1. No category

Protein Synthesis and Degradation:
Regulation of GATA binding protein 2
levels via ubiquitin-dependent degradation
by Fbw7: involvement of cyclin
B-cyclin-dependent kinase 1-mediated
phosphorylation of Thr176 in GATA
binding protein 2
J. Biol. Chem. published online February 10, 2015
Access the most updated version of this article at doi: 10.1074/jbc.M114.613018
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Tomomi Nakajima, Kyoko Kitagawa, Tatsuya
Ohhata, Satoshi Sakai, Chiharu Uchida,
Kiyashi Shibata, Naoko Minegishi, Kanae
Yumimoto, Keiichi I. Nakayama, Kazuma
Masumoto, Fuminori Katou, Hiroyuki Niida
and Masatoshi Kitagawa
JBC Papers in Press. Published on February 10, 2015 as Manuscript M114.613018
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M114.613018
Degradation of GATA2 by Fbw7 Regulation of GATA binding protein 2 levels via ubiquitin-dependent degradation by Fbw7: involvement
of cyclin B-cyclin-dependent kinase 1-mediated phosphorylation of Thr-176 in GATA binding protein 2
Tomomi Nakajima,1, 2, # Kyoko Kitagawa,1, # Tatsuya Ohhata,1 Satoshi Sakai,1 Chiharu Uchida3,
Kiyoshi Shibata3, Naoko Minegishi,4 Kanae Yumimoto,5 Keiichi I. Nakayama,5 Kazuma Masumoto,2
Fuminori Katou,2 Hiroyuki Niida,1 and Masatoshi Kitagawa1*
1
Department of Molecular Biology, Hamamatsu University School of Medicine, 1-20-1 Handayama,
Higashi-ku, Hamamatsu, Shizuoka 431-3192, Japan
2
Department of Oral and Maxillofacial Surgery, Hamamatsu University School of Medicine, 1-20-1
3
Research Equipment Center, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku,
Hamamatsu, Shizuoka 431-3192, Japan
4
Biobank and Life Science, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai,
Miyagi 980-8575, Japan
5
Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University,
3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
Running Title: Degradation of GATA2 by Fbw7
*To whom correspondence should be addressed: Masatoshi Kitagawa; Department of Molecular Biology,
Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu, Shizuoka
431-3192, Japan. Tel.:+81-53-435-2322; Fax: +81-53-435-2322 ; E-mail: [email protected]
#
Tomomi Nakajima and Kyoko Kitagawa equally contributed to this work.
Keywords: Ubiquitin-proteasome; ubiquitin ligase; GATA transcription factor; cyclin B-cyclin
dependent kinase1 (CDK1); phosphorylation
Background: SCF-Fbw7 participates in stability
kinase 3, an E3 ligase.
control of several Cdc4-phosphodegron-containing
Results:
proteins phosphorylated by glycogen synthase
GATA binding protein 2 is promoted by Fbw7, is
Ubiquitin-dependent
degradation
1
Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
of
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Handayama, Higashi-ku, Hamamatsu, Shizuoka 431-3192, Japan
Degradation of GATA2 by Fbw7 cyclin
B-CDK
1
Thr-176-phosphorylation
mediated
the phosphorylation of Thr-176, was cyclin
and
B-cyclin dependent kinase (CDK) 1. Moreover,
dependent,
influences hematopoietic cell differentiation.
depletion
of
endogenous
Fbw7
stabilized
Conclusion: GATA binding protein 2 is a novel
endogenous GATA2 in K562 cells. Conditional
target for Fbw7.
Fbw7-depletion in mice increased GATA2
of
levels in hematopoietic stem cells and myeloid
post-transcriptional control of GATA binding
progenitors at the early stage. Increased
protein 2 is clarified.
GATA2 levels in Fbw7-conditional knockout
Significance:
The
molecular
mechanism
mice were correlated with a decrease in a c-Kit
ABSTRACT
high
expressing
population
of
myeloid
progenitor cells. Our results suggest that Fbw7
GATA binding protein 2 (GATA2) participates
is a bona fide E3 ubiquitin ligase for GATA2 in
in cell growth and differentiation of various
vivo.
cells such as hematopoietic stem cells. Although
Ubiquitin-proteasome systems control the stability
its
by
of many cellular proteins and participate in
proteolytic
regulation of various biological processes such as
expression
transcriptional
level
is
induction
controlled
and
degradation, the responsible E3 ligase has not
cell
been identified. Here, we demonstrate that
transduction and apoptosis (1,2). Poly-ubiquitin
F-box/WD
7
chain conjugation to substrate proteins is mediated
(Fbw7/Fbxw7), a component of Skp1, Cullin 1,
by E1, E2 and E3. E3 ubiquitin ligases are of the
F-box containing complex (SCF)-type E3 ligase,
RING finger-type (3), HECT-type (4) and U-box
is an E3 ligase for GATA2. GATA2 contains a
type (5), bind to and poly-ubiquitylate their
cell
(CDC)
specific substrates. F-box/WD repeat-containing
4-phosphodegron (CPD), a consensus motif for
protein 7 (Fbw7; also called Fbxw7, cdc4 and
ubiquitylation
includes
Sel10), an F-box protein, is a substrate recognition
Fbw7
molecule of Skp1, Cullin, F-box containing
repeat-containing
division
Thr-176.
control
by
Fbw7,
Ectopic
protein
protein
which
expression
destabilized
GATA2
proteasomal
degradation.
cell
differentiation,
signal
promoted
its
complex (SCF)-type E3 ubiquitin ligase (6). It has
Substitution
of
been reported that Fbw7 targets cyclin E (7),
threonine 176 to alanine in GATA2 inhibited
c-Myc (8,9), c-Jun (10,11), Notch1 (12,13),
binding with Fbw7 and the ubiquitylation and
SREBP (14,15), mTOR (16), c-Myb (17-19),
degradation
was
MCL1 (20,21), NFκB2 (22) and GATA3 (23) for
suppressed. The CPD kinase, which mediates
ubiquitin-mediated proteasomal degradation (24,
of
and
of
proliferation,
GATA2
by
Fbw7
2
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A GATA family transcription factor
Degradation of GATA2 by Fbw7 25). Ablation of the Fbw7 gene was reported in
Fbw7 binds to a high-affinity recognition motif
human breast carcinomas, colon cancers and T cell
termed the Cdc4 phosphodegron (CPD), with a
acute lymphoblastic leukemias (26,27). Moreover,
consensus sequence of T/S(PO3)-P-X-X-S/T/D/E
conditional
(where X indicates an arbitrary residue) and often
Fbw7-knockout
mice
developed
thymus enlargement, thymic lymphomas, and
promotes
defects of bone marrow (BM) hematopoietic stem
phosphorylation of the CPD (18), (32). We found
cells (HSCs) (27). Because Fbw7 promotes
a CPD motif in GATA3 amino acid (aa) sequences
destabilization of many oncogenic proteins and
and
cell differentiation regulators, it is important to
ubiquitylation
identify unknown substrates of Fbw7 to aid
Thr-156 in CPD in GATA3 (23). We also found
understanding of hematopoietic cell differentiation
the CPD motif in GATA binding protein 1
and Fbw7-associated cancer development.
(GATA1) and GATA binding protein 2 (GATA2)
the
turnover
demonstrated
required
of
that
substrates
via
Fbw7-mediated
phosphorylation
of
suggesting that they might be targets for Fbw7.
transcription factor GATA binding protein 3
Among the GATA family, GATA1, 2 and 3 are
(GATA3) is a novel target for Fbw7 (23). GATA3
classified as hematopoietic GATA factors, based
is a member of the GATA family transcription
on their ability to regulate distinct and overlapping
factors that consist of GATA1, 2, 3, 4, 5 and 6
aspects of hematopoiesis. Especially, aa sequences
(28,29). We found conditional inactivation of
among GATA3 and GATA2 are highly conserved.
Fbw7 in mouse T-cell development skewed
GATA3 is expressed in HSCs in addition to
thymic CD8 single positive lineage differentiation,
T-lymphocytes (33). GATA2 is also expressed in
which exhibited a higher incidence of apoptosis
HSCs, and in hematopoietic progenitors, erythroid
(23). Similar perturbations during development of
precursors,
CD8 positive cells were studied with transgenic
(28,29). GATA2 participates in proliferation and
mice, in which GATA3 expression was enforced
differentiation of hematopoietic cell lineages.
throughout T-cell development. Excess GATA3
Although GATA1 is also expressed in erythroid
induced thymic lymphomas in the transgenic mice
precursors, megakaryocytes and eosinophils (34),
(30).
also
the identity of aa among GATA3 or GATA2 and
developed in mice when Fbw7 was conditionally
GATA1 is high in Zinc finger domains but low in
ablated in the T-cell lineage alone (31). It is
other regions. It was reported that mutations of
speculated that uncontrolled GATA3 protein
one allele of GATA2 participate in hematopoietic
levels result in the formation of lymphoblastoid
or immune system diseases (35,36). Therefore, it
tumors at a specific stage of thymic development.
is important to clarify the molecular mechanisms
Interestingly,
thymic
lymphomas
3
megakaryocytes
and
eosinophils
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Recently, we reported hematopoietic
Degradation of GATA2 by Fbw7 aphidicolin for 16 h and then released from arrest
Although cellular GATA2 levels are regulated by
by washing with fresh medium for 10 h (G1/S and
transcriptional control and proteasome-mediated
S phase cells). Then, cells were treated with
degradation (37,38), ubiquitin E3 ligase, which
aphidicolin for 16 h (G1/S phase cells) then
ubiquitylates GATA2 to promote degradation via
released from arrest by washing with fresh
the ubiquitin-proteasome system, has not been
medium for 5 h and harvested by treating trypsin
identified. In the present study, we demonstrated
(S phase cells). M phase cells were treated with 1
that GATA2 is a novel CPD-dependent substrate
µg/ml aphidicolin for 16 h, released from arrest by
for
the
washing with fresh medium for 4 h, treated with
involvement of cyclin B-cyclin-dependent kinase
100 ng/ml nocodazole for 16 h and harvested by
1 (CDK1), which is different from the CPD
adding trypsin. To determine contribution of
kinases identified for known substrates. We also
cyclin B-CDK1, cells were treated with CDK
demonstrated
inhibitor such as butyrolactone I (39,40) or
Fbw7.
Furthermore,
the
we
identified
physiological functions
of
RO-3306 (Calbiochem).
Fbw7-dependent control of GATA2 using cultured
cells and Fbw7-conditional knockout mice.
Antibodies - The antibodies used in this study were
EXPERIMENTAL PROCEDURES
anti-Myc 9E10 (Roche), anti-FLAG M2 (Sigma),
Cell Lines, Cell Culture and Synchronization -
anti-HA 3F10 (MBL), anti-GATA2 PA5-17368
HEK293 and HeLa cells were obtained from
(Thermo), ab109241 (Abcam) and RC1.1.1 (38),
American Type Culture Collection and cultured in
anti-Fbw7 A 301-720A-1 (Bethyl), anti-cyclin B
Dulbecco’s
medium
v-152 (Santa Cruz Biotechnology) and anti-β actin
supplemented with 10% FBS, penicillin (100
AC15 (Sigma). PE-Cy5 conjugated anti-c-Kit
U/ml), and streptomycin (100 µg/ml) at 37°C.
(2B8) was purchased from Biolegend. Biotinylated
Neuro2A cells were maintained in DMEM
anti-CD127 (B12-1) and mouse lineage depletion
supplemented with 10% FBS, 1 mM sodium
cocktail and streptavidin particles Plus, which are
pyruvate, 2 mM L-glutamine, 10 ml/l nonessential
streptavidin conjugated magnetic nanoparticles,
amino acids and the above-described antibiotics.
were purchased from BD Bioscience. Alexa
K562 cells were obtained from RIKEN Cell Bank
Fluor® 488 conjugated anti-GATA2 was prepared
and cultured in RPMI1640 supplemented with
by Alexa Fluor® 488 monoclonal antibody
10% FBS and the above-described antibiotics. To
labeling
obtain cell lysates synchronized at G1/S, S, and M
phosphorylated
phases, HeLa cells were treated with 1 µg/ml
(anti-p-T176-GATA2) was raised against keyhole
modified
Eagle’s
4
kit
(Zenon).
Thr-176
Anti-human
GATA2
polyclonal
antibody
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involved in the control of GATA2 levels.
Degradation of GATA2 by Fbw7 limpet hemocyanin (KLH)-conjugated chemically
leupeptin, and trypsin inhibitor and 2.5 µg/ml of
synthesized
chymostatin, phosphatase inhibitor mix) (23)
phosphorylated
Thr-176
peptide,
corresponding to the CPD region of GATA2 (aa
RNA Interference - siRNA oligonucleotides for
from an immunized Guinea Pig was bound to
GATA2 or Fbw7 or control siRNAs were
column chromatography conjugated with P-T176
transfected into HeLa cells using RNAiMax (Life
peptide. The affinity purified anti-P-T176 GATA2
Technologies), according to the manufacturer’s
was then passed through a column conjugated to
protocol. ON-TARGETplus Human SMARTpool
nonphosphorylated Thr-176 (NP-T176) peptide (aa
for GATA2 (L-009024-00-0005) was purchased
residues 172-181 of GATA2) to deplete antibodies
from Thermoscientific Dharmacon. The nucleotide
against nonphosphorylated antigen. The specific
sequences
binding ability of the purified antibody to P-T176
-GUGUGGAAUGCAGAGACUGGAGA-3′
peptide was confirmed by ELISA.
(Fbw7-A),
of
Fbw7
siRNAs
were
5′
5′-
AAUGAAAGCACAUAGAGUGCCAACU-3′
Plasmids and Transfection - Human GATA2
(Fbw7-B), 5′-ACAGGACAGUGUUUACAAA-3′
cDNA
(Fbw7-C)
was
cloned
into
pcDNA3.1/myc-His
and
(Invitrogen). Substitution of Thr-176 to Alanine in
5′-ACCUUCUCUGGAGAGAGAAAUGC-3′
GATA2
(Fbw7-D).
was
generated
using
PCR-based
mutagenesis. cDNAs encoding mouse Fbw7
(Fbxw7α) or its ΔF mutant were subcloned into
Immunoprecipitation and Immunoblotting - Total
p3×FLAG-CMV 7.1 (Sigma). cDNAs encoding
cell lysates were immunoprecipitated with 2 µg of
human Fbw7 were cloned into pCGN with an
antibodies and protein G+ Sepharose 4FF (GE
HA-tag or pcDNA3 with a FLAG-Tag. The
healthcare) at 4°C for 2 h. Immunocomplexes were
expression plasmid for ubiquitin (pCGN-HA-Ub)
washed
was previously described (41). Plasmids were
Immunoprecipitated samples and original cell
transfected into HEK293 or HeLa cells using the
lysates (input) were separated by SDS-PAGE and
calcium phosphate method or X-treme GENE 9
transferred from the gel onto a PVDF membrane
(Roche), respectively. Cells were harvested by a
(Millipore), followed by immunoblotting (IB).
scraper and lysed with lysis buffer (50 mM
Proteins were visualized using an enhanced
Tris-HCl, pH 7.5, 300 mM NaCl, 0.5% Triton
chemiluminescence system (Bio-Rad).
four
times
with
lysis
buffer.
X-100, 10 µg/ml each of antipain, pepstatin, E-64,
Immunoprecipitation under denaturing condition 5
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residues 172-181) (MBL). Anti-serum obtained
Degradation of GATA2 by Fbw7 Cells lysates which were prepared with lysis buffer
bead-bound proteins were then eluted with the
as described above were added to equal volume of
FLAG peptide (500 µg/ml, Sigma), precipitated
2x denaturing IP buffer (100 mM Tris-HCl, pH 7.5,
with ice-cold 20% trichloroacetic acid, and washed
2%SDS, 10 mM dithiothreitol) and incubated at
with
100°C for 8 min and then centrifuged for 13,000
immunoaffinity-purified proteins were dissolved in
rpm for 10 min. The supernatants were diluted with
SDS sample buffer, fractionated by SDS-PAGE,
five
and
and stained with silver. Individual lanes of the
immunoprecipitated with 2 µg of antibodies and
stained gel were sliced into 12 pieces and proteins
protein G+ Sepharose 4FF (GE healthcare) at 4°C
within these pieces were subjected to in-gel
for 2 h. Immunocomplexes were washed four times
digestion with trypsin as described previously (43).
with lysis buffer. Immunoprecipitated samples
The resulting peptides were analyzed by an
were separated by SDS-PAGE and transferred
ion-trap mass spectrometer (LTQ-XL, Thermo
from the gel onto a PVDF membrane (Millipore),
Finnigan).
followed by IB.
lcq_dta.exe (Thermo Finnigan) and were compared
volumes
of
lysis
buffer
acetone.
lists
were
concentrated
generated
with
with the use of the MASCOT algorithm (version
DiPIUS-NL – DiPIUS-NL analysis was performed
2.2.1) with the “Target-decoy” Mouse IPI database
as
cells
(version 3.4.4, released in June 2008; with 55,078
expressing mouse Fbw7α or its ΔF mutant were
target sequences, searched against a total of
incubated for 6 h in the presence of the proteasome
110,156 sequences [target and reverse/decoy]),
inhibitor MG132 (10 µM, Peptide Institute) and
maintained
were then lysed in 8 ml of a solution containing 20
Institute. Assigned high-scoring peptide sequences
mM HEPES-NaOH (pH 7.5), 150 mM NaCl, 1%
(MASCOT score >35) were processed with
digitonin, 10 mM NaF, 10 mM Na4P2O7, 0.4 mM
in-house software. If the MASCOT score was <45
Na3VO4, 0.4 mM EDTA, leupeptin (20 µg/ml),
(peptides for which the MS2 score was above the
aprotinin (10 µg/ml), and 1 mM PMSF. The lysates
95th
were centrifuged at 2200 ×g for 20 min at 4°C. The
sequences
supernatants (20 mg of protein in 8 ml of solution)
comparison
were incubated for 1 h at 4°C with 120 µl of beads
collision-induced dissociation spectra on the basis
conjugated with M2 antibodies to FLAG. The
of the following criteria: (i) a delta score of >15 or
beads were washed three times with 4 ml of a
(ii) at least six successive matches for y- or b-ions,
solution containing 10 mM HEPES-NaOH (pH
or at least three blocks of three successive matches
7.5), 150 mM NaCl, and 0.1% Triton X-100, and
for
described
(42).
Briefly,
Neuro2A
6
by
the
percentile
y-
or
European
of
were
with
b-ions.
Bioinformatics
significance),
manually
the
Identified
assigned
confirmed
by
corresponding
peptides
from
Downloaded from http://www.jbc.org/ by guest on February 16, 2015
Peak
The
Degradation of GATA2 by Fbw7 independent experiments were integrated and
B1-CDK1 (Millipore) or synchronized HeLa cell
regrouped by IPI accession number. Proteins
lysates at 30°C for 30 min in reaction buffer
identified in only one experiment or with a
containing 1 mM
single-peptide assignment were removed from
terminated by boiling with SDS-sample buffer for
spectral counting data. Estimated false discovery
8 min. For in vitro phosphorylation following
rates were zero at the protein level.
binding assays, phosphorylated mixtures were
ATP. The reaction was
incubated for an additional hour at 4°C with lysates
In
vivo
Degradation
Assay
from HEK293 cells exogenously expressing Fbw7.
or
GST-fused proteins were then precipitated using
was
glutathione-Sepharose beads. The mixtures were
transfected with or without pcDNA3-FLAG-Fbw7
treated with precision protease (Pharmacia biotech)
into HeLa cells. To analyze the effect of Fbw7 on
for 30 min to cleave GATA2 from the GST-tag.
the stability of endogenous GATA2, siRNA for
All reaction mixtures were analyzed by IB with the
Fbw7 or control siRNA was transfected into K562
indicated antibodies.
pcDNA3.1-GATA2-myc-His
pcDNA3.1-GATA2-T176A-myc-His
cells using RNAiMax. After 48 h, cells were
treated with 20 µg/ml of cycloheximide for the
Quantitative real time (qRT)-PCR analysis-Total
indicated times. Cell lysates were subjected to IB
RNA was isolated from cells using RNAiso
with the indicated antibodies. The intensity of the
(Takara), and subjected to reverse transcription
bands was measured using image analysis software
with
Image J, and the signal intensity of GATA2 in
SuperScript Reverse Transcriptase II (Invitrogen).
samples was normalized using levels of β-actin as a
The resulting complementary DNA was subjected
loading control.
to qRT-PCR using the Rotor-Gene 3000 system
random
hexanucleotide
primers
and
(Corbett Research) and the SYBR premix Ex Taq
In vitro Phosphorylation Assay - GST-fused WT or
kit (Takara). The sequences of PCR primers were
T176A mutant of GATA2 was produced in
as
Escherichia
-CCCACCTACCCCTCCTATGT-3′ (sense) and
coli
BL21,
and
purified
using
follows:
5
′
glutathione-Sepharose beads (GE healthcare). In
5
vitro phosphorylation was described previously
(antisense)
(23,44). Each recombinant GATA2 incubated with
-GTAACCCGTTGAACCCCATT-3′ (sense) and
the indicated kinase sources including recombinant
5
cyclin-D3-CDK4
(Abcam),
(antisense)
for
18S-rRNA.
The
(Abcam),
A2-CDK2
transcripts
was
normalized
against
cyclin
cyclin
E1-CDK2
(Abcam),
cyclin
7
′
′
-TGCCCATTCATCTTGTGGTA-3
for
GATA2
and
5
-CCATCCAATCGGTAGTAGCG-3
amount
that
′
′
′
of
of
Downloaded from http://www.jbc.org/ by guest on February 16, 2015
-
Degradation of GATA2 by Fbw7 18S-rRNA as an internal standard.
c-Kit
antibody,
permeabilized
Lin− cells
with
the
were
Foxp3
fixed
and
staining
kit
Conditional knockout mice - Generation of
(eBioscience) prior to intracellular staining with
Mx1-Cre/Fbw7Flx/Flx mice was described previously
the labeled anti-GATA2 antibody. The labeled Lin−
(45). Fbw7Flx/Flx mice were used as controls.
cells were scored and sorted by FACS Aria
Expression of Cre recombinase in transplant
instruments (BD).
recipients was induced by intraperitoneal injection
of polyinosinic-polycytidylic acid (pIpC) (Sigma)
Statistical Analysis - Quantitative data were
to 8 and 9 weeks of age at a dose of 20 mg per
presented as means ± SD and were analyzed by the
kilogram of body weight on 7 alternate days. A
Student’s t-test.
isolated 1 week after the last injection of pIpC. All
RESULTS
mice were treated according to the protocols
Fbw7 binds to and ubiquitylates GATA2 in a
approved by the Hamamatsu University School of
CPD-dependent manner. In many cases, Fbw7
Medicine Animal Care Committees at the Center
recognizes phosphorylation of Ser/Thr residues in
Animal Care facility. Genotype of gene targeted
CDC4-phosphodegron
mice and deletion of exon 5 of the floxed Fbw7
consensus sequence for recognition by Fbw7 in
allele were verified by PCR with DNA subjects
the selective substrates (19,32). The CPD is
prepared by CellEase Tissue II (Biocosm), and the
speculated
primers 5′-GTGTTCTTCACTTGGGAAGTGC-3′
pSer/pThr-Pro-X-X-pSer/pThr/Glu/Asp
(forward)
indicates an arbitrary residue). It has been reported
and
5′-TGAACAGACGCAGACGCATTCT-3′
(reverse)
for
Fbw7
allele
5′-AGGTTCGTTCACTCATGGA-3′
(CPD),
which
is
the
be
to
(X
that Fbw7 targets many substrates such as c-Myc
(8,9), c-Jun(10,11), cyclin E (7), NFκB2 (22),
and
(forward)
c-Myb
(17-19)
and
Notch
(12,13),
in
a
and 5′-TCGACCAGTTTAGTTACCC-3′ (reverse)
CPD-dependent manner (2,32). For c-Myc, c-Jun,
for Cre recombinase transgene.
cyclin E and NFκB2, their secondary Ser residues
participate as the priming phosphorylation sites for
FACS analysis - Isolation of mononuclear cells
GSK3-mediated phosphorylation of the first
from BM was performed by Ficoll-Paque PLUS
Ser/Thr residues in the CPD (18,23). Recently, we
(GE Healthcare). Subsequently, lineage cells and
found
common lymphoid progenitor populations were
transcriptional factor, has a CPD motif and is an
magnetically depleted. After staining with anti
Fbw7 target (23). Furthermore, putative CPD
8
that
GATA3,
a
GATA
family
Downloaded from http://www.jbc.org/ by guest on February 16, 2015
Lin− fraction of bone marrow (BM) cells was
Degradation of GATA2 by Fbw7 motifs were identified in both mouse and human
To determine whether endogenous GATA2
GATA1 and GATA2 (Fig. 1A). Therefore, it was
bound to Fbw7 in cells, we used two different
speculated that they were also the substrates for
approaches: differential proteomics analysis and
SCF-Fbw7. We investigated whether Fbw7 binds
immunoprecipitation
to GATA1 and GATA2. HEK293 cells were
immunoblotting (IP/IB) assay. To isolate the
transfected with GATA1 or GATA2 in the
substrates for a given F-box protein, we used a
presence or absence of Fbw7, and were treated
differential
with MG132. Fbw7 or GATAs were reciprocally
DiPIUS-NL
immunoprecipitated
identification
following
immunoblotting
analysis
proteomics
approach
(differential
of
following
termed
proteomics-based
ubiquitylation
substrates
-
nonlabeling) (42). A substrate is expected to be
substrates. As shown in Figure 1B, Fbw7 bound to
ubiquitylated and degraded on its recognition by a
wild type (WT)-GATA2 but not to GATA1.
ubiquitin ligase, resulting in a decrease in its
Among GATA family members, GATA2 has the
cellular
highest homology with GATA3 by aa sequence.
F-box-deleted ligase is expected to retain the
Especially, a 14 aa sequence around the CPD
ability to associate with a substrate but to have lost
region (Thr 156-160) of GATA3 that is recognized
the ability to mediate ubiquitin conjugation,
by Fbw7 in a phosphorylation-dependent manner,
resulting in accumulation of the substrate in the
is almost completely conserved in GATA2.
cell. Both WT Fbw7 and ΔF mutant of Fbw7 were
Therefore, we focused on a region (Thr 176-180)
tagged at their NH2-termini with the FLAG
of GATA2, corresponding to CPD of GATA3. To
epitope and expressed separately in Neuro2A cells.
validate the importance of Thr-176, which might
Cell
be phosphorylated, we prepared a GATA2 mutant
immunoprecipitation with antibodies to FLAG,
with aa substitution (T176A), in which the
and
Thr-176 residue was replaced by Ala. As shown in
analyzed by LC–MS/MS. We identified peptides
Figure 1B, the T176A-GATA2 did not bind to
coding m-GATA2 protein as a binding protein to
Fbw7. This result suggests that Fbw7 binds to
ΔF mutant of Fbw7 (Fig. 2A). The abundance of
GATA2
manner.
proteins that bound to the WT or mutant F-box
Moreover, we found that GATA2 interacted with
proteins in three independent experiments were
Skp1, Cul1 and Roc1/Rbx1 in the presence of wild
compared by semiquantitative spectral counting,
type Fbw7 but not its mutant form lacking the
respectively (42). The known Fbw7 substrates
entire F-box domain (ΔF) (Fig. 1C). This indicated
such as c-Myc and c-Myb were identified (42).
that GATA2 binds to the SCFFbw7 complex.
Moreover, this assay reproducibly identified
in
a
Thr-176-dependent
9
concentration.
lysates
the
In
were
immunoprecipitated
contrast,
subjected
proteins
an
to
were
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(IP/IB) to evaluate the binding of E3 and the
Degradation of GATA2 by Fbw7 GATA2 in the three independent experiments (Fig.
manner.
2B). These results strongly suggest that Fbw7
targets GATA2 in cells.
Fbw7 promotes proteasome-mediated degradation
An IP/IB assay to evaluate the interaction
of GATA2 - We next investigated whether Fbw7
between endogenous GATA2 and Fbw7 was
destabilized
performed. K562 cells treated with MG132 were
(TA)-GATA2-myc-His was transfected with or
lysed in the presence of proteasome inhibitors,
without HA-Fbw7 into HeLa cells with or without
protease inhibitors and phosphatase inhibitors.
MG132 treatment. Forced expression of WT-Fbw7
Cell
but
lysates
were
anti-GATA2
immunoprecipitated
antibody
suppressed
T176A
WT-GATA2
was recovered by MG132 treatment (Fig. 4A, lane
antibodies was performed. This result clarified the
3 vs 4). Expression of T176A-GATA2 was not
binding of endogenous Fbw7 to endogenous
decreased by the co-expression of Fbw7 (Fig. 4A,
GATA2 (Fig. 2C).
lane 7 vs 9). We also confirmed that expression of
the
GATA3 but not that of GATA1 was suppressed by
Fbw7 ubiquitylates GATA2 in a CPD-dependent
Fbw7 (Fig. 4B and C). Moreover, we evaluated
manner
Fbw7
the effect of Fbw7 on GATA2 stability using
ubiquitylated GATA2 using IP/IB. WT-GATA2
cycloheximide (CHX) to inhibit protein synthesis.
but not T176A-GATA2 was ubiquitylated by
Forced
Fbw7 (Fig. 3A). Furthermore, we performed a
WT-GATA2 in HeLa cells (Fig. 4D and E). In
ubiquitylation assay under denaturing conditions
contrast, the T176A-GATA2 was not affected by
to
-
avoid
We
the
investigated
detection
whether
expression
of
Fbw7
destabilized
of
ubiquitylated
co-expression of Fbw7 (Fig. 4F). There was a
by
Fbw7
(see
tendency for a higher turnover of the T176A
EXPERIMENTAL PROCEDURES). Under these
mutant than WT-GATA2. Although we evaluated
conditions,
the
whether the tendency was statistically significant
ubiquitylation of GATA2, whereas GATA2 was
by Student’s t-test, it was not significant.
only slightly ubiquitylated in cells without the
Therefore, degradation speeds of wild type
ectopic expression of Fbw7 (Fig. 3B). However,
GATA2
ubiquitylation
not
significantly different. These results suggested that
improved by Fbw7 (Fig. 3C). These results
Fbw7 promotes proteasome-mediated degradation
strongly
of GATA2 in a CPD-dependent manner.
GATA2-associated
proteins
Fbw7
clearly
levels
suggest
that
of
enhanced
GATA1
Fbw7
were
promotes
the
ubiquitylation of GATA2 in a CPD-dependent
10
and
GATA2-T176A
were
not
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indicated
with
IgG
ΔF-Fbw7
or
expression (Fig. 4A, lane 1 vs 3 and 5), and that
analysis
control
not
WT-
and
immunoblotting
or
with
GATA2.
Degradation of GATA2 by Fbw7 Cyclin
B-CDK1
phosphorylates
Thr-176
of
phosphorylation
assay
using
GATA2 - Fbw7 often recognizes phosphorylated
GST-GATA2
CPD in substrates it binds to. We speculated that
cyclin-CDK
as for other Fbw7 substrates, the regulation of
Thr-176 by kinases including cyclin D3-CDK4
GATA2 by Fbw7 would be mediated by
and cyclin E1-CDK2 was not detected, whereas all
phosphorylation of Thr-176 in its CPD. To
used CDK complex phosphorylated RB protein at
evaluate
almost the same efficiency (Fig. 5D, E). Cyclin
whether
Thr-176
of
GATA2
was
protein
purified
and
complexes.
Phosphorylation
specific antibody against phosphorylated-Thr-176
slightly phosphorylated GATA2 at Thr-176 (Fig.
of GATA2 (anti-p-T176-GATA2) as described in
5D). We also examined whether recombinant
the
PROCEDURES.
GSK3 phosphorylates GST-GATA2 in vitro (Fig.
recognized
5F). The slow migrating form of GST-GATA2
phospho-Thr-176 containing antigen peptides but
detected with anti-GATA2 was observed in the
not unphosphorylated-Thr-176 containing peptides
presence
by
GSK3β (Fig.
ELISA
expressed
(data
in
not
shown).
5F
B-CDK1
lower
panel).
but
not
of
Furthermore,
with
GST-GATA2 was phosphorylated at Thr-176 by
inhibitors
was
cyclin B-CDK1 but not GSK3β (Fig. 5F upper
detected by anti-p-T176-GATA2 antibody, but no
panel), although glutamic acid at position +4 in
signal
the
T176-P-P-K-E might function as a mimic for
T176A-GATA2 (Fig. 5A). Thus, the Thr-176 of
priming phosphorylation required for GSK3β. We
exogenous GATA2 is phosphorylated in vivo.
determined that GSK3β did not phosphorylate
Furthermore, we detected endogenous GATA2
Thr-176 in GATA2 in vitro, and thus might not be
and its phosphorylation on T176 in HeLa cells
the responsible kinase for CPD of GATA2. We
transfected with control siRNA but not with
further investigated whether phosphorylation of
GATA2
that
Thr-176 of GATA2 was inhibited by CDK
at
inhibitors in intact cells. Phosphorylation of
Thr-176 in HeLa cells. The CPD motif in GATA2,
Thr-176 in HEK293 cells was inhibited by
Thr176-Pro-Pro-Lys-Glu,
corresponds
to
a
RO-3306, a selective CDK1 inhibitor, as well as
consensus
CDKs
contain
by butyrolactone I (39,40), which inhibits CDK1,
Ser/Thr-Pro-X-Lys/Arg (Fig. 5C). To determine
2, 3 and 5 (Fig. 5G). Thus, cyclin B-CDK1 might
the kinase responsible for phosphorylation of
be a major kinase of Thr-176 of GATA2.
and
was
proteasome
detected
siRNA
endogenous
cells
cyclin
A2-CDK2
treated
phosphatase
HEK293
WT-GATA2
of
cyclin
(Fig.
GATA2
motif
for
when
5B),
was
using
suggesting
phosphorylated
that
GATA2 Thr-176, we performed an in vitro
Furthermore,
11
we
investigated
whether
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B1-CDK1
Anti-p-T176-GATA2
and
of
phosphorylated in intact cells, we generated a
EXPERIMENTAL
efficiently
recombinant
Degradation of GATA2 by Fbw7 total cell lysates from synchronized HeLa cells at
required for recognition by Fbw7 using a
G1/S or M phase or asynchronized HeLa cells and
GST-pull down assay. Purified GST-WT-GATA2
incubated with recombinant WT-GATA2 or
or GST-T176A-GATA2 was phosphorylated by
T176A-GATA2. Thr-176 of WT-GATA2 was
cyclin B1-CDK1 and incubated with cell lysates
phosphorylated only when incubated with M phase
expressing FLAG-Fbw7. Then the mixtures were
cell lysates, whereas that of T176A-GATA2 did
pulled down with glutathione beads following IB
not respond to any cell lysates tested (Fig. 6B).
analysis with anti-FLAG to detect the binding of
Moreover, endogenous GATA2 protein level was
Fbw7 to GATA2. In addition to T176, because
low in the M phase, during which cyclin B is
there are some putative phosphorylation sites for
expressed (Fig. 6C). GATA2 mRNA levels in the
CDK1 in GATA2, it is speculated that GATA2
M phase were moderately increased compared
underwent multiple phosphorylations by Cyclin
with that in the asynchronized cells, but were
B-CDK1 in vitro. GATA2 in the presence of
similar to that in G1/S phase cells, whereas
Cyclin
the
GATA2 protein levels in the M phase were
unphosphorylated form of GATA2, even when
dramatically reduced (Fig. 6C). Therefore, this
T176 was substituted to alanine (T176A) (Fig. 6A
suggested that the enhanced post-transcriptional
bottom panel). Finally, we found that Fbw7 bound
degradation of GATA2 was involved in decreased
to GST-WT-GATA2 which was detected by
GATA2 levels in the M phase. In addition, the low
anti-p-T176-GATA2 antibody after treatment with
GATA2 protein levels in the M phase were
cyclin B1-CDK1 (Fig. 6A middle panel), but not
increased by butyrolactone I treatment (Fig. 6D).
to GST-GATA2 without cyclin B1-CDK1 or not
In
to GST-T176A-GATA2 with or without cyclin
butyrolactone
B1-CDK1 (Fig. 6A top panel). These results
B-CDK1, although it inhibits both cyclin B-CDK1
suggested that phosphorylation of Thr-176 in
and cyclin A/E-CDK2 in asynchronized cells.
GATA2 by cyclin B-CDK1 participates in the
Therefore, this data suggests that inhibition of
recognition by Fbw7.
cyclin B-CDK1 activity by butyrolactone I
B-CDK1
migrated
slower
than
M
phase
cells,
I
it
was
selectively
expected
inhibited
that
cyclin
increased the stability of GATA2 in the M phase.
GATA2 is phosphorylated and destabilized in the
Together, this suggests that GATA2 protein is
M phase during cell cycle - Cyclin B-CDK1 is
degraded in the M phase in a cyclin-B-CDK1
activated in the M phase and phosphorylates its
dependent manner.
substrates.
To
determine
when
Thr-176
is
phosphorylated during the cell cycle, we prepared
Depletion of Fbw7 stabilizes endogenous GATA2 12
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phosphorylation of Thr-176 in GATA2 was
Degradation of GATA2 by Fbw7 Next, we addressed whether Fbw7 participated in
Effects of Fbw7-depletion in hematopoietic stem
stability control of endogenous GATA2 in intact
cells - Next, we addressed whether Fbw7
cells. K562 cells endogenously expressed both
participated in stability control of GATA2 in mice.
GATA2
significantly
GATA2 is highly expressed in HSCs and
accumulated in K562 cells treated with a
progenitors, but its expression declines after
proteasome
erythroid commitment of progenitors (28). To
and
Fbw7.
inhibitor
endogenous
GATA2
MG132,
GATA2
suggesting
address the in vivo contribution of Fbw7 to
proteasome-dependent (Fig. 7A and B). Next, the
GATA2 stability during the early development of
contribution of Fbw7 on GATA2 stability was
hematopoietic cells, we used conditional knockout
evaluated. Endogenous GATA2 was accumulated
Mx1-Cre/Fbw7F/F mice, in which Fbw7 expression
by depletion of Fbw7 using various siRNAs for
is lost by genetic deletion using Cre recombinase
Fbw7 in K562 cells (Fig. 7C). In addition, we
under control of the Mx1 gene promoter (Fig. 8A).
repeated
using
Fbw7F/F mice were used for controls. Both groups
siRNA-Fbw7-A, -B and -C (Fig. 7D and 7E). All
were injected with pIpC on 7 alternate days to
siRNAs had a tendency to accumulate GATA2
yield the Fbw7/ genotype. One week after the
protein, especially siRNA-Fbw7-A and Fbw7-B,
final injection of pIpC, we confirmed that almost
which significantly accumulated GATA2 protein
all floxed alleles of Fbw7 in Mx1-Cre/Fbw7F/F
in K562 cells. To exclude the possibility that
mice were exchanged to Fbw7/ (Fig. 8B).
Fbw7-depletion may increase the transcription of
Lineage negative (Lin-) populations derived from
GATA2,
of
BM monocyte were subjected to FACS analysis. A
Fbw7-depletion on GATA2 mRNA expression. As
histogram of c-Kit analysis indicated two peaks
shown in Figure 7F, GATA2 mRNA expression
(c-Kithi and c-Kitlo/−) in control mice, besides the
was not significantly influenced by treatment with
peak pattern derived from Fbw7/ mice were
siRNAs for Fbw7. We attempted to confirm the
classified
CHX chase experiment result. Although GATA2
Fbw7/-D). The expression pattern of c-Kit in
was degraded in a time-dependent manner by
Lin− cells from three of six Fbw7/ mice
treating CHX, depletion of Fbw7 decelerated its
(Fbw7/-C) corresponded with that from control
degradation rate in K562 cells (Fig. 7G). These
mice (Fig. 8C top). Meanwhile, a significant
results strongly suggest that endogenous Fbw7
decrease in the c-Kithi subpopulation in Lin−
functions as an E3 ligase for the degradation of
population was observed in the remaining three
GATA2 in intact cells.
Fbw7/ mice (Fbw7/-D) compared with that in
we
knockdown
evaluated
experiments
the
effects
in
two
types
(Fbw7/-C
and
other mice (Fig. 8C top). The expression of
13
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is
the
degradation
Degradation of GATA2 by Fbw7 GATA2 in the Lin− population was examined by
from Fbw7/ mice compared with control mice,
FACS and IB analysis of sorted cells. The c-Kithi
although its difference was not prominent. The
fraction had a higher expression of GATA2 than
depletion of Fbw7 did not increase GATA2
the c-Kitlo/− fraction in all examined subjects (Fig.
mRNA in either c-Kithi or c-Kitlo/- fractions (Fig.
8C middle, bottom left and table). The tendency
8D). Taken together, we speculate that the
was similar between three subgroups of mice (Fig.
depletion of Fbw7 caused increased GATA2 levels
8C middle, bottom left). Nevertheless, GATA2
at
levels in both fractions were higher in Fbw7/
development, and subsequently, when amounts of
mice compared with those of control mice (Fig.
GATA2 exceed the threshold, the cells may not be
8C bottom left). Interestingly, the ratio of GATA2
sustained in an undifferentiated state.
the
early
stages
of
hematopoietic
cell
DISCUSSION
significantly increased in the fraction from
Fbw7/ mice compared with that of control mice,
This study identified cyclin B-CDK1 as the
from
CPD-kinase responsible for phosphorylation of
Fbw7/-D mice was significantly higher than
Thr-176 in GATA2 in the M phase. Moreover,
from Fbw7/-C mice (Fig. 8C table). There are
Fbw7 participated in stability control of GATA2
two explanations for the increased GATA2 ratio.
via ubiquitin-proteasome system in both cultured
First, GATA2 levels might be repressed in c-Kitlo/−
cell lines and mouse HSCs. Therefore, we propose
cells from Fbw7/ mice, although there was a
Fbw7 is a bona fide E3 ligase for GATA2.
moreover,
the
ratio
in
the
fraction
tendency towards increased GATA2 levels in both
Many reported substrates of Fbw7 do not
c-Kit fractions of Fbw7/ mice. Second, a large
contain a lysine residue in their CPD and some
quantity of GATA2 might exist during the c-Kithi
reports indicate the existence of a lysine residue in
stage derived from Fbw7/ mice. In addition,
the CPD does not enhance recognition by Fbw7.
c-Kithi subpopulation was significantly repressed
However, conflicting findings have been reported.
in
Fbw7/-D
mice
(Fig.
8C
top
right).
The sequence of h-cyclin E contains two CPD
Conditional depletion of Fbw7 in BM cells often
motifs. One of them codes T62-P-D-K-E, which
resulted in the enhanced accumulation of GATA2.
contains a lysine residue, and is an active degron
It might depend on the undifferentiated degree in
recognized by Fbw7 (46-48). Furthermore, we
the progenitors. Therefore, excess GATA2 beyond
detected a functional CPD site in h-GATA3,
the threshold in a primitive progenitor might cause
which belongs to the substrates of Fbw7 (23). It
the consumption of c-Kithi cells. We observed a
codes T156-P-P-K-D, which is similar to that of
higher accumulation of GATA2 in c-Kithi cells
h-cyclin E. In this report, we detected CPD in
14
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levels in c-Kithi cells to c-Kitlo/− cells was
Degradation of GATA2 by Fbw7 h-GATA2, which codes T176-P-P-K-E. This is
B-CDK1 and SCFFbw7 contributes to protein
similar to that of h-GATA3. Therefore, these
degradation in the M phase.
findings provide compelling evidence that CPD
Koga et al. previously reported the cell
functions as an Fbw7 recognition motif even if a
cycle-dependent regulation of GATA2 expression.
lysine residue is present. Furthermore, the capacity
In their study, GATA2 levels were high in the S
of CPD we detected in h-GATA2 is supported by
phase but low in the G1/S and M phases (38).
this data. It has been reported that GSK3β
GATA2
phosphorylates CPD motifs in the cognate
sequences
substrates
of
Fbw7
protein
contains
some
consensus
(Ser/Thr-Pro-X-Lys/Arg)
for
GSK3β
phosphorylation by CDKs. In vitro GATA2
threonine
at
the
phosphorylation assays suggested that cyclin
for
GSK3β
D1-CDK4, cyclin A-CDK2 and cyclin B1-cdc2
after
priming
(CDK1) were candidates for the responsible
phosphorylation of the later Ser/Thr residue by
kinases, although the individual phosphorylation
other kinases. In addition, GSK3β functions on
sites were not identified. They also described that
Ser/Thr-X-X-X-Glu/Asp
priming
the 1-70, 153-256 and 412-480 aa regions were
phosphorylation as for mouse c-Myb (18).
important for degradation of GATA2 and that
Because the CPD motif overlaps with the GSK3β
Ser-227 participated in destabilizing GATA2 in
recognition consensus sequence, CPD motifs in
the M phase. They speculated that CDKs mediated
Fbw7-substrates are often phosphorylated by
phosphorylation of Ser-227, but the specific CDKs
GSK3β. In contrast, our studies indicated that
were not identified. Furthermore, it was not
CPDs in GATA2 and GATA3 (23) were
clarified why degradation of GATA2 was required
phosphorylated by cyclin B-CDK1 and cyclin
in the M phase. The current study also did not
A-CDK2, respectively. The aa residue before the
answer this question, thus further studies are
priming phosphorylation site in GATA2 and
required. Because sequences around Ser-227 do
GATA3 is lysine, consistent with a consensus
not contain a CPD motif, it is not expected that
sequence
for
Fbw7 recognizes phosphorylated Ser-227 in
In contrast to
GATA2 for ubiquitin-dependent degradation. Our
GSK3β responding to signal transduction (32),
study is the first to identify an E3 ligase for
CDKs are activated by the expression of specific
GATA2 and clarify the recognition mechanism of
cyclins in a cell cycle-dependent manner (49) and
GATA2 by Fbw7. Cellular proteins regulated by
cyclin B-CDK1 functions in the M phase. This is
the ubiquitin-proteasome system are often targeted
the first report that collaboration between cyclin
by E3 ligases. Moreover, depletion of Fbw7
phosphorylates
serine
consensus
sequence
or
(Ser/Thr-X-X-X-pSer/pThr)
without
(Ser/Thr-Pro-X-Lys/Arg)
phosphorylation by CDK (44).
15
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(25,32).
Degradation of GATA2 by Fbw7 increased the stability of GATA2 although it was
of premature depletion of normal BM HSCs when
still degraded. Accordingly, Fbw7 and other E3
Fbw7 was depleted. In addition to c-Myc and
ligases might target other sites including Ser-227
Notch1, GATA2 also functions as a cell cycle
for
accelerator
the
ubiquitin-dependent
degradation
of
in
HSCs.
We
observed
its
GATA2. Each E3 ligase might be specific for
accumulation in the Lin− fraction of Fbw7/ mice,
various cell types.
and speculated that c-Kithi cells might exhaust
themselves by excessive GATA2 expression.
cell cycle accelerators in hematopoietic cells.
However, it was also reported that enforced high
Fbw7 may coordinate their cellular levels via the
expression of GATA2 inhibited proliferation, cell
ubiquitin-proteasome
systemically
cycle entry from a quiescent stage and functions of
control proliferation and differentiation of HSCs.
stem and progenitor cells (51-53). Therefore,
Matsuoka et al. studied the influence of depletion
accumulation of GATA2 in Fbw7/ mice might
of Fbw7 in the development of BM HSCs using
inhibit cell cycle entry and cell proliferation of
conditional knockout Mx1-Cre/Fbw7F/F mice (50).
HSCs
In their report, leukocytes, hemoglobin and
preferentially decreased. In addition, the depletion
platelets from peripheral blood cells, Lin− fraction
of Fbw7 promotes c-Myc accumulation and
of BM and Lin−Sca-1+c-Kit+ (LSK) CD34- HSCs,
eliminates leukemia-initiating cells via apoptosis
respectively, were decreased in number in Fbw7
(45,54). The Fbw7-mediated control of GATA2
deleted mice compared with that in control mice.
and other Fbw7-substrates including c-Myc might
This suggests that disturbance of HSCs cause a
participate in self-renewal and maintenance of
diminution of differentiated cells. Moreover,
hematopoietic/leukemic stem cells. Accordingly,
increased c-Myc and Notch1 proteins involved in
GATA2 as well as c-Myc and Notch1 in HSCs
cell cycle progression, were present in LSK cells
from Fbw7/ mice might perturb cell homeostasis
from Fbw7 deleted mice. The authors explained
because of their excessive accumulation.
system
to
that cell cycle promotion of HSCs was one cause
16
and
thereby
c-Kithi
HSC
might
be
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Fbw7 participates in the degradation of many
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members of our laboratory for useful discussions. This work was supported in part by grants from the
Ministry of Education, Culture, Sports, Science and Technology of Japan grants-in-aid 24570151 (K.
Kitagawa), 25112508 (M. Kitagawa).
22
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Acknowledgments - We thank M. Matsumoto, M. Yoshida and M. Hakamata for technical support and
Degradation of GATA2 by Fbw7 FIGURE LEGENDS
FIGURE 1. Fbw7 binds to GATA2 in a Thr-176-dependent manner. A. Cdc4 phosphodegron (CPD), the
consensus sequence for recognition by Fbw7, is indicated in the top panel. Sequences surrounding
CPD-like motif of GATA1 and GATA2 were aligned to CPD motifs in the reported Fbw7-substrates (*).
Conserved amino acid residues within CPD are shown in bold. B. Fbw7 binds to WT-GATA2 but not
T176A-GATA2 and GATA1. HEK293 cells were transfected with indicated plasmids and then were
incubated with MG132 for 6h. Total cell lysates were subjected to immunoprecipitation (IP) with
indicated antibody, followed by immunoblotting (IB) with indicated antibodies. The original cell lysates
(input) were subjected to IB with indicated antibody to confirm protein expression. C. GATA2 binds to
incubated with MG132 for 6 h. Total cell lysates were subjected to IP with anti His antibodies, followed
by IB with the indicated antibodies.
FIGURE 2. Endogenous GATA2 binds to Fbw7. A and B. Differential proteomic analysis termed
DiPIUS-NL indicated that GATA2 is a potential target of Fbw7. Neuro2A cells expressing mouse
3xFLAG-Fbw7 (WT) or its ΔF mutant (ΔF) were treated with MG132. The lysates were
immunoprecipitated with anti-FLAG antibody and then eluted with FLAG peptide. The immunopurified
proteins were separated SDS-PAGE and the silver-stained gel was sliced and subjected to in-gel digestion
with trypsin. The resulting peptides were analyzed by LC–MS/MS. Indicated peptides bound to Fbw7-ΔF
mutant were identified as m-GATA2 derived peptide using MASCOT analysis (A). The abundance of
proteins that bound to the WT or mutant F-box proteins in three independent experiments were compared
by semiquantitative spectral counting, respectively (B) GATA2 was reproducibly identified as a strong
candidate of Fbw7 substrate. C. Endogenous GATA2 binds to endogenous Fbw7 in K562 cells. K562
cells treated with MG132 were lysed with proteasome inhibitors, protease inhibitors and phosphatase
inhibitors. Cell lysates were immunoprecipitated with anti-GATA2 rabbit monoclonal antibody
(ab109241) or control rabbit IgG following immunoblotting with anti-Fbw7 rabbit polyclonal antibody
(301-720A-1) or anti-GATA2 rat monoclonal antibody (RC1.1.1). Endogenous Fbw7 was co-precipitated
with endogenous GATA2.
FIGURE 3. Fbw7 promotes ubiquitylation of GATA2 but not GATA1. HEK293 cells were transfected
with indicated expression plasmids, and were then incubated with MG132 for 6 h. Total cell lysates were
23
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the SCFFbw7 complex. HEK293 cells were transfected with the indicated plasmids and then were
Degradation of GATA2 by Fbw7 subjected to immunoprecipitation (IP) with the indicated antibodies under native (A) or denaturing
conditions (B and C), followed by immunoblotting with the indicated antibodies (see EXPERIMENTAL
PROCEDURES).
FIGURE 4. Fbw7-mediated degradation of GATA2 is Thr-176-dependent. A. Effects of Fbw7 on
GATA2 stability. HeLa cells were transfected with WT-GATA2-myc-His or T176A-GATA2-myc-His in
the absence or presence of Fbw7 or ΔF-box mutant (ΔF). After 41 h transfection, cells were treated with
or without 10 µM MG132 for 7 h and then harvested. Total cell lysates were subjected to immunoblotting
with indicated antibodies. B-C. Effects of Fbw7 on GATA3 and GATA1 stability. HeLa cells were
transfected with GATA3-myc-His or FLAG-GATA1 in the absence or presence of HA-Fbw7 and then
analyzed by immunoblotting with the indicated antibodies. D. Effects of ectopic expression of Fbw7 on
degradation of WT-GATA2 or T176A-GATA2. HeLa cells were transfected with WT-GATA2-myc-His
in the absence or presence of FLAG-Fbw7, treated with 20 µg/ml cycloheximide (CHX) for the indicated
periods and harvested. Total cell lysates were subjected to immunoblotting. The representative data of
WT-GATA2 is indicated (D). E-F. Levels of WT-GATA2 and T176A-GATA2 in the absence or presence
of FLAG-Fbw7 after the various chase times were quantitated by image analysis and normalized against
β-actin. The percentages of remaining GATA2 protein were calculated as the mean ± s.d. from three
independent experiments. The P value was determined by Student’s t-test. * p<0.05 ** p<0.01.
FIGURE 5. Cyclin B-CDK1 participates in phosphorylation of Thr-176 of GATA2. A. Evaluation of
antibodies
against
phospho-Thr-176-GATA2
(p-T176-GATA2).
WT-GATA2-myc-His
or
T176A-GATA2-myc-His were transiently expressed in HEK293 cells. Cell lysates prepared with lysis
buffer containing phosphatase inhibitors were subjected to immunoblotting with anti GATA2 or
anti-p-T176-GATA2. B. Thr-176 of endogenous GATA2 protein is phosphorylated in HeLa cells.
Endogenous GATA2 protein was depleted by GATA2 siRNA and lysates were subjected to
immunoblotting with the indicated antibodies. C. Putative consensus motif for phosphorylation by CDK1
and CDK2 in the CPD motif containing Thr-176 in GATA2. D-F. Thr-176 in GATA2 is efficiently
phosphorylated by cyclin B1-CDK1 in vitro. Recombinant GST-WT-GATA2 (D and F) or GST-RB (E)
was incubated with indicated recombinant kinases in reaction buffer with 1 mM ATP for 30 min, and then
subjected to immunoblotting with the indicated antibodies. G. Inhibition of Thr-176-phosphorylation of
GATA2 by CDK inhibitors. HEK293 cells were transfected with WT-GATA2-myc-His and then treated
24
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were treated with or without 10 µM MG132 for 7 h. Levels of GATA3 (B) and GATA1 (C) were
Degradation of GATA2 by Fbw7 with CDK inhibitors, 1 µM RO-3306 or 1 µM butyrolactone I for 5 h. Cell lysates were subjected to
immunoblotting with anti p-T176-GATA2 or anti GATA2.
FIGURE 6. Phosphorylation of Thr-176 of GATA2 participates in binding with Fbw7 and GATA2 is
decreased in M phase. A. Thr-176-phosphorylated GATA2 binds Fbw7 in vitro. Purified GST fused WTor T176A- GATA2 or GST protein using glutathione beads was incubated with or without recombinant
cyclin B1-CDK1 in reaction buffer containing 1 mM ATP for 30 min. The reaction mixtures were
incubated with lysates from HEK293 cells expressing FLAG-Fbw7, then precipitated using
glutathione-Sepharose beads and subjected to immunoblotting (IB) with the indicated antibodies. B.
Thr-176 of WT-GATA2 protein was phosphorylated by cell lysates prepared from synchronized HeLa
glutathione beads were incubated with lysates prepared from HeLa cells arrested in G1/S or M phase or
asynchronized (AS) HeLa cells in reaction buffer containing 1 mM ATP for 30 min. The mixtures were
treated with precision protease for 30 min to cleave out GATA2 from the GST-tag and then subjected to
immunoblotting with indicated antibodies. C. Endogenous GATA2 is decreased in the M phase. Lysates
prepared from HeLa cells arrested at G1/S, S or M phase or asynchronized HeLa cells were subjected to
immunoblotting with indicated antibodies. mRNA levels in synchronized cells were measured by
qRT-PCR. Total RNA was isolated from cells, and subjected to reverse transcription with random
hexanucleotide primers. The resulting complementary DNA was subjected to qRT-PCR. The amount of
transcripts was normalized against that of 18s-rRNA as an internal standard. Relative protein and mRNA
levels of GATA2 are indicated below. D. Effect of CDK inhibitor treatment on GATA2 levels in M phase
cells. HeLa cells synchronized at M phase were treated with 10 µM butyrolactone I for 6 h and then
GATA2 levels were analyzed by immunoblotting.
FIGURE 7. Depletion of endogenous Fbw7 stabilizes endogenous GATA2 protein in K562 cells. A-B.
GATA2 is degraded in a proteasome-dependent manner. K562 cells were treated with or without 20 µM
MG132 for 6 h. The cells were harvested and the lysates were subjected to immunoblotting with the
indicated antibodies (A). GATA2 levels normalized to β-actin levels were calculated from three
independent experiments (B). C–F. Effects of Fbw7-depletion on GATA2 stability. The lysates were
subjected to immunoblotting with the indicated antibodies. K562 cells were transfected with the indicated
siRNA or control siRNA for 48 h and harvested. The lysates were subjected to immunoblotting with the
indicated antibodies. Representative data are shown (D). Relative GATA2 levels in the absence or
25
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cells at the M phase in vitro. Purified GST fused WT- or T176A- GATA2 or GST proteins with
Degradation of GATA2 by Fbw7 presence of siRNA for Fbw7 were normalized to β-actin levels and calculated from four independent
experiments (E). Relative mRNA levels of GATA2 were measured by qRT-PCR (F). Total RNA was
isolated from cells and subjected to reverse transcription with random hexanucleotide primers. The
resulting complementary DNA was subjected to qRT-PCR. The amount of transcripts was normalized
against that of 18s-rRNA as an internal standard. G. K562 cells were transfected with siRNA for Fbw7 or
control siRNA for 48 h and then treated with 20 µg/ml CHX for the indicated times. Cells were harvested
and lysates were subjected to immunoblotting with the indicated antibodies. P values were determined
using Student’s t-test. * p<0.05 ** p<0.01 (B, E and F).
FIGURE 8. GATA2 increases in Lin−/c-Kithi BM cells by Fbw7-depletion. A. Schematic representations
of exon 5 by Cre recombinase (ΔE5). Triangles within Flx and ΔE5 alleles indicate LoxP genes. The
positions of forward (F) and reverse (R) primer annealing corresponding to WT, Flx and ΔE5 alleles are
indicated by arrows, and those corresponding to the Cre recombinase gene are indicated by arrowheads,
with each PCR product size. B. PCR analysis of genomic DNA from mouse tails (left) and mononuclear
cells isolated from BM one week after the last injection of pIpC (right) were performed for genotyping of
transgenic mice and verifying deletion of exon 5 (Fbw7/) in Cre+FbwFlx/Flx mice after pIpC treatment,
respectively. C. Top graphs show representative profiles of c-Kit staining in Lin− BM cells from
pIpC-injected mice. Cells were classified by expression levels of c-Kit, into c-Kithi and c-Kitlo/− subsets
and subsequently each subpopulation was analyzed for expression of GATA2 protein by FACS (middle)
or subjected to immunoblot analysis (bottom right). Fbw7/ mice were categorized into two classes
based upon c-Kit profiles. Those with same levels as control mice and those with significantly decreased
c-Kithi populations were named Fbw7/-C and Fbw7/-D, respectively (top right). Mean counts of
GATA2 Alexa488 (bottom left) and ratio of GATA2 levels in c-Kithi cells to c-Kitlo/− cells (table) were
compared among the three subgroups. Data are means + SD from three mice of each subgroup. D.
GATA2 mRNA does not increase in Lin− BM cells by Fbw7-depletion. c-Kithi and c-Kitlo/− subsets from
Lin− BM cells of control and Fbw7/ mice were sorted and were analyzed for expression of GATA2
mRNA by qRT-PCR. Data are means + SD from four mice of each genotype.
26
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of the wild type mouse Fbw7 allele (WT), floxed Fbw7 allele (Flx), and floxed Fbw7 allele after removal
Nakajima et al.
A
C
Consensus seq.
for Fbw7 targets
*h-NFkB2
*m-c-Myb
L- P - T-P- P- L -S- P
G- E - T-P- P- L -S- P
L- I - T-P-D-K -E-D
L- L - T-P- P-Q-S-G
L- P -S-P- P- T -S-D
L-M- T-P- V-S -E-D
HA-Fbw7
WT-GATA2-myc-His
HA-Skp1
Myc-Cul1
HA-Roc1
anti HA
IP: anti His
*h-c-Myc
*h-c-Jun
*h-cyclin E
S/T -P -X-X- S/T/D/E
T156(h)/T155(m)
*m/h-GATA3 P- P- T- P- P- K- D-V
m/h-GATA2 P- P- T- P- P- K- E -V
T176
anti Myc
- WT DF
+ + +
+ + +
+ + +
+ + +
Fbw7
GATA2
anti HA
Skp1
anti Myc
Cul1
anti HA
Roc1
anti HA
Fbw7
m/h-GATA1 F - F - S-P - T- G- S - V
-
IP: anti Myc IP: anti FLAG
-
-
Fbw7
anti FLAG
Fbw7
anti Myc
anti Myc
GATA2
anti HA
Skp1
anti Myc
Cul1
anti HA
Roc1
GATA2
GATA1
anti FLAG
anti FLAG
input
+ - + - +
+ - - - - + + - -
anti Myc
+
-
+ +
FLAG-Fbw7
WT-GATA2-myc-His
T176A-GATA2-myc-His
GATA1-myc-His
input
anti Myc
B
GATA2
GATA1
Fbw7
GATA2
GATA1
Figure 1
Nakajima et al.
A
ECVNCGATATPLWR (GATA2: 294-307)
Name
IPI00224113.1
IPI00124047.1
Fbw7 (bait)
Cul1
IPI00331163.9
IPI00114226.2
IPI00131999.2
IPI00225178.3
Skp1
GATA2
c-Myc
c-Myb
Exp. 3
IPI number
Exp. 2
Exp. 1
B
GAECFEELSK (GATA2: 410-419)
DF only (substrate)
0.5  DF/WT  2
DF/WT < 0.5
WT only
Not detected
C
K562 cells
input (lysate)
IB:
anti-Fbw7
anti-GATA2
IP
C-IgG anti GATA2
- Fbw7
- GATA2
Figure 2
Nakajima et al.
A
FLAG-Fbw7 +
WT-GATA2-myc-His T176A-GATA2-myc-His MG132 +
HA-Ub +
+
+
+
+
+
+
+
+
+
+
+
+
+
+
KD
115
input
IP: anti Myc
anti HA
B
82
64
anti Myc
64
anti FLAG
115
anti Myc
64
anti FLAG
115
C
Denaturing IP: Myc
FLAG-Fbw7
WT-GATA2-myc-His
HA-Ub
+
-
+
+
+
+
+
Denaturing IP: FLAG
HA-Fbw7
FLAG-GATA1
HA-Ub
+
-
- +
+ +
+ +
KD
anti HA
115
anti Ub
KD
115
64
82
64
anti Myc
64
anti FLAG
64
Figure 3
GATA2-myc-His
-
WT WT
ΔF ΔF
WT WT WT WT WT WT
MG132
-
+
-
+
-
-
WT WT
D
TA TA TA TA
-
+
-
GATA2-myc-His
+
vector
anti Myc
(GATA2)
CHX (h)
anti HA
(Fbw7)
anti Myc
(GATA2)
anti β-actin
anti FLAG
(Fbw7)
HA-Fbw7
GATA3-myc-His
MG132
anti Myc
(GATA3)
- - + +
+ + + +
- + - +
anti HA
(Fbw7)
anti β-actin
C
+
-
HA-Fbw7
FLAG-GATA1
MG132
anti FLAG
(GATA1)
anti HA
(Fbw7)
- - + +
+ + + +
- + - +
0 0.5
FLAG-Fbw7
1 2
4
0 0.5 1
anti β-actin
E
Relative GATA2 protein level
B
-
F
1.5-
n=3
**
*
*
WT
1-
0.5-
00
WT+Fbw7
1
2
3
CHX (h)
4
Relative GATA2 protein level
HA-Fbw7
Downloaded from http://www.jbc.org/ by guest on February 16, 2015
A
Nakajima et al.
2
1.5-
4
n=3
1T176A
0.5T176A+Fbw7
00
1
2
3
4
CHX (h)
anti β-actin
Figure 4
anti p-T176
-GATA2
anti p-T176
-GATA2
anti GATA2
GST-p-GATA2
anti GATA2
GST-GATA2
anti RB
RB
GST-RB
B/CDK1
GSK3β
Butyrolactone Ⅰ
p-RB
anti β-actin
C
inhibitor
RO-3306
115
GATA2
DMSO
49
p-GATA2
GATA2-myc-His
+
+
+
G
p-S807
-RB
anti p-S807
-RB
anti GATA2
82
p-T176GATA2
GST-GATA2
B/CDK1
A/CDK2
E/CDK2
KD
115
D/CDK4
-
E
KD
49
anti p-T176
-GATA2
82
115
82
115
GST-GATA2
GATA2
control
siRNA
GST-p-T176
-GATA2
82
B
KD
115
anti p-T176
-GATA2
82
anti GATA2
F
B/CDK1
KD
115
A/CDK2
KD
82
E/CDK2
-
T176A
WT
GATA2-myc-His
vector
D
Downloaded from http://www.jbc.org/ by guest on February 16, 2015
A
D/CDK4
Nakajima et al.
anti p-T176
-GATA2
CPD motif
anti GATA2
GATA2 P-P-T-P-P-K-E-V
Consensus Seq.
for CDK1/2
S/T-P-X-K/R
P
Figure 5
GST pull down
+
+
+
+
+
+
T176A
+
Fbw7
p-T176GATA2
anti GATA2
p-GATA2
GATA2
anti p-T176
-GATA2
anti GATA2
D
inhibitor
AS
G1/S
AS
Relative GATA2 1.11 1.71 0.22 1.00
protein level
Relative GATA2
1.67 2.13 1.52 1.00
mRNA level
T176A
AS
M
G1/S
WT
AS
G1/S
cell lysates
M
-
GST-GATA2
M
anti β-actin
anti p-T176
-GATA2
B
S
anti Fbw7
anti Fbw7
IB
anti GATA2
anti cyclin B
+
+
M
cyclin B/CDK1
Fbw7
WT
p-T176GATA2
GATA2
Butyrolactone Ⅰ
-
GST-GATA2
C
DMSO
A
Downloaded from http://www.jbc.org/ by guest on February 16, 2015
G1/S
Nakajima et al.
anti GATA2
anti β-actin
Figure 6
6
4
anti Fbw7
2
β-actin
0
MG132
-
+
Fbw7-B
control
Fbw7-B
control
Fbw7-C
Fbw7-A
control
control
Fbw7-C
E
Fbw7-A
D
siRNA
anti GATA2
anti Fbw7
G
1.5
n=4
4
*
n=4
3
2
1
0
siRNA
siRNA
CHX (h)
4
3
2
4
SMART
POOL
Fbw7-C
Fbw7-B
Fbw7-A
2
1
0
control
0
**
n=4
Fbw7-B
6
8
0
2
4
6
8
1
anti GATA2
0.5
Fbw7-C
Fbw7-B
0
siRNA
Fbw7-A
anti-Fbw7
control
Relative GATA2 mRNA level
anti β-actin
F
control
anti GATA2
Fbw7-B
anti β-actin
siRNA
control
anti Fbw7
8
n=3
Fbw7-D
anti GATA2
*
Relative GATA2 protein level
- +
Fbw7-C
- +
C
10
Fbw7-A
- +
B
control
exp.3
Relative GATA2 protein level
exp.2
Relative GATA2 level
MG132
exp.1
Downloaded from http://www.jbc.org/ by guest on February 16, 2015
A
Nakajima et al.
anti β-actin
Figure 7
B
Fbw7Flx/WT
734bp
Flx
984bp
DE5
pI/pC injection (Cre exp.)
Control
(Fbw7Flx/Flx )
Fbw7D/D
Flx
WT
304bp
F
Cre
Nakajima et al.
Cre+Fbw7Flx/Flx
Pro
Cre
Cre
R
230bp
Fbw7D/D-C
control
（not stained)
Fbw7D/D-D
30
* p<0.05
lo/-
lo/-
hi
c-Kit PE-Cy5
c-Kithi
GATA2 Alexa488 (count)
control
F/F
Fbw7D/D-C
Mx1Cre+F/F-A
Fbw7D/D-D
Mx1Cre+F/F-B
15000
1.5
10000
1.0
1.5
0.15
Ratio of GATA2 level in
c-Kithi
cells to
control
1.99+0.01
Fbw7D/D-C
2.17+0.05
p<0.05 vs control
Fbw7D/D-D
2.37+0.07
p<0.05 vs control, Fbw7D/D-C
c-Kithi c-Kitlo/-
c-Kithi
cKit+
c-Kitlo/cKitlo/-
anti GATA2
1.5
1.9
control
control
cKO
Fbw7D/D-D
1.0
0.10
5000
0.5
0
c-Kitlo/- cells
Relative GATA2 mRNA level
20000
2.0
DE5
Cre
10
D
GATA2 Alexa488
x104
20
0
c-Kitlo/-
Flx
*
*
Fbw7D/D-C
hi
control
lo/-
hi
Population of c-Kithi cells (%)
C
Downloaded from http://www.jbc.org/ by guest on February 16, 2015
R
Fbw7Flx/Flx
Fbw7D/D-D
E6
Fbw7Flx/Flx
E5
F
WT
Cre+Fbw7Flx/Flx
E4
A
sorted cells (x105)
0.5
0.05
0.000
hi
c-Kit
Hi
lo/c-Kit
Lo/-
Figure 8