (C) or
FLAG–Romo1 (N), the cells
were treated with MG132.
Ubiquitylated proteins were
immunoprecipitated with antiHA antibody for western blot
analysis with anti-Myc antibody.
(F)After HeLa cells were
transfected with Myc, Ubiquitin–
HA, FLAG–Romo1 and FLAG–
Skp2, the cells were treated with
MG132 and H2O2 (200M, 2
hours). Ubiquitylated proteins
were immunoprecipitated with
anti-HA antibody for western
blot analysis with anti-Myc
antibody. (G)After HeLa cells
were transfected with FLAG–
Romo1 (upper panel) or cotransfected with Myc and
FLAG–Romo1 (lower panel), the
cells were treated with
cycloheximide (CHX, 20g/ml)
or trolox. Quantification of the
Myc levels following CHX
treatment was carried out by
densitometric scanning in the
ImageJ program.
1922
Journal of Cell Science 124 (11)
Invitrogen), respectively. All media contained 10% heat-inactivated FBS (GibcoInvitrogen), sodium bicarbonate (2 mg/ml; Sigma-Aldrich, St Louis, MO), penicillin
(100 units/ml), and streptomycin (100 g/ml; Gibco-Invitrogen). PD98059, U0126,
LY294002, Wortmannin, SP600215 and SB203580 were purchased from StressGen
(Victoria, BC, Canada). 3-[[6-(3-amino-phenyl)-1H-pyrrolo[2,3-d] pyrimidin-4yl]oxy]-phenol (TWS119) was obtained from Calbiochem (La Jolla, CA). N-acetylnocodazol,
cysteine
(NAC),
hydrogen
peroxide
(H2O2),
6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), stigmatellin,
myxothiazol, malonate, rotenone, sodium azide and N-carbobenzoxy-l-leucinyl-lleucinyl-l-norleucinal (MG132) were purchased from Sigma-Aldrich. 2⬘,7⬘dichlorofluorescein diacetate (DCF-DA) and MitoSOX were obtained from Molecular
Probes (Eugene, OR).
Plasmids
Journal of Cell Science
Fig. 9. A proposed model for Romo1-mediated Myc degradation in a
negative-feedback mechanism.
ERK activation and induction of p27Kip1 expression, demonstrating
that ROS originating from the mitochondria play a key role as
signaling mediators (Chung et al., 2009; Na et al., 2008). In the
present study, we also showed that Romo1 knockdown inhibits
ERK activation and Myc expression (Fig. 3A), resulting in cell
cycle arrest at G1 (Fig. 3C). Therefore, we suggest that the basal
level of ROS derived from the steady-state level of Romo1 is
required for ERK activation and Myc stabilization. In contrast,
excessive ROS produced from increased Romo1 expression induced
by Myc trigger Myc degradation. This suggestion is supported by
Fig. 3B and Fig. 4F. When the cells were treated with a low
amount of H2O2, Myc expression was increased; however, treatment
with additional H2O2 down-regulated Myc expression.
Myc is known to play an important role in cell proliferation and
its level is controlled transcriptionally, post-transcriptionally or
post-translationally. Abnormal regulation of Myc contributes to
tumor formation. In the present study, we identified a novel pathway
of negative feedback regulation for Myc during G1 phase. Upon
mitogenic stimulation, Myc expression is increased for cell cycle
progression. Myc induces Romo1 expression to enhance cellular
ROS levels. The ROS promote the cytoplasmic translocation of
Skp2 to cause Myc ubiquitylation, resulting in Myc degradation
(Fig. 9). Myc has also been reported to stimulate ROS generation,
which induces DNA damage (Vafa et al., 2002). Enhanced ROS
levels were also observed in Myc transgenic animals. We showed
that Myc up-regulation, which was induced by serum stimulation,
increased the ROS level via Romo1 expression. Our results
presented in this study are the first to elucidate the mechanism of
Myc-induced ROS production. An ROS imbalance can cause
cellular DNA damage and genomic instability, which can contribute
to the initiation, promotion and malignancy of tumors (Finkel and
Holbrook, 2000). Therefore, the results presented in this study
provide important information regarding the mechanism of Mycstimulated oncogenesis associated with ROS.
Materials and Methods
Cell culture and reagents
The human lung fibroblast IMR-90 and WI-38 cells were obtained from the American
Type Culture Collection (ATCC, Manassas, VA) and cells ranging from 29 to 34 in
population doubling level (PDL) were used. WI-38 VA-13 cells were cultured in
Eagle’s minimal essential media (EMEM, Gibco-Invitrogen, Grand Island, NY).
Human embryonic kidney (HEK) 293 cells, HeLa cervix carcinoma cells and Huh7 human hepatocarcinoma cells were cultured in Dulbecco’s modified Eagle’s media
(DMEM, Gibco-Invitrogen). Human non-small cell lung cancer (NSCLC) cell lines
A549 and H1299 were cultured in Ham’s F12 and RPMI 1640 medium (Gibco-
cDNAs encoding FLAG–Romo1 Wild-type (Wt) and deletion mutants, N and C,
were prepared in our laboratory and have been validated previously (Kim et al.,
2010). Complementary DNA encoding Myc (human) was cloned into pcDNA3
(Invitrogen). pCGN-HA–Myc (Wt) and deletion mutants (A, B, C, D, E, F
and G) were described previously (Herbst et al., 2004; Tworkowski et al., 2002).
pCl-FLAG–Myc (Wt) and substitution mutants (T58A, S62A and T58AS62A) and
the pCGN-HA–Fbw7 construct were kindly provided by Keiichi I. Nakayama and
Masaki Matsumoto (Department of Molecular and Cellular Biology, Kyushu
University, Japan) and have been described earlier (Yada et al., 2004). The p3FLAG–Myc-Fbw7 was kindly provided by Professor Bruce E. Clurman (Fred
Hutchinson Cancer Research Center, University of Washington School of Medicine,
Seattle) and was described previously (Welcker et al., 2004). CMV-FLAG–Skp2 was
kindly provided by Professor Tae Jun Park (Department of Biochemistry and
Molecular Biology, Ajou University, Republic of Korea) and was described previously
(Park et al., 2009).
siRNA
The sequences of Romo1 siRNA were unique to their intended targets, based on
BLAST searches. The Romo1 siRNA sequence was 5⬘-GGGCUUCGUGAUGGGUUG-3⬘ (sense strand). The other siRNA against Romo1 was described
previously (Hwang et al., 2007). The Myc siRNA sequences (Grandori et al., 2005),
Skp2 siRNA sequences (Carrano et al., 1999; Nishitani et al., 2006; Zhang et al.,
2004), and control siRNA sequence (Chung et al., 2009) were described previously.
siRNAs were purchased from Bioneer (Taejon, Republic of Korea).
Antibodies
Antibodies were: anti-Myc mouse monoclonal (Santa Cruz Biotechnology, Santa
Cruz, CA) and rabbit polyclonal (Santa Cruz Biotechnology), anti-p27kip1 mouse
monoclonal (BD Pharmingen, San Diego, CA) and rabbit polyclonal (Zymed
Laboratories, San Francisco, CA), anti-phospho-ERK rabbit polyclonal (Cell
Signaling Technology, Beverly, MA), anti-ERK rabbit polyclonal (Cell Signaling
Technology), anti-phospho-Akt rabbit polyclonal (Cell Signaling Technology), antiAkt rabbit polyclonal (Cell Signaling Technology), anti-Skp2 rabbit polyclonal
(Santa Cruz Biotechnology) and anti-Lamin B1 rabbit polyclonal (Santa Cruz
Biotechnology), -actin mouse monoclonal (Sigma-Aldrich), anti-FLAG (M2)
(Sigma-Aldrich) and anti-HA (Sigma-Aldrich). Mouse monoclonal antibody (mAb)
against Romo1 was described previously (Kim et al., 2010).
Serum deprivation and stimulation
For serum stimulation experiments, human lung primary fibroblast IMR-90 and WI38 cells and human embryo kidney (HEK) 293 cells were washed twice with serumfree media and further incubated in EMEM with 0.05% FBS for 48 h (Lee et al.,
2010). EMEM containing 30% FBS was then added and cells were collected at the
indicated time points.
Semi-quantitative RT-PCR and real-time PCR
Semi-quantitative RT-PCR analysis was performed as described previously (Chung
et al., 2008). SYBR Green PCR amplifications were performed using an iCycler iQ
Real-Time Detection System (Bio-Rad Laboratories, USA) associated with the
iCycler Optical System Interface software (version 2.3; Bio-Rad). All PCR
experiments were carried out in triplicate with a reaction volume of 25 l, using
iCycler IQ 96-well optical grade PCR plates (Bio-Rad) covered with iCycler opticalquality sealing film (Bio-Rad). Data analyses (calculations), including determining
the relative amounts of each target mRNA, were performed with the iCycler IQ realtime detection system (Bio-Rad).
Transfection, immunoprecipitation, and western blot analysis
Cells were transfected with the indicated constructs or siRNA using LipofectamineTM
(Gibco-Invitrogen). The immunoprecipitation and western blot analysis were
described previously (Kim et al., 2010).
Measurement of ROS production and immunofluorescence assay
Intracellular ROS production was measured using a fluorescence microscope
(Olympus LX71 microscope), and the images were analyzed using MetaMorph
software (Universal Imaging, Westchester, PA) for quantification purposes as
Myc regulation by Romo1
described earlier (Kim et al., 2010; Lee et al., 2010). For immunofluorescence
assays, cells were fixed in 3.7% paraformaldehyde (Sigma-Aldrich) for 10 minutes
at room temperature and stained using standard protocols. For quantification of
protein translocation, 100–200 cells were monitored in each experiment by
fluorescence microscopy and were validated as described previously (Gao et al.,
2009; Lin et al., 2009).
Flow cytometric analysis
For analysis of cell cycle profile by FACS, cells were harvested in a time-dependent
manner after induction, fixed with ethanol, stained with propidium iodide (PI, 50
g/ml, Sigma-Aldrich) containing RNase A (100 mg/ml, Sigma-Aldrich) for 30
minutes at room temperature. The DNA content was analyzed using a FACScan flow
cytometer (Becton Dickinson, San Jose, CA).
Cell fractionation assay
The Nuclear extract kit (California, USA) was used to perform cellular fractionation
in accordance with the manufacturer’s instructions. The purity of the extract was
confirmed by western blot analysis against anti-cytosol-specific--actin (SigmaAldrich) or anti-nuclear-specific-lamin B1.
Protein stabilization analysis and in vitro ubiquitylation assay
Journal of Cell Science
For protein stabilization analysis, HeLa cells were transfected with the indicated
constructs. After transfection for 48 h, cells were treated with cycloheximide (CHX,
20 g/ml). The cell lysates were prepared and analyzed by western blot analysis.
After CHX treatment, endogenous or exogenous Myc levels were quantified by
densitometric scanning in the image J program. For Myc ubiquitylation, cells were
transfected with Ubiquitin (Ub)-HA plasmid together with various constructs for 2
days and then treated with MG132 (10 M) for 6 h. The immunoprecipitates were
subjected to western blot analysis as described previously (Kim et al., 2010).
Statistical analysis
Each assay was performed in triplicate and independently repeated at least three
times. Statistical significance was defined as P<0.05. Means, SEs and Ps were
calculated using GraphPad PRISM version 4.02 for Windows (GraphPad Software,
San Diego, CA).
This research was supported by Basic Science Research Program
through the National Research Foundation of Korea (NRF) funded by
the Ministry of Education, Science and Technology (2010-0021371),
by a grant from the National R&D Program for Cancer Control,
Ministry for Health, Welfare and Family Affairs, Republic of Korea
(1020180), by National Nuclear R&D Program through the National
Research Foundation of Korea (NRF) funded by the Ministry of
Education, Science and Technology (20100018574) and by a grant of
the Korea Healthcare Technology R&D Project, Ministry for Health,
Welfare & Family Affairs, Republic of Korea (A084537-09020000100).
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/124/11/1911/DC1
References
Alvarez, E., Northwood, I. C., Gonzalez, F. A., Latour, D. A., Seth, A., Abate, C.,
Curran, T. and Davis, R. J. (1991). Pro-Leu-Ser/Thr-Pro is a consensus primary
sequence for substrate protein phosphorylation. Characterization of the phosphorylation
of Myc and c-jun proteins by an epidermal growth factor receptor threonine 669 protein
kinase. J. Biol. Chem. 266, 15277-15285.
Arnold, H. K. and Sears, R. C. (2008). A tumor suppressor role for PP2A-B56alpha
through negative regulation of c-Myc and other key oncoproteins. Cancer Metastasis
Rev. 27, 147-158.
Bader, J. P., Hausman, F. A. and Ray, D. A. (1986). Intranuclear degradation of the
transformation-inducing protein encoded by avian MC29 virus. J. Biol. Chem. 261,
8303-8308.
Bashir, T. and Pagano, M. (2003). Aberrant ubiquitin-mediated proteolysis of cell cycle
regulatory proteins and oncogenesis. Adv. Cancer Res. 88, 101-144.
Benassi, B., Fanciulli, M., Fiorentino, F., Porrello, A., Chiorino, G., Loda, M., Zupi,
G. and Biroccio, A. (2006). c-Myc phosphorylation is required for cellular response to
oxidative stress. Mol. Cell 21, 509-519.
Bhatia, K., Huppi, K., Spangler, G., Siwarski, D., Iyer, R. and Magrath, I. (1993).
Point mutations in the c-Myc transactivation domain are common in Burkitt’s lymphoma
and mouse plasmacytomas. Nat. Genet. 5, 56-61.
Burdon, R. H. and Rice-Evans, C. (1989). Free radicals and the regulation of mammalian
cell proliferation. Free Radic. Res. Commun. 6, 345-358.
Carrano, A. C., Eytan, E., Hershko, A. and Pagano, M. (1999). SKP2 is required for
ubiquitin-mediated degradation of the CDK inhibitor p27. Nat. Cell Biol. 1, 193-199.
Chung, J. S., Lee, S. B., Park, S. H., Kang, S. T., Na, A. R., Chang, T. S., Kim, H. J.
and Yoo, Y. D. (2009). Mitochondrial reactive oxygen species originating from Romo1
1923
exert an important role in normal cell cycle progression by regulating p27(Kip1)
expression. Free Radic. Res. 43, 729-737.
Chung, Y. M., Kim, J. S. and Yoo, Y. D. (2006). A novel protein, Romo1, induces ROS
production in the mitochondria. Biochem. Biophys. Res. Commun. 347, 649-655.
Chung, Y. M., Lee, S. B., Kim, H. J., Park, S. H., Kim, J. J., Chung, J. S. and Yoo, Y.
D. (2008). Replicative senescence induced by Romo1-derived reactive oxygen species.
J. Biol. Chem. 283, 33763-33771.
Cross, D. A., Alessi, D. R., Cohen, P., Andjelkovich, M. and Hemmings, B. A. (1995).
Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B.
Nature 378, 785-789.
Finkel, T. (2003). Oxidant signals and oxidative stress. Curr. Opin. Cell Biol. 15, 247-254.
Finkel, T. and Holbrook, N. J. (2000). Oxidants, oxidative stress and the biology of
ageing. Nature 408, 239-247.
Gao, D., Inuzuka, H., Tseng, A., Chin, R. Y., Toker, A. and Wei, W. (2009).
Phosphorylation by Akt1 promotes cytoplasmic localization of Skp2 and impairs
APCCdh1-mediated Skp2 destruction. Nat. Cell Biol. 11, 397-408.
Grandori, C., Gomez-Roman, N., Felton-Edkins, Z. A., Ngouenet, C., Galloway, D.
A., Eisenman, R. N. and White, R. J. (2005). c-Myc binds to human ribosomal DNA
and stimulates transcription of rRNA genes by RNA polymerase I. Nat. Cell Biol. 7,
311-318.
Gregory, M. A. and Hann, S. R. (2000). c-Myc proteolysis by the ubiquitin-proteasome
pathway: stabilization of c-Myc in Burkitt’s lymphoma cells. Mol. Cell. Biol. 20, 24232435.
Gross-Mesilaty, S., Reinstein, E., Bercovich, B., Tobias, K. E., Schwartz, A. L.,
Kahana, C. and Ciechanover, A. (1998). Basal and human papillomavirus E6
oncoprotein-induced degradation of Myc proteins by the ubiquitin pathway. Proc. Natl.
Acad. Sci. USA 95, 8058-8063.
Hann, S. R. (2006). Role of post-translational modifications in regulating c-Myc
proteolysis, transcriptional activity and biological function. Semin. Cancer Biol. 16,
288-302.
Hann, S. R. and Eisenman, R. N. (1984). Proteins encoded by the human c-myc
oncogene: differential expression in neoplastic cells. Mol. Cell. Biol. 4, 2486-2497.
Henriksson, M., Bakardjiev, A., Klein, G. and Luscher, B. (1993). Phosphorylation
sites mapping in the N-terminal domain of c-myc modulate its transforming potential.
Oncogene 8, 3199-3209.
Herbst, A., Salghetti, S. E., Kim, S. Y. and Tansey, W. P. (2004). Multiple cell-typespecific elements regulate Myc protein stability. Oncogene 23, 3863-3871.
Hiramatsu, Y., Kitagawa, K., Suzuki, T., Uchida, C., Hattori, T., Kikuchi, H., Oda,
T., Hatakeyama, S., Nakayama, K. I., Yamamoto, T. et al. (2006). Degradation of
Tob1 mediated by SCFSkp2-dependent ubiquitination. Cancer Res. 66, 8477-8483.
Hwang, I. T., Chung, Y. M., Kim, J. J., Chung, J. S., Kim, B. S., Kim, H. J., Kim, J.
S. and Yoo, Y. D. (2007). Drug resistance to 5-FU linked to reactive oxygen species
modulator 1. Biochem. Biophys. Res. Commun. 359, 304-310.
Iiyama, M., Kakihana, K., Kurosu, T. and Miura, O. (2006). Reactive oxygen species
generated by hematopoietic cytokines play roles in activation of receptor-mediated
signaling and in cell cycle progression. Cell. Signal. 18, 174-182.
Kamura, T., Hara, T., Kotoshiba, S., Yada, M., Ishida, N., Imaki, H., Hatakeyama, S.,
Nakayama, K. and Nakayama, K. I. (2003). Degradation of p57Kip2 mediated by
SCFSkp2-dependent ubiquitylation. Proc. Natl. Acad. Sci. USA 100, 10231-10236.
Kim, J. J., Lee, S. B., Park, J. K. and Yoo, Y. D. (2010). TNF-[alpha]-induced ROS
production triggering apoptosis is directly linked to Romo1 and Bcl-XL. Cell Death
Differ. 17, 1420-1434.
Kim, S. Y., Herbst, A., Tworkowski, K. A., Salghetti, S. E. and Tansey, W. P. (2003).
Skp2 regulates Myc protein stability and activity. Mol. Cell 11, 1177-1188.
Lee, S. B., Kim, J. J., Kim, T. W., Kim, B. S., Lee, M. S. and Yoo, Y. D. (2010). Serum
deprivation-induced reactive oxygen species production is mediated by Romo1.
Apoptosis 15, 204-218.
Lin, H. K., Wang, G., Chen, Z., Teruya-Feldstein, J., Liu, Y., Chan, C. H., Yang, W.
L., Erdjument-Bromage, H., Nakayama, K. I., Nimer, S. et al. (2009).
Phosphorylation-dependent regulation of cytosolic localization and oncogenic function
of Skp2 by Akt/PKB. Nat. Cell Biol. 11, 420-432.
Lisztwan, J., Marti, A., Sutterluty, H., Gstaiger, M., Wirbelauer, C. and Krek, W.
(1998). Association of human CUL-1 and ubiquitin-conjugating enzyme CDC34 with
the F-box protein p45(SKP2): evidence for evolutionary conservation in the subunit
composition of the CDC34-SCF pathway. EMBO J. 17, 368-383.
Miura, M., Hatakeyama, S., Hattori, K. and Nakayama, K. (1999). Structure and
expression of the gene encoding mouse F-box protein, Fwd2. Genomics 62, 50-58.
Na, A. R., Chung, Y. M., Lee, S. B., Park, S. H., Lee, M. S. and Yoo, Y. D. (2008). A
critical role for Romo1-derived ROS in cell proliferation. Biochem. Biophys. Res.
Commun. 369, 672-678.
Nishitani, H., Sugimoto, N., Roukos, V., Nakanishi, Y., Saijo, M., Obuse, C., Tsurimoto,
T., Nakayama, K. I., Nakayama, K., Fujita, M. et al. (2006). Two E3 ubiquitin
ligases, SCF-Skp2 and DDB1-Cul4, target human Cdt1 for proteolysis. EMBO J. 25,
1126-1136.
Park, T. J., Kim, J. Y., Park, S. H., Kim, H. S. and Lim, I. K. (2009). Skp2 enhances
polyubiquitination and degradation of TIS21/BTG2/PC3, tumor suppressor protein, at
the downstream of FoxM1. Exp. Cell Res. 315, 3152-3162.
Saksela, K., Makela, T. P., Hughes, K., Woodgett, J. R. and Alitalo, K. (1992).
Activation of protein kinase C increases phosphorylation of the L-myc trans-activator
domain at a GSK-3 target site. Oncogene 7, 347-353.
Salghetti, S. E., Kim, S. Y. and Tansey, W. P. (1999). Destruction of Myc by ubiquitinmediated proteolysis: cancer-associated and transforming mutations stabilize Myc.
EMBO J. 18, 717-726.
1924
Journal of Cell Science 124 (11)
Journal of Cell Science
Sears, R., Leone, G., DeGregori, J. and Nevins, J. R. (1999). Ras enhances Myc protein
stability. Mol. Cell 3, 169-179.
Sears, R., Nuckolls, F., Haura, E., Taya, Y., Tamai, K. and Nevins, J. R. (2000).
Multiple Ras-dependent phosphorylation pathways regulate Myc protein stability. Genes
Dev. 14, 2501-2514.
Seth, A., Alvarez, E., Gupta, S. and Davis, R. J. (1991). A phosphorylation site located
in the NH2-terminal domain of c-Myc increases transactivation of gene expression. J.
Biol. Chem. 266, 23521-23524.
Tedesco, D., Lukas, J. and Reed, S. I. (2002). The pRb-related protein p130 is regulated
by phosphorylation-dependent proteolysis via the protein-ubiquitin ligase SCF(Skp2).
Genes Dev. 16, 2946-2957.
Turrens, J. F. (2003). Mitochondrial formation of reactive oxygen species. J. Physiol. 552,
335-344.
Tworkowski, K. A., Salghetti, S. E. and Tansey, W. P. (2002). Stable and unstable pools
of Myc protein exist in human cells. Oncogene 21, 8515-8520.
Vafa, O., Wade, M., Kern, S., Beeche, M., Pandita, T. K., Hampton, G. M. and Wahl,
G. M. (2002). c-Myc can induce DNA damage, increase reactive oxygen species, and
mitigate p53 function: a mechanism for oncogene-induced genetic instability. Mol. Cell
9, 1031-1044.
von der Lehr, N., Johansson, S., Wu, S., Bahram, F., Castell, A., Cetinkaya, C.,
Hydbring, P., Weidung, I., Nakayama, K., Nakayama, K. I. et al. (2003). The F-box
protein Skp2 participates in c-Myc proteosomal degradation and acts as a cofactor for
c-Myc-regulated transcription. Mol. Cell 11, 1189-1200.
Welcker, M., Orian, A., Jin, J., Grim, J. E., Harper, J. W., Eisenman, R. N. and
Clurman, B. E. (2004). The Fbw7 tumor suppressor regulates glycogen synthase
kinase 3 phosphorylation-dependent c-Myc protein degradation. Proc. Natl. Acad. Sci.
USA 101, 9085-9090.
Yada, M., Hatakeyama, S., Kamura, T., Nishiyama, M., Tsunematsu, R., Imaki, H.,
Ishida, N., Okumura, F., Nakayama, K. and Nakayama, K. I. (2004).
Phosphorylation-dependent degradation of c-Myc is mediated by the F-box protein
Fbw7. EMBO J. 23, 2116-2125.
Yeh, E., Cunningham, M., Arnold, H., Chasse, D., Monteith, T., Ivaldi, G., Hahn, W.
C., Stukenberg, P. T., Shenolikar, S., Uchida, T. et al. (2004). A signalling pathway
controlling c-Myc degradation that impacts oncogenic transformation of human cells.
Nat. Cell Biol. 6, 308-318.
Zhang, G. J., Safran, M., Wei, W., Sorensen, E., Lassota, P., Zhelev, N., Neuberg, D.
S., Shapiro, G. and Kaelin, W. G., Jr (2004). Bioluminescent imaging of Cdk2
inhibition in vivo. Nat. Med. 10, 643-648.