Cell, Volume 126
Supplemental Data
TSC2 Integrates Wnt and Energy Signals
via a Coordinated Phosphorylation by
AMPK and GSK3 to Regulate Cell Growth Ken Inoki, Hongjiao Ouyang, Tianqing Zhu, Charlotta Lindvall, Yian Wang, Xiaojie Zhang, Qian Yang, Christina Bennett, Yuko Harada, Kryn Stankunas, Cun-yu Wang, Xi He, Ormond A. MacDougald, Ming You, Bart O. Williams, and Kun-Liang Guan
Figure S1. Wnt Activates mTOR Pathway
S1A. Wnt-1 does not activate ERK. Wnt-1 expressing RIE or Rat1 cells were treated
with various inhibitors as indicated. ERK protein and phospho-ERK were detected by
Western blots with specific antibodies.
S1B. Stimulation of S6K phosphorylation by Wnt-1 overexpression is blocked by
rapamycin. Wnt-1 or empty vector expressing HEK293T cells were treated with or
without rapamycin for 30 minutes. Phosphorylation of S6K and Akt was determined by
immunoblot analyses.
S1C. Activation of mTOR signaling by Wnt-1 conditioned media. 293T cells were
treated with Wnt-1 conditioned media or control media for one hour. Phosphorylation of
endogenous proteins was detected by Western blotting with indicated specific phospho-
antibodies. Wnt-1 conditioned media were derived from Wnt-1 stable expressing 293T
cells. Vector indicates the media from 293T cells stably transfected with empty vector.
S1D. Dose dependent activation of S6K by Wnt-3a. Serum starved MEF cells were
stimulated with various concentrations of Wnt-3A for 30 minutes as indicated.
S1E. Rapamycin blocks Wnt-3a-induced phosphorylation of 4EBP1 in MEF cells.
S1F. Time course of Wnt-3a stimulation. Serum starved MEF were treated with Wnt-
3a (300 ng/ml) for indicated times. Active β-catenin was detected by specific antibody
recognizing the unphosphorylated protein. Note that activation of S6K preceeded
accumulation of active β-catenin.
S1G. Wnt-3a stimulates phosphorylation of S6K in C2C12 cells. Serum starved C2C12
cells were treated with Wnt-3a (300 ng/ml) for the indicated times.
S1H. Wnt-3a stimulates S6K phosphorylation in TSC2 expressing LEF cells. Serum
starved LEF cells were treated with Wnt-3a (300 ng/ml) for 30 minutes.
Figure S2. Wnt Stimulates Cell Growth in Rat1 and RIE Cells
S2A. Wnt-1 increases size of Rat1 cells. Vector and Wnt-1 expressing Rat1 cells were
treated with or without rapamycin (10 nM for 48 hours). Cell size of the G1 population
was determined by FACS analysis. Rapamycin decreases cell size at a basal level and
diminishes the effect of Wnt-1 on cell size.
S2B. Wnt-1 increases size of RIE cells. Experiments are similar to panel S2A.
S2C. Wnt10b expression increases cell size. Wnt10b expressing ST2 cells were treated
with rapamycin (5 nM) for 48 hours. Cell size was determined by FACS analysis.
Figure S3. The Effect of Rapamycin on Wnt-1-Expressing Tumor Development in
Nude Mice
The arrows indicate injection of rapamycin. Closed squares and circles denote tumor
growth of mice injected with rapamycin and control, respectively.
Figure S4. Involvement of Wnt Pathway Components in S6K Phosphorylation
S4A. DKK1 does not inhibit insulin response. MC3T3E1 cells were treated with insulin
(200 nM for 10 minutes) in the presence or absence of soluble recombinant human DKK1
(hDKK1, 200 ng/ml, pretreatment for 2 hours) as indicated. Phosphorylation of S6K and
S6 were determined.
S4B. DKK1 inhibits Wnt-3a-induced mTOR signaling. MC3T3E1 cells were
pretreated with various concentrations of hDKK1 as indicated for 2 hours followed by
Wnt-3a treatment for 30 minutes. Phosphorylation of S6K was determined by
immunoblot.
S4C. Axin is important for Wnt-induced mTOR signaling. Hela cells expressing
LKB1were transfected with indicated RNAi. Wnt-3a (300 ng/ml, for 30 minutes)
stimulation was indicated. Phosphorylation of endogenous S6K and S6K protein levels
were shown.
S4D. APC is required for Wnt-induced mTOR signaling. The APC mutant HT29 cells
with inducible expression of β-galactosidase or APC were treated with ZnCl (100 µM) to
induce the expression of β-galactosidase or APC. Serum starved cells were treated with
Wnt-3a (400 ng/ml, 40 minutes) as indicated. Phosphorylation of S6 was determined.
Figure S5. Function of GSK3 in Wnt-Induced mTOR Activation
S5A. LiCl inhibits β-catenin phosphorylation and increases β-catenin protein levels.
HEK293 cells were treated with indicated concentrations of NaCl or LiCl for 2 hours.
Phosphorylation and protein levels of S6K, Akt, and β-caternin were determined by
Western blotting.
S5B. Phosphorylation of S6K by LiCl treatment in MEF and LEF cells.
S5C. GSK3-Inhibitor (GSKI) treatment increases S6K phosphorylation in LEF and
bone marrow stromal (ST2) cells. Cells were treated with 20 µM GSK-I for 6 hours.
S5D. GSK3 is not essential for insulin to stimulate S6K phosphorylation. RIE cells
(top two panels) and MEF cells (bottom two panels) were treated with 20 mM LiCl for 1
hour and then stimulated with 400 nM insulin for 30 minutes. Phosphorylation of S6K
and its protein level were determined by immunoblotting.
S5E. Increase of TSC2 mobility by LiCl, GSK-I, or Wnt-1. Serum starved
cementoblasts were treated with LiCl (20 mM) or GSK-I (20 µM) and mobility of
endogenous TSC2 was determined (top panel). Wnt-1 expressing HEK293 cells also
show increased TSC2 mobility (bottom panel).
Figure S6. Phosphorylation of TSC2 by AMPK and GSK3
S6A. S1341 and S1337 in TSC2 are required for phosphorylation by GSK3β but not
AMPK in vitro. Recombinant GST-TSC2 fragment containing intact or mutated
phosphorylation sites were incubated with immunoprecipitated AMPK in the presence of
32P-ATP (left panels). Phosphorylation of TSC2 F1 was detected by phosphoimager. The
experiments in the right panels were performed similarly to those in Fig. 6B. . Arrow
indicates the GSK3-phosphorylated TSC2 F1. Protein levels for HA-AMPK and HA-
GSK3b-S9A were detected by immunoblot while GST-TSC2 F1 protein levels detected
by coomassie staining. Mutation of S1345A, but not S1341/1337, completely eliminated
AMPK dependent phosphorylation. Mutation of either S1345A or S1341/1337A
abolished TSC2 phosphorylation by GSK3.
S6B. GSK3β phosphorylates TSC2 in a sequential manner. Phosphorylation of various
TSC2 mutants by GSK3β was performed similarly to those in the right panels of S6A.
Mutation of S1341A eliminated TSC2 phosphorylation by GSK3 while mutation of
S1337A decreased TSC2 phosphorylation by GSK3. Mutation of S1333A or T1329A
had minor effects on TSC2 phosphorylation. The lack of effect of two N-terminal
mutations (S1333A or T1329A) is likely due to the fact that the in vitro phosphorylation
is incomplete at the more N terminal residues.
S6C. 2D phosphopeptide mapping of in vitro phosphorylation site. Samples from Fig.
S6B were analyzed by 2D phosphopeptide mapping.
S6D. S1337 and S1341 are phosphorylated in vivo. TSC2 wild type and mutant (2AC)
were transfected into HEK293 cells and labeled with 32P-phosphate. TSC2 protein was
immunoprecipitated and 2D phosphopeptide mapping was performed. Comparison of in
panel a and panel b shows that the intensities of the circled spots (1 to 4) are missing or
reduced in the 2AC mutant . The in vitro phosphorylated TSC2 F1 by GSK3β (panel c)
was mixed with the in vivo labeled TSC2-2AC (panelb) to produce panel d. The arrow
head in panel b indicates a new phosphopeptide created by the mutation of
S1337A/S1341A on TSC2.
S6E. Inhibition of GSK3 by GSK-I decreases the retarded mobility of TSC2 caused by
2DG.
S6F. Schematic presentation of TSC2 mutants and recognition of phospho-TSC2
antibody.
S6G. Characterization of TSC2 phosphorylation by GSK3 in vivo. HEK293 cells were
co-transfected with indicated plasmids. HA-TSC2 was immunoprecipited and
phosphorylation of TSC2 was monitored by phospho-specific antibody (upper panels).
Co-expression of GSK3 increased TSC2 phosphorylation. Mutation of the
phosphorylation sites (2AC) completely eliminated the recognition, suggesting the
specificity of the antibody. The effect of 2DG on TSC2 phosphoryaltion was also
determined. Transfected cells were treated with 2DG (10 mM) for 30 minutes. TSC2
phosphorylation was determined by immunoblot analysis (lower panels).
Figure S7. Phosphorylation of TSC2 by SGK3 Is Important for Cellular Energy
Response
S7A. Inactivation of the mTOR pathway by energy depletion is compromised in
GSK3β-/- cells. Cells were treated with the indicated concentrations of 2DG for 30
minutes. Protein levels and phosphorylation were determined by immunoblots with
specific antibodies.
S7B. Elimination of the GSK3 phosphorylation sites in TSC2 decreases ability of TSC2
to inhibit mTOR signaling. Wild type (Wt) and TSC2-4A (4A) (see Fig.6A for details of
the mutations) were expressed in TSC2-/- LEF cells and stable clones with similar
expression were characterized.
S7C. Glucose deprivation stimulates apoptosis of LEF cells expressing TSC2-4A but
not wild type TSC2. Cells were cultured in medium containing 25 mM (glucose+) or 0
mM (glucose-) glucose medium for 48 hours. Phase contrast photomicrographs are
shown.
S7D. Glucose deprivation induces caspase 3 cleavage in LEF cells expressing vector or
TSC2-4A, but not TSC2. Cells were cultured in medium containing 0 or 25 mM glucose
for 20 hours.
Figure S8. TSC2 Integrates Signals from Energy via AMPK and Wnt via GSK3 to
Regulate Cell Growth
In this model, Wnt signals to stimulate the mTOR pathway via GSK3 and TSC2.
Phosphorylation of TSC2 by GSK3 requires priming phosphorylation by AMPK. TSC2
integrates inputs from growth factors via Akt, RSK, and ERK, cellular energy levels via
AMPK, and Wnt via GSK3 to regulate mTOR, a central cell growth regulator.
Supplemental Information
Supplemental Experimental Procedures
Antibodies, Plasmids, and Materials
Anti-S6K, anti-phospho S6K (T389, S421/424), anti-4EBP1, anti-phospho 4EBP1
(S65, T37), anti-S6, anti-phospho S6 (S240/244), anti-Akt, anti-phospho Akt (S473),
anti-AMPK, anti-phospho AMPK (T172), anti-mTOR, anti-phospho-mTOR (S2448),
anti-phospho GSK3-β (S9), anti-phospho-β-catenin (S33/37/T41), anti-cyclin D, and
anti-cleaved Caspase-3 were obtained from Cell Signaling (Beverly, MA). Anti-Actin,
anti-VEGF and anti-TSC2 antibodies were from Santa Cruz Biotechnololgy (Santa Cruz,
CA). Anti-active-β catenin and anti-GSK3 antibodies were obtained from Upstate
(Charlottesville, VA). Anti-β-catenin, anti-phospho ERK(T202/204), and anti-ERK
antibodies were from Transduction laboratories (Lexington KY). Anti-Axin was obtained
from Zymed (South San Francisco, CA). Anti-HA and anti-FLAG antibodies were from
Covance (Philadelphia, PA) and Sigma (St. Louis, MO), respectively. Anti-phospho-
TSC2 antibody (S1337/1341) was generated using phosphorylated peptide
(CFQPpSQPLpSKSS) as antigen (Covance). Horseradish peroxidase-conjugated IgG
secondary antibodies were obtained from Amersham (Buckinghamshire, UK).
The plasmids expressing HA-tagged S6K1(αII), HA-tagged TSC2, Myc-tagged
TSC1, HA-tagged AMPK (αI), HA-tagged AMPK (αI) D159A and GST-TSC2 (1300-
1367), Flag-Dvl-2 were described previously (Inoki et al., 2003; Tamai et al., 2000).
Flag-β-catenin, Axin, Flag-dn TCF, and TCF reporters were kindly provided by E. R.
Fearon (University of Michigan). Human GSK3β (HA-GSK3β) and GSK3β-S9A (HA-
GSK3β-S9A) constructs were kindly provided by J. R. Woodgett (Ontario Cancer
Institute). HA-GSK3β-K85M/K86I mutant and the other mutant constructs used in this
study were created by site-directed mutagenesis via the Stratagene QuickChange Kit (La
Jolla, CA) and were verified by DNA sequencing.
Recombinant mouse Wnt-3a was purchased from R&D Systems (Minneapolis,
MN). The AMPK inhibitor (compound C) and 5-aminoimidazole-4-carboxamide-1-D-
ribofuranoside (AICAR) were obtained from Merck (Whitehouse Station, NJ) and Tronto
Research Chemicals (North York, ON, Canada), respectively. GSK3 inhibitor (GSK
inhibitor-1/TDZD-8) was purchased from Calbiochem (La Jolla, CA).
RNA Interference
Smart Pool short interfering RNA oligonucleotides toward TSC2, GSK3α,
GSK3β, Axin1, and Axin2 were purchased from Dharmacon (Denver, CO). EGFP
siRNA was (5’-AAGACAAUCGGCUGCUCUGAU-3’) was synthesized by Dharmacon.
Oligonucleotides were transfected into HEK293 cells, and lysates were made 48 hours
post transfection.
Reporter Assay
For luciferase reporter assays, cells were plated in six-well plates. pTOPFLASH,
pFOPFLASH, CMV-β-gal and indicated plasmids were co-transfected as described
previously. TCF transcriptional activity was measured as the ratio of luciferase activity
from the pTOPFLASH vector to the pFOPFLASH vector. All luciferase activities were
normalized by β-galactosidase activity.
Metabolic Labeling and Two-Dimensional Phosphopeptide Mapping
HEK293 cells were co-transfected with HA-TSC2, Myc-TSC1 and indicated
plasmids. The transfected cells were incubated in phosphate and serum free medium for
1 hour before incubation with 0.5 mCi/ml 32P-orthophosphate for 6 hours. Before
harvesting, the labeling cells were treated with or without 40 mM 2-deoxy-glucose for 30
minutes. HA-TSC2 was immunoprecipitated, resolved by SDS-PAGE and transferred to
a PVDF membrane. Phosphorylated TSC2 was visualized by autoradiography.
Phosphopeptide mapping was then performed. In brief, the phosphorylated TSC2 bands
were excised, fixed in methanol and incubated in 500 µl of 0.5% polyvinylpyrrolidone-40
dissolved in 100 mM acetic acid for 30 minutes at 37oC. The samples were then digested
with 20 µg of TPCK-treated trypsin (Sigma) at 37oC in 75 mM ammonium bicarbonate
buffer (pH 8.0 containing 5% acetonitrile). After digestion, samples were dried under
vacuum and suspended in 10 µl of water containing 4% acetonitrile. Samples were
spotted onto a cellulose plate and first dimensional electrophoresis was performed using
1% ammonium bicarbonate buffer pH 8.9. The plates were chromatographed in the
second dimension in chromatography buffer (ie: n-butanol/ pyridine/ acetic acid/ water,
75: 50: 24: 50). The plates were dried, and phosphopeptides were visualized by
autoradiography.
Transgenic Mice
Animal studies were cared for by the Unit for Laboratory Animal Medicine at the
University of Michigan under the approval by the University Committee on Use and Care
of Animals. DNA purification and microinjection of bone specific promoter (osteocalcin)
driving Wnt10b transgene (OCN-Wnt10b) into fertilized C57Bl/6 mouse eggs were
performed by the transgenic animal model core facility at the University of Michigan.
Mice were screened for integration of the Wnt10b transgene by PCR. Wild type C57Bl/6
females were used to establish lines. The detail characterizations of Wnt10b transgenic
mice will be described elsewhere (O. MacDougald). Wnt1 transgenic and LRP6 knockout
mice have been reported previously (Li et al., 2000; Pinson et al., 2000).
Immunohistochemical Staining
The mandibular bone was immersed immediately in fixative-10% neutralized
formalin buffer (Fisher Scientific). Fixation was carried out for 24 hours followed by
demineralization in 0.5M EDTA (PH. 7.33). Serial 7 µM paraffin embedded longitudinal
sections of the mandibular bone from both Wnt10b and wild type animals were processed
in the Histology Core at the School of Dentistry, University of Michigan. The factor
VIII-related antigen Ab-1 (NeoMarkers) and normal goat IgG (R & D) were used as
positive and negative controls, respectively.
Tumor Growth in Nude Mice
A primary Wnt-1 expressing tumor cell line, G105, was established from a
mammary tumor of an MMTV-Wnt-1 transgenic female. The tumor tissue was finely
chopped and further digested in Trypsin-EDTA 0.05% for 40 minutes. The tumor cells
were then cultured in Dulbecco’s modified Eagle’s Medium (DMEM) (Invitrogen;
Carlsbad, CA) supplemented with L-glutamine (2 mM), penicillin (100
units/ml)/streptomycin (1µg/ml) and 15% (v/v) fetal bovine serum at 37°C and in 5%
CO2 atmosphere. The tumorigenicity of G105 cells was assayed by subcutaneous
injection of 1 x 106 cells suspended in 100 µL of serum-free DMEM into right flank of 4
to 6 weeks old female athymic mice (Hsd:Athymic Nude-nu (nu/nu), which were
obtained from the Harlan (Indianapolis, Indiana). Rapamycin was reconstituted in
absolute ethanol at 10 mg/ml and diluted in 5% Tween 80 and 5% Peg-400 before
injection. Tumor cells were allowed to grow for 3 days without treatment. On day 4 after
tumor cell injection, mice were randomized into 2 groups (5 animals per group), and
treatment of rapamycin was initiated. One group was treated with rapamycin,
administered at doses of 1.5 mg/kg/d intraperitoneally for 5 consecutive days. The second
group received daily injections of the carrier solution as controls. Tumor size was
measured with calipers in 3 dimensions at times as indicated. Tumor volume was
calculated using the formula for volume of an ellipsoid: 4/3 x L/2 x W/2 x H/2, where L
= length, W = width, and H = height. All mice were sacrificed by asphyxiation with CO2
on day 28, and tumors were removed and tumor weight (gram) measured.
Quantification and Statistical Analysis
Quantification of signals in Western blotting was performed by pixel analysis via
the NIH Image software suite. For statistical analysis in multiple groups, differences
between groups were assessed by one-way ANOVA followed by Scheffe's test among the
groups.