Activation of PI3K/Akt and MAPK pathways regulates
Myc-mediated transcription by phosphorylating and
promoting the degradation of Mad1
Jidong Zhu, John Blenis, and Junying Yuan*
Department of Cell Biology, Harvard Medical School, Boston, MA 02115
Communicated by Lewis C. Cantley, Harvard Institutes of Medicine, Boston, MA, March 19, 2008 (received for review January 11, 2008)
Mad1, a member of the Myc/Max/Mad family, suppresses Myc-
mediated transcriptional activity by competing with Myc for het-
erodimerization with its obligatory partner, Max. The expression
of Mad1 suppresses Myc-mediated cell proliferation and transfor-
mation. The levels of Mad1 protein are generally low in many
human cancers, and Mad1 protein has a very short half-life.
However, the mechanism that regulates the turnover of Mad1
protein is poorly understood. In this study, we showed that Mad1
is a substrate of p90 ribosomal kinase (RSK) and p70 S6 kinase
(S6K). Both RSK and S6K phosphorylate serine 145 of Mad1 upon
serum or insulin stimulation. Ser-145 phosphorylation of Mad1
accelerates the ubiquitination and degradation of Mad1 through
the 26S proteasome pathway, which in turn promotes the tran-
the growth factor signaling pathways regulated by PI3 kinase/Akt
and MAP kinases with Myc-mediated transcription.
phosphorylation ? RSK ? S6K1 ? ubiquitination ? proliferation
2), is recognized as an important cellular antagonist of Myc (1,
2). Mad1 antagonizes Myc function by recruiting Max and the
mSin3 repressor complex to Myc-responsive elements, directly
competing with the Myc–Max dimer for access to transcriptional
sites (1, 2). The half-life of Mad1 protein, like that of Myc
protein, is very short (1), and the levels of Mad1 are tightly
regulated during cell proliferation and differentiation (2). In
contrast, the levels of Max protein were found to be constitutive
under different conditions (3, 4). Therefore, because both Myc
and Mad1 compete for Max (1), the availability of Max to Myc
is profoundly dependent on the expression levels of Mad1
protein (and vice versa).
PtdIns3 kinase (PI3K) is activated in response to mitogen
stimulation of multiple receptor tyrosine kinases and G protein-
coupled receptors. PI3K phosphorylates phosphatidylinositol-
4, 5-bisphosphate (PIP2) to generate phosphatidylinositol-3,4, 5-
trisphosphate (PIP3) (5). PIP3 is critical in mediating the
activation of a number of important downstream effector mol-
ecules, including Ser/Thr protein kinase Akt/PKB, which further
activates mammalian target of rapamycin (mTOR). mTOR in
turn propagates the signals by its two important downstream
ribosomal S6 kinase 1 (S6K1). eIF4E and S6K1 mediate mTOR-
dependent cell size control (6), as well as contribute to efficient
G1cell cycle progression (7).
A number of observations suggest that Myc specifically co-
operates with activated PI3K in deregulated cell proliferation
and transformation. For example, coexpression of c-Myc and
PI3K was found to be sufficient to transform early passage
human mammary epithelial cells and fibroblasts in vitro in the
presence of hTERT and SV40 large T (8). This cooperation has
been shown to occur indirectly by regulating downstream target
gene transcription. For example, the phosphorylation of FoxO
transcription factors by Akt has been shown to alleviate the
ad1 (encoded by the gene mxd1), a member of the
Myc/Mad/Max family known as bHLH/LZip proteins (1,
inhibition of Myc target genes by FoxO transcriptional factors
and to facilitate Myc-mediated transcription and cellular trans-
formation (9). However, no direct interaction of the PI3K
pathway with Myc/Max/Mad family of proteins has been
In this study, we show that the levels of Mad1 are rapidly
decreased upon mitogen stimulation, and this degradation is
regulated by the MAPK and PI3K/Akt pathways. We provide
evidence that both RSK and S6K1 phosphorylate Mad1 and
phosphorylation of Mad1 accelerates its ubiquitination and
proteasome-mediated degradation, thereby inhibiting the tumor
suppressor function of Mad1. Our findings provide a novel
mechanism by which the growth factor signaling pathways
cooperate with Myc to promote proliferation and cellular
The Cellular Levels of Mad1 Are Rapidly Reduced upon Mitogen or
Insulin Stimulation. Because Myc and Mad1 have been shown to
antagonistically regulate G0–S progression and the levels of Myc
proteins are increased during G0–S transition (10, 11), we
examined the abundance of Mad1 during cell cycle reentry after
starvation. HeLa cells were made quiescent by serum starvation
for 48 h and then reactivated by the addition of serum. Inter-
estingly, the levels of nuclear Mad1 were increased in serum-
starved cells, but rapidly reduced upon serum stimulation (Fig.
1A). Our previous results show Mad1 is exclusively in nuclei even
in the presence of the proteasome inhibitor MG132 (12). This
reduction of nuclear Mad1 levels, however, was not due to
nuclear export because Mad1 was not detected in the cytosolic
fractions in serum-stimulated cells (Fig. 1A). To test whether the
reduction of Mad1 upon serum stimulation was due to increased
proteasomal degradation, we pretreated quiescent HeLa cells
prevented the disappearance of nuclear Mad1 upon serum
addition (Fig. 1B), suggesting that the reduction of Mad1 levels
in serum-stimulated cells was due to proteasomal degradation.
To further exclude the possibility that the reduction of Mad1
levels was due to transcriptional regulation, we generated a
stable HeLa cell line expressing Flag-tagged Mad1 (HeLa-Flag-
Mad1) driven by the murine phosphoglycerate kinase (PGK)
promoter. This polyclonal stable cell line was made by infecting
HeLa cells with virus expressing Flag-tagged Mad1 and selected
surviving cells as a pool. Similar to those of endogenous Mad1,
Author contributions: J.Z., J.B., and J.Y. designed research; J.Z. performed research; J.Z.,
J.B., and J.Y. analyzed data; and J.Z. and J.Y. wrote the paper.
The authors declare no conflict of interest.
*To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
May 6, 2008 ?
vol. 105 ?
serum stimulation also down-regulated the levels of Flag-tagged
Mad1 (Fig. 1C). Furthermore, the addition of insulin (10 ?g/ml)
to quiescent HeLa-Flag-Mad1 cells also reduced the levels of
Flag-tagged Mad1, which was inhibited by the presence of
MG132 (Fig. 1 C and D). Because serum or high concentration
MAPK pathways, we hypothesized that the activation of PI3K/
Akt or MAPK may be involved in mediating the degradation of
Mad1. Consistent with this possibility, the endogenous Mad1
levels were rapidly increased in serum-starved HeLa cells, and
correlated with the dephosphorylation of Akt and ERK
protein demonstrated reduced protein mobility on SDS-page
electrophoresis in cells stimulated with insulin in the presence of
MG132 (Fig. 1D), we hypothesized that Mad1 may be phos-
phorylated as a result of PI3K/Akt or MAPK pathway activation.
To test this hypothesis, we analyzed the Mad1 protein sequence
using the Scansite (http://scansite.mit.edu) and found a con-
served consensus phosphorylation site (RXRXXS) in Mad1 for
2A). Using an in vitro kinase assay, we found that both S6K1 and
RSK could robustly phosphorylate purified GST-Mad1, but not
GST alone (Fig. 2B). The phosphorylation was largely abolished
in Mad1 S145A mutant, suggesting that Ser-145 is a major site
of S6K1 and RSK phosphorylation in Mad1 (Fig. 2B). Similarly,
Akt could also phosphorylate purified GST-Mad1 mainly on
Ser-145 (Fig. 2C). These results indicated that all of the three
AGC kinases tested, Akt, RSK, and S6K, can phosphorylate
Mad1 in vitro.
We then tested whether overexpression of the three AGC
kinases leads to Mad1 phosphorylation in cells. The phosphor-
ylation of Mad1 at Ser-145 was monitored by Western blot
analysis with an anti-phospho-S145 Mad1-specific antibody. As
stimulation. (A) HeLa cells were cultured in the presence (S) or absence of serum
The lysates were fractionated and analyzed by Western blotting with the indi-
analyzed by Western blotting. (C) The experiment was performed as in A except
by using HeLa-Flag-Mad1 cells and stimulating with insulin (10 ?g/ml) as indi-
cated. (D) HeLa-Flag-Mad1 cells were starved overnight and pretreated with
min. The Mad1 levels were analyzed by Western blotting. (E) HeLa cells were
starved (SD) with serum-free medium for indicated amount of time. The cell
lysates were analyzed by using Western blotting with the indicated antibodies.
and previously reported AGC kinase substrates. (B) 293T cells were transfected with expression vector of Flag-S6K1. Cells were stimulated with insulin (10 ?g/ml) for
15 min. Activated Flag-S6K1 was immunoprecipitated from the cell lysate by using anti-Flag M2 beads and was eluted with Flag peptide. Purified GST, GST-Mad1, or
GST-Mad1S145A protein (1 ?g) was incubated with activated Flag-S6K1 or recombinant RSK1 in the presence of [?-32P] ATP.32P-labeled proteins were resolved by
as in B except by using recombinant activated Akt. (D) 293T cells were transfected with vectors encoding Flag-tagged Mad1 with kinase-dead, wild-type, or activated
?g/ml) for 15 min. Flag-Mad1 was immunoprecipitated and immunoblotted with anti-phospho-Mad1 and anti-Mad1 antibodies. Total cell extracts were analyzed by
Western blotting with the indicated antibodies. (E) 293T cells were transfected with vectors encoding Flag-tagged Mad1 wild-type or kinase-dead RSK1. After serum
starvation for 18 h, cells were treated with MG132 (10 ?M) for 1 h and stimulated with PMA (50 nM) for 15 min. Analysis was performed as in D. (F) 293T cells were
PD98059 (50 ?M) for 1 h and stimulated with PMA (50 nM) for 15 min. Analysis was performed as in D.
AGC kinases phosphorylates Mad1 in vitro and in cells. (A) Alignment of the amino acid sequences of the putative AGC kinase phosphorylation site in Mad1
Zhu et al.
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to an increase in basal phosphorylation of Mad1 compared with
control overexpression of a kinase-inactive S6K1 (containing a
K100R mutation) in the presence of proteasome inhibitor
MG132. The phosphorylation was increased by insulin (0.5
?g/ml, a weak stimulator of MAPK pathway at this dose)
treatment and was rapamycin-sensitive. Overexpression of an
activated allele of S6K1 (F5A-E389-R3A) led to constitutive
phosphorylation of Mad1 that was rapamycin-insensitive. To test
whether RSK can phosphorylate Mad1 in cells, we coexpressed
RSK1 with Mad1 in 293T cells, which resulted in a dramatic
increase in phosphorylation of Mad1 upon stimulation with
PMA (Fig. 2E). Moreover, when coexpressed in cells, kinase-
2E). The PMA-induced Mad1 phosphorylation was inhibited by
the MEK inhibitor PD98059 (Fig. 2F). Moreover, the mobility
shift of Mad1 induced by serum stimulation was completely
inhibited by S145A mutation [supporting information (SI) Fig.
S1], suggesting that Ser-145 is the major, if not the only,
phosphorylation site induced by serum stimulation. Overexpres-
sion of activated Akt also phosphorylated Mad1 in 293T cells
(data not shown). These results suggested that these AGC
kinases could phosphorylate Mad1 in vitro and in cells under
Mad1 is Phosphorylated by Endogenous RSK and S6K1 in Vivo upon
Serum Stimulation. Because Mad1 is degraded in cells upon
mitogen or insulin stimulation (Fig. 1), we tested whether
Ser-145 of Mad1 was also phosphorylated under these condi-
tions. Because of the low expression levels of Mad1 protein in all
of the cell lines that we have tested and because our phospho-
S145Mad1 antibody was not sensitive enough to detect phos-
phorylation of endogenous Mad1 in routine Western blot anal-
ysis, we first used a HeLa-Flag-Mad1 stable cell line to
investigate the regulation of Mad1 phosphorylation. As shown in
Fig. 3A, Ser-145 of Flag-tagged Mad1 was indeed phosphory-
lated upon serum or insulin (10 ?g/ml) stimulation in the
presence of proteasome inhibitor MG132. Consistent with the
role of AGC kinases in mediating Ser-145 phosphorylation of
Mad1 in vitro, phosphorylation of Flag-tagged Mad1 in HeLa
cells was also inhibited by the combination of PI3K inhibitor
LY294002 and MEK inhibitor PD98059. Interestingly, treatment
of LY294002 or PD98059 alone was not sufficient to inhibit the
Ser-145 phosphorylation of Mad1. This result suggests that both
ERK/RSK and PI3K/Akt signaling modules are involved in
Ser-145 phosphorylation of Mad1. Consistent with this proposal,
serum stimulation-induced Mad1 Ser-145 phosphorylation was
prevented only by the expression of both dominant negative
mutants of Akt (Akt-DN) and RSK1 (RSK1?/?). Expression of
either inactive Akt or inactive RSK alone, however, was not
sufficient to block the phosphorylation of Ser-145 Mad1 (Fig.
3B), suggesting that Akt and RSK may act in a redundant fashion
on Mad1 phosphorylation.
S6K1 is a downstream effector kinase of Akt upon serum or
insulin stimulation, and is phosphorylated and activated in a
rapamycin-sensitive manner by mTOR (13). S6K1 activation is
also mediated by the Ras pathway via RSK-dependent phos-
was immunoprecipitated with anti-Flag antibody and immunoblotted with anti-phospho-Mad1 and anti-Mad1 antibodies. Total cell extracts were analyzed by
(RSK1?/?). The cells were serum-starved for 18 h, pretreated with MG132 (10 ?M) for 1 h and stimulated with serum for 15 min. Analysis was performed as in
A. (C) The experiment was performed as in B except that the cells were transfected with kinase-dead S6K1 (K/R) as indicated. (D) The experiment was performed
as in A except with rapamycin (20 nM) as indicated. (E) HeLa-Flag-Mad1 cells were transfected with siRNAs targeting S6K1 and/or RSK1/2 as indicated. The total
amount of siRNA was normalized by addition of a nontarget siRNA. Forty hours after transfection, cells were starved for 18 h, pretreated with MG132 (10 ?M),
and stimulated with serum for 15 min. Analysis was performed as in A. (F) HeLa cells (?108) were starved, pretreated, and stimulated as in A. Mad1 was
immunoprecipitated by using anti-Mad1 antibody. The immunoprecipitates and cell lysates were analyzed by Western blotting with the indicated antibodies.
PD, PD98059; LY, LY 294004; Rap, rapamycin.
Mad1 is phosphorylated by endogenous RSK and S6K1 in vivo upon serum stimulation. (A) HeLa-Flag-Mad1 cells were starved for 18 h, pretreated with
www.pnas.org?cgi?doi?10.1073?pnas.0802785105 Zhu et al.
phorylation and inactivation of the Rheb GTPase-activating
protein TSC2 (14). To investigate whether Mad1 phosphoryla-
tion by RSK or Akt is mediated by S6K1, we coexpressed Mad1
with the kinase-inactive S6K1 (S6K1K/R) and/or kinase-inactive
RSK1 (RSK1?/?) in 293T cells. Expression of inactive RSK1 or
inactive S6K1 alone cannot inhibit Ser-145 Mad1 phosphoryla-
tion (Fig. 3C). However, coexpression of inactive RSK1 and
inactive S6K1 together, which had no effect on the activation of
Akt upon serum stimulation, totally prevented Mad1 phosphor-
ylation (Fig. 3C). This result suggests that both RSK and S6K1
can independently phosphorylate Mad1 after serum stimulation.
On the other hand, although Akt can phosphorylate Mad1 in
vitro (Fig. 2C), activated Akt is not sufficient to mediate Ser-145
phosphorylation of Mad1 (Fig. 3C), and thus Akt is not likely to
directly phosphorylate Mad1 in cells. To further confirm this
point, we treated HeLa-Flag-Mad1 cells with PD98059 and
rapamycin before serum stimulation. Consistent with the result
inhibited the activation of S6K1 and RSK and Ser-145 phos-
phorylation of Mad1, without affecting the activation of Akt
(Fig. 3D), whereas rapamycin alone is not sufficient to inhibit
Mad1 phosphorylation induced by serum (Fig. S2). Finally, using
siRNA, we found that knockdown of both RSK1/2 and S6K1 is
required for inhibiting S145 phosphorylation of Mad1 (Fig. 3E).
These results suggest that both endogenous RSK and S6K can
directly phosphorylate Mad1 on Ser-145 upon serum stimula-
tion, whereas Akt is likely not a physiological kinase for Mad1.
To examine whether Ser-145 of endogenous Mad1 is phos-
phorylated, we immunoprecipitated Mad1 in lysate prepared
from ?108HeLa cells. We found that anti-phospho-S145Mad1
recognized Mad1 in the immunoprecipitates from the cells
stimulated with serum, but not in those from unstimulated cells
or cells treated with PD98059 and rapamycin (Fig. 3F). This
provides further support for our hypothesis that both endoge-
nous RSK and S6K1 may mediate Ser-145 phosphorylation of
endogenous Mad1 in cells in response to serum stimulation.
Ser-145 Phosphorylation of Mad1 Promotes Serum-Induced Mad1
Degradation.We next tested whether phosphorylation of Mad1 at
Ser-145 was important for Mad1 degradation induced by serum
stimulation. As shown in Fig. 4A, serum stimulation quickly
induced polyubiquitination of Mad1, corresponding to the ap-
pearance of Mad1 phosphorylation at Ser-145. Furthermore, the
treatment of PD98059 and rapamycin together inhibited both
phosphorylation and ubiquitination of Mad1 (Fig. 4B), suggest-
ing that Ser-145 phosphorylation of Mad1 is important for its
degradation. To further test this hypothesis, we stimulated
mitogen-deprived HeLa-Flag-Mad1 cells with serum in the
PD98059 alone could not inhibit the degradation of Mad1, the
combination of these two inhibitors efficiently prevented the
proteolysis of Mad1 (Fig. 4C).
To determine the effect of S145 phosphorylation on the
stability of Mad1, we next analyzed the levels of S145A mutant
Mad1 after serum stimulation. We generated two polyclonal
stable HeLa cell lines expressing wild-type or S145A mutant
Mad1 by infecting HeLa cells with virus expressing wild-type or
S145A mutant Mad1 and selecting the survival cells as a pool.
Cells stably expressing wild-type or S145A Mad1 were deprived
of serum for 24 h and then restimulated with serum. The cell
lysates were harvested for analysis after different periods of
serum stimulation. The level of wild-type Mad1 was significantly
decreased within 30 min, whereas that of S145A mutant Mad1
remained unchanged upon serum addition (Fig. 4D). These data
indicate that Ser-145 phosphorylation of Mad1 is required for
serum-induced Mad1 degradation. To further confirm this, we
coexpressed Mad1 with activated S6K1 or wild-type RSK1 in
293T cells. Overexpression of S6K1 or RSK1 dramatically re-
duced the level of wild-type Mad1, but not that of S145A mutant
Mad1 (Fig. 4E), suggesting that S6K1 or RSK promoted Mad1
degradation by Mad1 phosphorylation at Ser-145.
We have reported that c-IAP1 is the E3 ligase for Mad1 and
promotes Mad1 ubiquitination and degradation (12). To test
whether c-IAP1 is mediated in this serum-induced Mad1 deg-
radation, we knocked down c-IAP1 in HeLa-Flag-Mad1 cells.
Serum stimulation induced Mad1 ubiquitination to a similar
extent in both control RNAi and c-IAP1 RNAi cells (Fig. S3).
These results indicated that the degradation of Mad1 induced by
mitogen stimulation is independent of c-IAP1.
S145A Mad1 Mutant is More Stable than Wild-Type Mad1. To further
explore the significance of Ser-145 phosphorylation, we used
retroviral infection to generate stable MCF10A and HeLa cells
expressing wild-type or S145A mutant Mad1. Consistent with a
destabilizing effect of Ser-145 phosphorylation, S145A Mad1
Mad1 in both MCF10A and HeLa stable cell lines (Fig. 5A and
data not shown). However, the mRNA levels of wild-type and
mutant Mad1 were not significantly different, ruling out the
possible effects of differential transcription or mRNA stability
(Fig. 5A). To confirm that the reduced wild-type Mad1 protein
levels were due to accelerated protein degradation, we used
cycloheximide (CHX) to inhibit de novo protein synthesis in
HeLa-Flag-Mad1 stable cells. Wild-type Mad1 was degraded
degradation. (A) HeLa-Flag-Mad1 cells were serum-starved for 18 h, pre-
treated with MG132 (10 ?M) for 1 h, and stimulated with serum for indicated
amount of time. Flag-Mad1 was immunoprecipitated and eluted with Flag
peptide. The elution was analyzed by Western blotting with the indicated
antibodies. (B) The experiment was performed as in A except that cells were
pretreated with MG132 and with or without a combination of PD98059 (50
?M) and rapamycin (20 nM). The immunoprecipitates and lysates were ana-
lyzed by Western blotting with the indicated antibodies. (C) HeLa-Flag-Mad1
cells were starved (SD) for 24 h, pretreated with various drugs as indicated for
1 h, and stimulated with serum (SS) for the indicated times. The lysates were
analyzed by Western blotting with the indicated antibodies. (D) HeLa cells
expressing wild-type or S145A mutant Mad1 were starved in serum-free
medium overnight. The cells were then stimulated with serum for the indi-
indicated antibodies. (E) 293T cells were transfected with vectors encoding
wild-type or S145A mutant Mad1 and S6K1 or RSK1 as indicated. After 24 h,
cells were lysed and the lysates were analyzed by Western blotting with the
indicated antibodies. LY, LY294002; PD, PD98059; Rap, rapamycin.
Ser-145 phosphorylation of Mad1 promotes serum-induced Mad1
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quickly, with an estimated half-life of ?10 min; on the other
upon CHX treatment for 10 min, the remaining Mad1 pool was
stable after CHX treatment for 60 min (Fig. 5B). These results
suggest that the degradation of Mad1 may be regulated by both
phosphorylation-dependent and phosphorylation-independent
To investigate whether ubiquitination of S145A Mad1 mutant
was reduced compared to that of wild-type Mad1, we coex-
pressed wild-type or S145A Mad1 with ubiquitin in 293T cells.
The polyubiquitination of S145A Mad1 mutant was significantly
reduced in both the presence and absence of MG132 compared
to that of wild-type Mad1, indicating that phosphorylation of
Mad1 at Ser-145 promotes Mad1 ubiquitination (Fig. 5C). In
contrast to that of up-regulation of endogenous Mad1 after
serum starvation (Fig. 1E), the levels of mutant Mad1 were
unchanged by serum starvation (Fig. 5D), indicating that Ser-145
phosphorylation of Mad1 is a key regulatory event in response
to mitogen stimulation.
Phosphorylation of Mad1 by RSK and S6K1 Promotes Cell Proliferation
and Myc-Mediated Transcription. The expression of Mad1 nega-
tively regulates Myc-mediated tumorigenesis by inhibiting cell
proliferation (15) and transformation (16). Consistent with these
reports, the expression of wild-type Mad1 in MCF10A cells led
to a reduced rate of cell proliferation, whereas cells expressing
S145A Mad1 mutant showed a slower growth rate than those
expressing wild-type Mad1 (Fig. 6A). Cell cycle analysis revealed
that MCF10A cells expressing mutant Mad1 had a larger pop-
ulation of G1-arrested cells and fewer S phase cells than those
expressing wild-type Mad1 (Fig. 6B).
The transcription of ornithine decarboxylase-1 (ODC1) and
Cyclin D2 (CCND2), two Myc-responsive genes, is repressed by
overexpression of Mad1 (17, 18). Consistent with these reports,
we found that the mRNA levels of ODC1 and CCND2 were
decreased in cells stably expressing Mad1. Importantly, the
repression by Mad1 was further enhanced in cells stably express-
ing the S145A Mad1 mutant (Fig. 6C).
Activation of Myc promotes cellular transformation, whereas
overexpression of Mad1 suppresses Myc/Ras-mediated transfor-
mation (16). To examine whether phosphorylation of Mad1 at
Ser-145 can affect the ability of Mad1 to suppress transforma-
tion, we tested the efficiency of foci formation in cells expressing
wild-type and S145A Mad1. MCF7 cells were infected with
retrovirus encoding vector alone, wild-type Mad1, or S145A
Mad1 mutant. After selection, cells were cultured for 12 days to
allow foci formation. MCF7 cells expressing wild-type Mad1
showed a modest reduction in the number of foci compared to
formed a dramatic reduction in foci formation (Fig. 6D). Taken
together, these results indicate that by phosphorylating Ser-145
of Mad1 and promoting Mad1 degradation, RSK and S6K1
alleviate the repression of Mad1 on its target genes to promote
cell proliferation and transformation.
The results presented here suggest that both PI3K/Akt/mTOR
and MAPK pathways regulate the Myc/Max/Mad1 network by
phosphorylating Mad1 and promoting its degradation (Fig. 6E).
Moreover, we show that S145A mutant Mad1 has an elevated
effect on inhibiting cell proliferation and transformation. These
were prepared from MCF10A cells stably expressing empty vector (MSCV),
wild-type Mad1, or S145A mutant Mad1 and analyzed by using Western
blotting with the indicated antibodies. The mRNA levels of Mad1 and ?-actin
were analyzed by RT-PCR. (B) HeLa cells expressing Mad1 or S145A mutant
anti-Mad1 and anti-tubulin antibodies. (C) 293T cells were transfected with
vectors encoding Flag-tagged Mad1 (wt) or S145A mutant Mad1 (sa) and
HA-tagged ubiquitin for 20 h and then treated with or without MG132 (10
?M) for 4 h before harvesting. Anti-Flag immunoprecipitates were analyzed
by immunoblotting with the indicated antibodies. (D) HeLa cells expressing
Mad1 or S145A mutant Mad1 were starved with serum-free medium (SD) and
harvested at indicated time points. The cell lysates were analyzed by Western
blotting with the indicated antibodies.
S145A Mad1 mutant is more stable than wild-type Mad1. (A) Lysates
tion and Myc-mediated transcription. (A) Equal numbers of MCF10A cells
stably expressing indicated proteins were seeded, and cell proliferation was
measured by counting cell number with a Levy hemacytometer. Data are
presented as the mean ? SD of three independent experiments. (B) The cell
or S145A mutant Mad1 were analyzed by FACS. (C) The total RNA from
MCF10A cells stably expressing empty vector, wild-type Mad1, or S145A
mutant Mad1 were extracted and analyzed by semiquantitative RT-PCR for
the levels of ODC1 or CCND2 mRNAs. ?-actin was used as an internal control.
(D) MCF7 stable cells expressing empty vector, wild-type Mad1, or S145A
mutant Mad1 were seeded at equal numbers (5 ? 104cells per plate) and
cultured for 12 days. The colonies were stained with crystal violet. The exper-
iment was repeated three times, and the representative images were shown.
c-IAP1 promotes phosphorylation-independent ubiquitination and degrada-
tion of Mad1.
Phosphorylation of Mad1 by RSK and S6K1 promotes cell prolifera-
www.pnas.org?cgi?doi?10.1073?pnas.0802785105Zhu et al.
findings provide a new strategy to antagonize Myc transcription Download full-text
activity by inhibiting both PI3K/Akt/mTOR and MAPK
Overexpression or amplification of the myc gene has been
detected in numerous solid tumors and blood malignancies (19).
In addition, earlier studies showed that Myc protein is required
for transformation by the oncogenic tyrosine kinases, including
BCR-Abl (20, 21) and Src (22). Expression of nonreceptor
tyrosine kinase v-Src results in tyrosine phosphorylation of
numerous substrates and activation of multiple signaling path-
ways. Several studies showed that although inhibition of Ras/
MAPK kinase pathway by expression of a dominant-negative
mutant of Ras was not sufficient to prevent transformation by
v-Src (23, 24), simultaneous inhibition of signaling by the Ras/
MAPK pathway and the PI3K-mTOR pathway essentially
blocked v-Src-induced transformation (25). These results are
consistent with our hypothesis that inhibition of both the Ras/
MAPK and PI3K/mTOR pathways is required to stabilize Mad1
protein and thereby antagonize Myc transcription activity.
Mad1 is a very short-lived protein, suggesting its level is tightly
regulated in cells. We have identified c-IAP1 as the E3 ligase for
Mad1. Overexpression of c-IAP1 decreases the level of Mad1
and thus facilitates Myc-induced transformation (12). However,
in this study we found that c-IAP1 is not required for Mad1
degradation induced by serum or insulin stimulation, suggesting
that multiple mechanisms exist in cells to regulate Mad1 level
(Fig. 6E). Consistent with this possibility, we found that ?50%
S145A mutant Mad1 was still degraded quickly upon cyclohex-
imide treatment, whereas the remaining pool of S145A mutant
persists for a long time (Fig. 5B). Interestingly, although c-IAP1
is not required for Mad1 degradation induced by serum stimu-
lation, our data suggest that inhibition of c-IAP1 may contribute
to the increase of Mad1 levels after starvation because the
binding between c-IAP1 and Mad1 is weakened after starvation
(data not shown), suggesting that Mad1 is stabilized during
starvation by keeping it away from its E3s, including c-IAP1.
Taken together, our study provides a mechanism to link
between the growth factor signaling pathways regulated by PI3
kinase/Akt and MAP kinases with Myc-mediated transcription.
Identification of the molecular mechanism that regulates the
protein levels of Mad1 provides a new strategy to suppress
Myc-mediated cellular transformation and tumorigenesis by
inhibiting both RSK and S6K1.
Materials and Methods
siRNAs. siRNA sequences targeting RSK1, RSK2, and S6K1 (Qiagen) are 5?-
TGCCACGTACTCCGCACTCAA-3?, 5?-CCGAGTGAGATCGAAGATGGA-3?, and
Antibodies. The following antibodies were used: anti-Mad1 C19 (Santa Cruz
Biotechnology); anti-Akt, anti-phospho-Akt (Ser-473), anti-ERK1/2, anti-
phospho-ERK1/2 (Thr-202/Tyr-204), anti-p90RSK, anti-phospho-p90RSK (Ser-
anti-HA (Covance); anti-ubiquitin (DAKO); anti-Flag M2 and anti-tubulin
(Sigma). Phospho-Mad1 antibody was raised against peptide ERIRMD-pS-
IGSTVSS and was custom-made by Proteintech Group.
In Vitro Kinase Assay. GST fusion proteins (1 ?g) were incubated with recom-
binant active Akt (50 ng, Upstate Biotechnology) or RSK1 (50 ng, Echelon
Biosciences) in 25 ?l of kinase buffer [8 mM Mops/NaOH (pH 7.0), 0.2 mM
EDTA, 10 mM MgAc and 0.1 mM ATP] at 30°C for 30 min in the presence of
5 ?Ci [?-32P]ATP. The reaction products were resolved by SDS/PAGE and
32P-labeled proteins visualized by autoradiography.
Foci Formation Assay. MCF7 stable cells were seeded to 6-cm diameter culture
with crystal violet.
ACKNOWLEDGMENTS. We thank Drs. R. N. Eisenman, A. Degterev, and Y. Lin
for plasmids. This work was supported in part by the Innovator’s Award from
the Department of Defense (5DP10D580) and National Institute on Aging
Merit Award 5 R37 AG012859 (to J.Y.).
1. Ayer DE, Kretzner L, Eisenman RN (1993) Mad: A heterodimeric partner for Max that
antagonizes Myc transcriptional activity. Cell 72:211–222.
2. Rottmann S, Lu ¨scher B (2006) The Mad side of the Max network: Antagonizing the
function of Myc and more. Curr Top Microbiol Immunol 302:63–122.
3. LarssonLG,PetterssonM,ObergF,NilssonK,Lu ¨scherB(1994)Expressionofmad,mxi1,
max and c-myc during induced differentiation of hematopoietic cells: Opposite regu-
lation of mad and c-myc. Oncogene 9:1247–1252.
4. Wagner AJ, Le BM, Diaz MO, Hay N (1992) Expression, regulation, and chromosomal
localization of the Max gene. Proc Natl Acad Sci USA 89:3111–3115.
5. Vanhaesebroeck B, et al. (2001) Synthesis and function of 3-phosphorylated inositol
lipids. Ann Rev Biochem 70:535–602.
6. Fingar DC, Salama S, Tsou C, Harlow E, Blenis J (2002) Mammalian cell size is controlled
by mTOR and its downstream targets S6K1 and 4EBP1/eIF4E. Genes Dev 16:1472–1487.
7. Fingar DC, et al. (2004) mTOR controls cell cycle progression through its cell growth
effectors S6K1 and 4E-BP1/eukaryotic translation initiation factor 4E. Mol Cell Biol
8. Zhao JJ, et al. (2003) Human mammary epithelial cell transformation through the
activation of phosphatidylinositol 3-kinase. Cancer Cell 3:483–495.
and transformation required Akt-mediated phosphorylation of FoxO proteins. EMBO
cellularity and impaired mitogen-induced proliferation. Oncogene 20:1164–1175.
11. Walker W, Zhou ZQ, Ota S, Wynshaw-Boris A, Hurlin PJ (2005) Mnt-Max to Myc-Max
complex switching regulates cell cycle entry. J Cell Biol 169:405–413.
12. Xu L, et al. (2007) c-IAP1 cooperates with Myc by acting as a ubiquitin ligase for Mad1.
Mol Cell 28:914–922.
13. Burnett PE, Barrow RK, Cohen NA, Snyder SH, Sabatini DM (1998) RAFT1 phosphory-
lation of the translational regulators p70 S6 kinase and 4E-BP1. Proc Natl Acad Sci USA
14. Roux PP, Ballif BA, Anjum R, Gygi SP, Blenis J (2004) Tumor-promoting phorbol esters
and activated Ras inactivate the tuberous sclerosis tumor suppressor complex via p90
ribosomal S6 kinase. Proc Natl Acad Sci USA 101:13489–13494.
15. Roussel MF, Ashmun RA, SHerr CJ, Eisenman RN, Ayer DE (1996) Inhibition of cell
proliferation by the Mad1 transcriptional repressor. Mol Cell Biol 16:2796–2801.
16. Lahoz EG, Xu L, Schreiber-Agus N, DePinho RA (1994) Suppression of Myc, but not E1a,
transformation activity by Max-associated proteins, Mad and Mxi1. Proc Natl Acad Sci
17. Auvinen M, et al. (2003) Transcriptional regulation of the ornithine decarboxylase
gene by c-Myc/Max/Mad network and retinoblastoma protein interacting with c-Myc.
Int J Biochem Cell Biol 35:496–521.
network: Myc-dependent TRRAP recruitment and histone acetylation at the cyclin D2
promotor. Genes Dev 15:2042–2047.
20. Sawyers CL, Callahan W, Witte ON (1992) Dominant negative MYC blocks transforma-
tion by ABL oncogenes. Cell 70:901–910.
21. Afar DE, Goga A, McLaughlin J, Witte ON, Sawyers CL (1994) Differential complemen-
tation of Bcr-Abl point mutants with c-Myc. Science 264:424–426.
22. Bowman T, et al. (2001) Stat3-mediated Myc expression is required for Src transfor-
mation and PDGF-induced mitogenesis. Proc Natl Acad Sci USA 98:7319–7324.
Acad Sci USA 94:3028–3033.
24. Oldham SM, et al. (1998) Ras, but not Src, transformation of RIE-1 epithelial cells
depends on activation of the mitogen-activated protein kinase cascade. Oncogene
25. Penuel E, Martin GS (1999) Transformation by v-Src: Ras-MAPK and PI3K-mTOR medi-
ate parallel pathways. Mol Biol Cell 10:1693–1703.
Zhu et al.
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