mTOR Generates an Auto-Amplification Loop
by Triggering the bTrCP- and CK1a-Dependent
Degradation of DEPTOR
Shanshan Duan,1,2Jeffrey R. Skaar,1,2Shafi Kuchay,1,2Alfredo Toschi,1Naama Kanarek,3Yinon Ben-Neriah,3
and Michele Pagano1,2,*
1Department of Pathology, NYU Cancer Institute, New York University School of Medicine, 522 First Avenue, SRB 1107, New York,
NY 10016, USA
2Howard Hughes Medical Institute
3The Lautenberg Center for Immunology, Hebrew University, Jerusalem 91120, Israel
DEPTOR is a recently identified inhibitor of the
mTOR kinase that is highly regulated at the post-
that DEPTOR was rapidly phosphorylated on three
serines in a conserved degron, facilitating binding
and ubiquitylation by the F box protein bTrCP, with
consequent proteasomal degradation of DEPTOR.
Phosphorylation of the bTrCP degron in DEPTOR is
executed by CK1a after a priming phosphorylation
event mediated by either the mTORC1 or mTORC2
complexes. Blocking the bTrCP-dependent degra-
dation of DEPTOR via bTrCP knockdown or expres-
sion of a stable DEPTOR mutant that is unable to
bind bTrCP results in mTOR inhibition. Our findings
reveal that mTOR cooperates with CK1a and bTrCP
to generate an auto-amplification loop to promote
its own full activation. Moreover, our results suggest
that pharmacologic inhibition of CK1 may be a viable
therapeutic option for the treatment of cancers char-
acterized by activation of mTOR-signaling pathways.
The mammalian target of rapamycin (mTOR) kinase controls
many aspects of the response to growth factor and nutrient
signaling (Laplante and Sabatini, 2009; Zoncu et al., 2011).
mTOR complex 1 (TORC1) and TORC2 share the mTOR and
mLST8/GbL proteins, but the complexes also feature distinct
subunits, with RAPTOR and PRAS40 in TORC1 and RICTOR,
PROTOR, and mSin1 in TORC2 (Laplante and Sabatini, 2009).
In general, TORC1 controls mRNA translation, ribosome biogen-
esis, cell growth, and autophagy through substrates such as
S6K1 and 4E-BP1, whereas TORC2 controls cell proliferation,
cell survival, and the cytoskeleton through substrates such as
Akt, SGK1, and PGCa (Dancey, 2010; Guertin and Sabatini,
2007; Sengupta et al., 2010).
The pathways controlled by TORC1 and TORC2 are
frequently activated in tumors by mutations in upstream sig-
naling factors (e.g., growth factor receptors, PI3K regulators,
or PTEN), and mTOR inhibitors have been used successfully in
the treatment of several cancers (Dancey, 2010; Hay, 2005).
However, direct activating mutations of mTOR have not been
observed in cancer, and in some settings, mTOR has been
shown to possess tumor-suppressive properties, likely due to
negative feedback loops that control the TORC1 or TORC2
pathways (Laplante and Sabatini, 2009).
Both mTOR complexes are directly inhibited by DEPTOR,
which binds and inhibits mTOR through a PDZ domain (Peterson
et al., 2009). DEPTOR is downregulated in many tumors, sug-
gesting a tumor suppressor function, which is consistent with
the activation of mTOR in many tumors. However, DEPTOR is
overexpressed in multiple myeloma via transcription or copy
number amplifications, and this overexpression is necessary for
Akt activation and cell survival, which is likely mediated through
the feedback inhibition of PI3K (Carrasco et al., 2006; Peterson
et al., 2009). Notably, despite a general downregulation of
DEPTOR across other tumor types, amplification of the genomic
region containing the DEPTOR locus is an indicator of poor
prognosis or tumor progression in tumor subsets from multiple
cancers, including breast cancer, prostate cancer, lung cancer,
and CML. DEPTOR is overexpressed in many of these tumors
The impact of DEPTOR in cancer makes it vital to understand
the regulation of DEPTOR. DEPTOR activity appears to be regu-
lated largely through the control of DEPTOR levels, which are
tightly controlled both transcriptionally and posttranslationally
in response to growth factor signaling (Peterson et al., 2009).
Although DEPTOR levels are high in the absence of serum, in
response to serum, transcription of DEPTOR decreases and
DEPTOR protein is rapidly phosphorylated on as many as 13
sites. Many of these phosphorylations are mTOR dependent,
and nonphosphorylated mutants of DEPTOR bind mTOR more
efficiently than wild-type DEPTOR, indicating that phosphoryla-
tion of DEPTOR inhibits binding to mTOR. mTOR activity also
correlates with DEPTOR degradation, suggesting that these
two processes are linked. However, the precise mechanisms
for this regulation remain unclear.
Molecular Cell 44, 317–324, October 21, 2011 ª2011 Elsevier Inc. 317
Skp1/Cul1/F box protein (SCF) ubiquitin ligase complexes
control the degradation of many important regulatory proteins
(Cardozo and Pagano, 2004). In mammals, there are 69 SCF
ing subunit (Jin et al., 2004). In this study, we identify SCFbTrCP
as the ubiquitin ligase for DEPTOR and demonstrate that
SCFbTrCPmediates the mTOR- and CK1a-dependent degrada-
tion of DEPTOR.
The expression of Cul1(1–252), adominant negativeCul1 mutant
that binds Skp1 and F box proteins but cannot recruit an E2
ubiquitin conjugating enzyme, results in the accumulation of
SCF substrates (Piva et al., 2002; Yen and Elledge, 2008).
To identify new SCF substrates, we transiently transfected
Cul1(1–252) into HeLa cells and analyzed cell extracts for the
levels of several regulators of cell proliferation by immunoblot-
ting. The level of DEPTOR increased compared to mock trans-
fected controls (Figure S1A available online), suggesting that
DEPTOR is an SCF substrate. Therefore, we investigated
which F box protein targets DEPTOR to the SCF, using a library
of F box protein cDNAs. Screening of the FBXW (F box proteins
with WD40 repeats) family proteins, as well as Cdc20 and Cdh1
(WD40 domain-containing subunits of an SCF-like ubiquitin
ligase), revealed that endogenous DEPTOR specifically interacts
with bTrCP1 and bTrCP2 (Figure 1A), paralogous F box proteins
that share identical biochemical properties and substrates.
(In this article, bTrCP will refer to both, unless specified.)
Significantly, the binding of DEPTOR to transiently expressed
bTrCP was dependent on the substrate-binding domain, as
Figure 1. DEPTOR Is a Serum-Dependent Sub-
strate of bTrCP
(A) DEPTOR binds bTrCP1 and bTrCP2. HEK293T cells
proteins. Forty-eight hours posttransfection, after a serum
starvation of 16 hr, cells were restimulated with media
containing serum and MG132 for 3 hr prior to harvesting
for immunoprecipitations and immunoblotting as indi-
cated. Asterisks indicate the position of exogenously ex-
(B) HEK293T cells were transfected with an empty vector
(EV) or FLAG-tagged DEPTOR. Forty-eight hours post-
transfection, after a serum starvation of 24 hr, cells were
pretreated with the indicated drugs for 2 hr and then
stimulated with serum-containing media (SR) for 3 hr prior
to harvesting for immunoprecipitations and immunoblot-
ting as indicated. WCL, whole cell lysate.
(C) During a serum starvation of 72 hr, T98G cells were
transfected with siRNAs targeting either LacZ or bTrCP1
and bTrCP2. Cells were subsequently restimulated with
media containing serum and cycloheximide (SR+CHX),
and samples were harvested at the indicated time points
demonstrated by the inability of a previously
established substrate-binding point mutant,
bTrCP2(R434A), or a WD40 repeat deletion
mutant, bTrCP2(DWD40) (Suzuki et al., 2000;
Wu et al., 2003), to bind endogenous DEPTOR (Figure S1B).
Serum starvation of HeLa cells induces accumulation of
DEPTOR, whereas serum stimulation results in DEPTOR degra-
dation (Peterson et al., 2009). After confirming these results
in T98G cells (Figure S1C), we found that serum stimulation
induced a significant increase in the binding of DEPTOR to
endogenous bTrCP1 (Figure 1B).
To investigate the hypothesis that bTrCP controls the degra-
dation of DEPTOR in serum-stimulated cells, we reduced
the expression of both bTrCP1 and bTrCP2 in T98G and
HeLa cells using a validated siRNA (Dehan et al., 2009; Dorrello
et al., 2006; Fong and Sun, 2002; Guardavaccaro et al., 2008;
Peschiaroli et al., 2006). Figures 1C and S1D show that
silencing bTrCP increased the DEPTOR half-life upon stimula-
tion with serum, demonstrating that bTrCP controls DEPTOR
Next, we mapped the bTrCP binding motif in human DEPTOR.
Using deletion mutants, the binding motif was mapped to
a region of DEPTOR between amino acids 241 and 340 (Fig-
ure S2A). bTrCP binds substrates via phosphorylated residues
in conserved degradation motifs (degrons), typically including
the consensus sequence DpSGXXpS or similar variants, such
as pS/TpSGXXpS (Figure S2B). The bTrCP-binding region of
DEPTOR contains a conserved286SSGYFS291motif, matching
other bTrCP substrate degrons. To investigate whether DEPTOR
binds bTrCP via this motif, we generated serine to alanine
mutants and tested their binding to endogenous bTrCP1. Single
mutations of Ser286, Ser287, and Ser291 to Ala or a triple muta-
tion of Ser286/287/291 to Ala inhibited the interaction between
DEPTOR and bTrCP1, although the mutations did not affect
DEPTOR binding to endogenous mTOR (Figure 2A).
DEPTOR Degradation Requires SCFbTrCP
318 Molecular Cell 44, 317–324, October 21, 2011 ª2011 Elsevier Inc.
To confirm the role of phosphorylation in the interaction of
DEPTOR with bTrCP, we used immobilized, synthetic peptides
containing the candidate degron sequence to test binding to
bTrCP1. Although a peptide containing phosphorylated Ser286,
Ser287, and Ser291 efficiently bound bTrCP1 (but not Fbxw5 or
to bind bTrCP1 (Figure 2B). Accordingly, l-phosphatase treat-
ment of bTrCP1 immunoprecipitates abolished the interaction
with DEPTOR (Figure S2C). These results, together with the
analysis of point mutants (Figure 2A), the crystal structure of the
bTrCP1-b-catenin complex (Wu et al., 2003), and the modeling
indicate that phosphorylation of all three serine residues in the
DEPTOR degron (Ser286, Ser287, and Ser291) is necessary
for—and directly mediates—the interaction with bTrCP.
Figure 2. The DEPTOR Degron Is Controlled by Phosphorylation
(A)Ser286, Ser287,and Ser291arerequired fortheinteraction ofDEPTOR withbTrCP.HEK293Tcellsweretransfected withan empty vector(EV) ortheindicated
HA-DEPTOR constructs. Forty-eight hours posttransfection, after a serum starvation of 24 hr, cells were restimulated with media containing serum and MG132
for 3 hr prior to harvesting for HA immunoprecipitation and western blotting as indicated. WCL, whole cell lysate.
(B) The DEPTOR degron requires phosphorylation to bind bTrCP1. HEK293T lysates were used in binding reactions with beads coupled to a peptide containing
the sequence SSGYFS (lane 2) or the phosphomotif pSpSGYFpS (lane 3). Beads were washed with lysis buffer, and bound proteins were eluted and subjected
to SDS-PAGE and immunoblotting.
(C) In vivo phosphorylation of DEPTOR on Ser286/287/291/299 is induced by mitogens. HEK293T cells were transfected with the indicated HA-tagged DEPTOR
constructs. Following aserum deprivation(SD) of 24 hr, cells were stimulated with serum (SR) for 3hrin thepresenceor absenceof PP242 or D4476as indicated.
Whole cell lysates (WCL) were immunoprecipitated and immunoblotted as indicated.
or the indicated HA-DEPTOR constructs. Forty-eight hours posttransfection, after a serum starvation of 24 hr, cells were restimulated with media containing
serum and MG132 for 3 hr prior to harvesting for immunoprecipitations and immunoblotting as indicated. WCL, whole cell lysate.
DEPTOR Degradation Requires SCFbTrCP
Molecular Cell 44, 317–324, October 21, 2011 ª2011 Elsevier Inc. 319
To further investigate DEPTOR phosphorylation, we used a
phosphospecific antibody against the pSpSGYFpS degron
motif. This antibody recognized wild-type DEPTOR, but not a
DEPTOR(S286/287/291A) mutant (Figure 2A). Additionally,
DEPTOR point mutants displayed decreasing levels of detec-
tion, suggesting that all three serines are phosphorylated and
contribute to recognition by this antibody. Significantly, we
found that DEPTOR was phosphorylated on its degron in
HEK293T cells in response to stimulation with serum, but it
was poorly phosphorylated in serum-starved HEK293T cells
Several bTrCP substrates, such as b-catenin, Cdc25A, Emi1,
Snail, Wee1, and YAP, are phosphorylated on their degrons
only after an initial phosphorylation event that either allows
binding to or exposure of a previously masked site for a second
kinase (Frescas and Pagano, 2008; Hunter, 2007). To investigate
whether a similar mechanism controls phosphorylation of the
DEPTOR degron, we mutated a number of residues flanking
the degron. Mutation of Ser279, Ser280, Ser292, Thr295,
Ser297, and Ser298 to Ala, singly or in combination, did not
inhibit DEPTOR binding to bTrCP1 (Figures 2A and S2D). In
contrast, mutation of Ser293 or Ser299, or both, strongly
reduced the interaction between bTrCP and DEPTOR, even in
serum-stimulated cells (Figures 2D and S2D). Additionally,
a DEPTOR phosphomimic mutant, in which Ser286, Ser287,
and Ser291 in the degron are mutated to Asp (DEPTOR(S286/
287/291D)), retains the ability to bind bTrCP1 even when
Ser293 and Ser299 are mutated to Ala (Figure 2D). The ability
of the phosphomimic degron mutant of DEPTOR to bind bTrCP,
together with the phosphopeptide experiment in Figure 2B,
demonstrates that Ser293 and Ser299 are dispensable for
binding a prephosphorylated degron and suggests that phos-
phorylation of Ser293 and Ser299 may function to prime the
phosphorylation of Ser286, Ser287, and Ser291.
We also used a phosphospecific antibody generated against
a DEPTOR peptide C terminal to the degron, with Ser299 phos-
phorylated. This antibody recognized wild-type DEPTOR, but
not DEPTOR(S299A) or DEPTOR(S293/299A) (Figure 2C and
data not shown). We found that DEPTOR was phosphorylated
on Ser299 in HEK293T cells stimulated with serum, but this
site was poorly phosphorylated in serum-starved cells (Fig-
ure 2C). Interestingly, of the five serines in DEPTOR that are
important for binding to bTrCP, four (Ser286, Ser287, Ser293,
and Ser 299) have been previously identified as phosphorylation
sites (Peterson et al., 2009; Ville ´n et al., 2007). Additionally,
a different study also identified these four serines as sites of
phosphorylation that are enriched after proteasome inhibition
(Gao et al., 2011 [this issue of Molecular Cell]).
To identify the kinase(s) involved in the phosphorylation and
degradation of DEPTOR, we performed a candidate search
using pharmacologic inhibition. We found that D4476 (a CK1
inhibitor) and PP242 (an mTOR inhibitor) counteracted the
destabilizing effect of serum on DEPTOR, whereas GSK3i IX
(a GSK3 inhibitor), U0126 (a MEK inhibitor), and API-2 (an Akt
inhibitor) had no effect (Figures S3A and S3B). Importantly,
D4476 and PP242, but not U0126 and GSK3i IX, inhibited the
interaction between DEPTOR and bTrCP and the phosphoryla-
tion of the DEPTOR degron (Figures 1B and 2C and data not
shown). In agreement with the involvement of mTOR in DEPTOR
degradation, we observed that low doses of rapamycin (an
mTORC1 inhibitor) and high doses of wortmannin (a PI3K inhib-
itor that, at high concentrations, inhibits mTOR) induced
DEPTOR stabilization (Figures S3B and S3C). We also found
that knockdown of mTOR or CK1a (but not CK1d or CK1ε)
resulted in accumulation of DEPTOR (Figures 3A and 3B).
Furthermore, silencing of either RAPTOR or RICTOR inhibited
the serum-dependent destabilization of DEPTOR, although to
a lesser extent than mTOR depletion (Figure 3A), indicating
that both mTORC1 and mTORC2 control DEPTOR turnover.
Wethenusedphosphomimic mutantsof DEPTORto studythe
hierarchy of mTOR- and CK1a-mediated phosphorylation of
DEPTOR. The binding of wild-type DEPTOR to endogenous
bTrCP is inhibited by either PP242 or D4476 (Figures 1B and
3C), but DEPTOR phoshomimic mutants are differentially
responsive to these inhibitors. The binding of DEPTOR(S286/
287/291D) to bTrCP is not inhibited by either D4476 or PP242
(Figure 3C). In contrast, the binding of DEPTOR(S293/299D)
to bTrCP is not inhibited by PP242 but is still inhibited by D4476
(Figure 3C). These findings suggest that mTOR phosphorylates
Ser293 and Ser299 to promote degron phosphorylation by
CK1a. Accordingly, although PP242 inhibited the phosphoryla-
tion of DEPTOR on both Ser299 and the degron, D4476 was
ure 2C). Finally, CK1a-mediated stimulation of the DEPTOR-
bTrCP interaction was inhibited by PP242 (Figure S3D).
To test whether CK1 and mTOR can phosphorylate DEPTOR
on its degron, we performed in vitro kinase assays using re-
combinant, bacterially expressed, and purified DEPTOR and
kinases. CK1 phosphorylated the degron of DEPTOR as shown
by western blotting with the phosphospecific antibody (Figures
S3E and S3F). In contrast, mTOR alone was unable to induce
phosphorylation of DEPTOR on Ser286, Ser287, and Ser291.
Importantly, incubation with mTOR enhanced the CK1-depen-
dent phosphorylation of DEPTOR, likely due to mTOR-depen-
dent phosphorylation of Ser293 and Ser299, as no mTOR-
dependent enhancement of phosphorylation was observed
with DEPTOR(S293/299A). Finally, mTOR, but not CK1, was
able to phosphorylate DEPTOR on Ser299 in vitro (Figures 3D
and 3E). Accordingly, Torin, a highly specific mTOR inhibitor,
inhibits in vivo phosphorylation of Ser293 and Ser299 (Peterson
et al., 2009); mTOR phosphorylates Ser293 and Ser299 in vitro;
and prephosphorylation of DEPTOR by mTOR enhances its
CK1-dependent in vitro phosphorylation (Gao et al., 2011).
Finally, we reconstituted the ubiquitylation of DEPTOR
in vitro. Wild-type DEPTOR, but not DEPTOR(S286/287/291A)
or DEPTOR(S293/299A), was ubiquitylated only when both
bTrCP1 and CK1 were present in the reaction (Figures 3D and
S3G). Moreover, in agreement with the phosphorylation data,
mTOR enhanced the bTrCP1- and CK1-dependent ubiquityla-
tion of DEPTOR.
Because the above results indicate that mTOR promotes
phosphorylation of the DEPTOR degron by CK1a, we further
with PP242 inhibits this binding. Additionally, purified recombi-
nant mTOR strongly stimulates the in vitro binding of CK1a to
DEPTOR Degradation Requires SCFbTrCP
320 Molecular Cell 44, 317–324, October 21, 2011 ª2011 Elsevier Inc.
wild-type DEPTOR, but not to DEPTOR(S293/299A) (Figure 3E),
suggesting that phosphorylation of Ser293 and Ser299 in
DEPTOR by mTOR generates a binding site for CK1a, thus
promoting DEPTOR phosphorylation by CK1a.
Altogether, these data strongly support a model in which,
in response to mitogenic stimulation, mTOR phosphory-
lates DEPTOR on Ser293 and Ser299, thus promoting the
CK1a-mediated phosphorylation ofDEPTOR on Ser286,
Ser287, and Ser291, facilitating bTrCP binding, SCFbTrCP-medi-
ated ubiquitylation, and consequent degradation. Therefore,
inhibition of bTrCP-mediated degradation of DEPTOR should
lead to increased DEPTOR levels and decreased mTOR activity.
This hypothesis was tested in three ways. First, T98G cells were
transfected with siRNAs against LacZ or bTrCP and synchro-
ulation with serum, DEPTOR levels rapidly decreased in the
Figure 3. mTOR and CK1a Are Required for DEPTOR Degradation
(A) Inhibition of TORC1 and TORC2 blocks DEPTOR degradation. During a serum starvation of 72 hr, T98G cells were transfected with siRNAs targeting LacZ,
RAPTOR, RICTOR, or mTOR. Cells were subsequently restimulated with media containing serum (SR), and samples were harvested at the indicated time points
(B) Silencing of CK1a, but not CK1d or CK1ε, induces DEPTOR accumulation. HeLa cells were infected with an empty lentivirus (EV) or lentiviruses containing
shRNA targeting CK1a, CK1d, or CK1ε. Seven days postinfection, samples were harvested at the indicated time points for immunoblotting.
(C) Differential sensitivity of wild-type DEPTOR and DEPTOR mutants to inhibition of CK1 and mTOR. HEK293T cells were transfected with the indicated HA-
DEPTOR constructs. Forty-eight hours posttransfection, after a serum starvation of 24 hr, cells were restimulated with media containing serum and MG132 for
3 hr in the presence or absence of PP242 or D4476 as indicated. Cell lysates were immunoprecipitated and immunoblotted as indicated. WCL, whole cell lysate.
(D) In vitro ubiquitylation assays of recombinant DEPTOR (WT or DEPTOR(S287/287/291A)) were conducted in the presence of the indicated proteins. Samples
were incubated at 30?C for 90 min. The bracket (top left) marks a ladder of bands corresponding to polyubiquitylated DEPTOR.
alone was incubated with ATP in the presence or absence of purified recombinant mTOR. Reaction products were then diluted, incubated with FLAG-tagged
CK1a, purified with GST Sepharose 4B beads, and immunoblotted as indicated.
DEPTOR Degradation Requires SCFbTrCP
Molecular Cell 44, 317–324, October 21, 2011 ª2011 Elsevier Inc. 321
siLacZ-transfected cells but decreased muchless in the sibTrCP
cells. In accordance with the increased DEPTOR levels in the
bTrCP knockdown samples, the induction of phosphorylated
S6K1 (Thr389) was severely blunted, demonstrating a decrease
in mTOR activation. Second, to confirm that the observed effect
of bTrCP knockdown on mTOR activity was mediated through
increased DEPTOR levels, we transiently transfected either
wild-type DEPTOR or DEPTOR(S286/287/291A) into HeLa cells,
which were subsequently serum starved for 24 hr before restim-
ulation with serum. As predicted, in contrast to wild-type
DEPTOR, DEPTOR(S286/287/291A) was not degraded when
cells were exposed to serum, and the mTOR-mediated phos-
phorylation of S6K1 in response to serum was strongly inhibited
(Figure S4A). Third, virtually identical results were obtained using
retroviruses that express DEPTOR(S286/287/291A) at near
physiological levels in T98G cells (Figure 4B). Significantly, after
serum stimulation, cells expressing DEPTOR(S286/287/291A)
displayed reduced cell size relative to cells expressing wild-
Proper regulation of mTOR activity is essential to blocking
tumorigenesis, and deregulation of the mTOR pathway at the
level of DEPTOR appears common (Peterson et al., 2009). Our
study demonstrates that DEPTOR is regulated at the posttrans-
lational level by the SCFbTrCPubiquitin ligase in an mTOR- and
CK1a-dependent manner, generating a positive feedback loop
that facilitates full activation of mTOR. The mTOR dependence
of this auto-amplification loop is reminiscent of the SCFSkp2-
mediated degradation of the CDK1/2 inhibitor p27 following
phosphorylation of p27 by CDK1 or CDK2 (Frescas and Pagano,
2008), suggesting a common mechanism for the regulation of
kinase inhibitors. Conversely, the observed effects of CK1a on
DEPTOR demonstrate a noncanonical mechanism for CK1,
which typically requires priming phosphorylation at the ?3
position. The negative charge of phosphorylated Ser293 and
Ser299 may function as acidic-like C-terminal residues to prime
the phosphorylation of Ser286/287/91, similar to other CK1
substrates (Marin et al., 2003).
Finally, our results show that pharmacologic inhibition of CK1
increases DEPTOR levels and inhibits mTOR signaling, suggest-
ing that CK1 inhibition may be a viable therapeutic option for the
treatment of cancers characterized by low DEPTOR levels and
activation of mTOR. Paradoxically, although mTOR activity is
required for DEPTOR degradation, multiple myelomas appear
to retain both high mTOR activity and high DEPTOR levels. It
remains undetermined whether the elevated levels of DEPTOR
Figure 4. Failure to Degrade DEPTOR Results in mTOR Activation Defects
(A) During a serum starvation of 72 hr, T98G cells were transfected with siRNAs targeting either LacZ or bTrCP1 and bTrCP2. Cells were subsequently
restimulated with media containing serum (SR), and samples were harvested at the indicated time points for immunoblotting as indicated.
(B) T98G cells were infected with an empty virus (EV), a retrovirus expressing wild-type DEPTOR, or DEPTOR(S286/287/291A).After a serum deprivation of 72 hr,
cells were restimulated with serum (SR) for the indicated times, harvested, and analyzed by immunoblotting.
(C) The experiment was performed as in (B), and cell size was determined by FACS (forward scatter) in cells deprived of serum (SD) and 24 hr after serum
DEPTOR Degradation Requires SCFbTrCP
322 Molecular Cell 44, 317–324, October 21, 2011 ª2011 Elsevier Inc.
in multiple myelomas result solely from transcriptional regulation
or whether the bTrCP-mediated degradation of DEPTOR is also
Extract Generation and Western Blotting
Extract preparation, immunoprecipitation, and western blotting were per-
formed as previously described (Dehan et al., 2009; Dorrello et al., 2006).
containing the phosphodegron sequence pSpSGYFpS. Fbxw5, Fbxw9, and
cyclin A rabbit polyclonal antibodies were generated/characterized by the
Pagano laboratory. Commercial mouse antibodies included S6K(Thr389)
(Cell Signaling), CK1ε (BD), b-Catenin (BD), Actin (Sigma), FLAG (Sigma), HA
(Covance), GST (Invitrogen), and Claspin (Peschiaroli etal.,2006).Commercial
rabbit antibodies included bTrCP1 (Cell Signaling), Akt (Ser473) (Cell
Signaling), Akt (Cell Signaling), S6K (Cell Signaling), GAPDH (Cell Signaling),
mTOR (Cell Signaling), DEPTOR (Millipore), DEPTOR(Ser299) (Cell Signaling),
CK1a (Cell Signaling), RICTOR (Bethyl), RAPTOR (Millipore), b-Catenin (Ser33/
37/Thr41) (Cell Signaling), and Skp1 (Invitrogen).
Plasmids, siRNAs, and shRNAs
DEPTOR mutants were generated using QuikChange Site-Directed Mutagen-
esis Kits (Stratagene). All cDNAs were completely sequenced. Transient
transfections of HEK293T cells were performed using polyethylenimine (PEI).
Additional cell lines were transfected using Lipofectamine 2000 (Invitrogen).
siRNAs were transfected using Metafectene Pro (Biontex). The LacZ and
bTrCP siRNAs were previously validated and described (Dehan et al., 2009;
Dorrello et al., 2006; Fong and Sun, 2002). RAPTOR, RICTOR, mTOR
siRNAs, and CK1a were from Sigma (SASI_Hs02_00366683, SASI_Hs01_
00048380, SASI_Hs01_00203144, and TRCN0000006042, respectively). The
Precision-LentiORF shRNA vector targeting CK1d and CK1ε contained the
sequence GGGCTTCTCCTATGACTAC. Retrovirus-mediated gene transfer
Cell Lines, Serum Starvation, and Drug Treatments
Human embryonic kidney 293T (HEK293T), HeLa, and T98G cells (ATCC) were
used as indicated. All cell lines were cultivated in Dulbecco’s modified Eagle’s
medium (DMEM), supplemented with 10% fetal bovine serum (Hyclone) and
antibiotics. All cells were starved for the indicated time periods in 0.1% serum.
The following drugs were used: MG132 (Peptides International; 10mM), cyclo-
heximide (Sigma; 100 mg/ml, PP242 (Sigma; 2.5 mM), D4476 (Calbiochem;
50 mM), GSK3i (Calbiochem; 5 mM), Rapamycin (LC labs, 200 nM), Akt inhibitor
API-2 (Tocris Bioscience, 1 mM), and U0126 (Calbiochem; 10 mM).
Supplemental Information includes Supplemental Experimental Procedures
and four figures and can be found with this article online at doi:10.1016/j.
The authors thank D. Foster, D. Sabatini, and W. Wei for reagents and W.
Harper and W. Wei for sharing unpublished results. M.P. is grateful to T.M.
Thor and K.E. Davidson for continuous support. J.R.S. is supported by the
Mr. and Mrs. William G. Campbell Postdoctoral Fellowship in Memory of
Carolyn Cabott from the American Cancer Society. This work was funded by
grants to M.P. from the National Institutes of Health (R01-GM057587,
R37-CA076584, and R21-CA161108) and the Multiple Myeloma Research
Foundation. M.P. is an Investigator with the Howard Hughes Medical Institute.
Received: April 5, 2011
Revised: July 5, 2011
Accepted: August 16, 2011
Published: October 20, 2011
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