Article

Nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase is regulated by acetylation

Department of Cell Biology, Immunology and Neurosciences, Faculty of Medicine, University of Barcelona, Barcelona, Spain.
The international journal of biochemistry & cell biology (Impact Factor: 4.05). 10/2010; 42(10):1672-80. DOI: 10.1016/j.biocel.2010.06.014
Source: PubMed

ABSTRACT

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is considered a housekeeping glycolitic enzyme that recently has been implicated in cell signaling. Under apoptotic stresses, cells activate nitric oxide formation leading to S-nitrosylation of GAPDH that binds to Siah and translocates to the nucleus. The GAPDH-Siah interaction depends on the integrity of lysine 227 in human GAPDH, being the mutant K227A unable to associate with Siah. As lysine residues are susceptible to be modified by acetylation, we aimed to analyze whether acetylation could mediate transport of GAPDH from cytoplasm to the nucleus. We observed that the acetyltransferase P300/CBP-associated factor (PCAF) interacts with and acetylates GAPDH. We also found that over-expression of PCAF induces the nuclear translocation of GAPDH and that for this translocation its intact acetylase activity is needed. Finally, the knocking down of PCAF reduces nuclear translocation of GAPDH induced by apoptotic stimuli. By spot mapping analysis we first identified Lys 117 and 251 as the putative GAPDH residues that could be acetylated by PCAF. We further demonstrated that both Lys were necessary but not sufficient for nuclear translocation of GAPDH after apoptotic stimulation. Finally, we identified Lys 227 as a third GAPDH residue whose acetylation is needed for its transport from cytoplasm to the nucleus. Thus, results reported here indicate that nuclear translocation of GAPDH is mediated by acetylation of three specific Lys residues (117, 227 and 251 in human cells). Our results also revealed that PCAF participates in the GAPDH acetylation that leads to its translocation to the nucleus.

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The International Journal of Biochemistry & Cell Biology 42 (2010) 1672–1680
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The International Journal of Biochemistry
& Cell Biology
journal homepage: www.elsevier.com/locate/biocel
Nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase is
regulated by acetylation
Mireia Ventura
a
, Francesca Mateo
a
, Joan Serratosa
b
, Ignasi Salaet
a
, Sonia Carujo
a
,
Oriol Bachs
a,
, María Jesús Pujol
a,
a
Department of Cell Biology, Immunology and Neurosciences, Faculty of Medicine, University of Barcelona, 08036 Barcelona, Spain
b
Department of Brain Ischemia and Neurodegeneration, Instituto de Investigaciones Biomédicas de Barcelona,
Consejo de Investigaciones Científicas, (IIBB-CSIC-IDIBAPS), 08036 Barcelona, Spain
article info
Article history:
Received 4 February 2010
Received in revised form 2 June 2010
Accepted 16 June 2010
Available online 25 June 2010
Keywords:
GAPDH
PCAF
Acetylation
Nuclear translocation
abstract
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is considered a housekeeping glycolitic enzyme
that recently has been implicated in cell signaling. Under apoptotic stresses, cells activate nitric oxide
formation leading to S-nitrosylation of GAPDH that binds to Siah and translocates to the nucleus. The
GAPDH–Siah interaction depends on the integrity of lysine 227 in human GAPDH, being the mutant
K227A unable to associate with Siah. As lysine residues are susceptible to be modified by acetylation,
we aimed to analyze whether acetylation could mediate transport of GAPDH from cytoplasm to the
nucleus. We observed that the acetyltransferase P300/CBP-associated factor (PCAF) interacts with and
acetylates GAPDH. We also found that over-expression of PCAF induces the nuclear translocation of
GAPDH and that for this translocation its intact acetylase activity is needed. Finally, the knocking down
of PCAF reduces nuclear translocation of GAPDH induced by apoptotic stimuli. By spot mapping analysis
we first identified Lys 117 and 251 as the putative GAPDH residues that could be acetylated by PCAF. We
further demonstrated that both Lys were necessary but not sufficient for nuclear translocation of GAPDH
after apoptotic stimulation. Finally, we identified Lys 227 as a third GAPDH residue whose acetylation is
needed for its transport from cytoplasm to the nucleus. Thus, results reported here indicate that nuclear
translocation of GAPDH is mediated by acetylation of three specific Lys residues (117, 227 and 251 in
human cells). Our results also revealed that PCAF participates in the GAPDH acetylation that leads to its
translocation to the nucleus.
© 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is con-
ventionally considered a housekeeping glycolytic enzyme (Sirover,
2005). It forms a homo-tetramer complex that catalyzes a cru-
cial reaction in the glycolytic pathway. It uses NAD
+
and converts
glyceraldehyde-3-phosphate to 1,3 bisphosphoglycerate with the
release of NADH (Chuang et al., 2005). Besides its conventional
metabolic role, it participates in diverse cellular functions (Colell et
al., 2009). It is involved in nuclear membrane assembly and in mem-
brane transport from ER to Golgi (Nakagawa et al., 2003; Tisdale
et al., 2004). It binds to microtubules and modulates cytoskeleton
organisation (Cueille et al., 2007; Tisdale et al., 2009). Moreover,
GAPDH is an RNA-binding protein that regulates mRNA stability
(Rodriguez-Pascual et al., 2008; Zhou et al., 2008).
Corresponding authors. Tel.: +34 93 403 52 86; fax: +34 93 402 19 07.
E-mail addresses: obachs@ub.edu (O. Bachs), mjpujol@ub.edu (M.J. Pujol).
It addition to its cytosolic and membrane roles, GAPDH shows
several nuclear functions as the regulation of transcription (Harada
et al., 2007; Kim et al., 2007; Dai et al., 2008) and DNA repair (Azam
et al., 2008) but also in the maintenance of telomeric structure
(Sundararaj et al., 2004). Moreover, GAPDH participates in apopto-
sis and in neurodegenerative disorders (Tatton et al., 2000; Mazzola
and Sirover, 2003; Chuang et al., 2005; Du et al., 2007; Sen et al.,
2008).
GAPDH is mainly located in the cytoplasm. However, it can
translocate to the nucleus under different circumstances as for
instance during cell cycle progression or after apoptotic stimulation
(Sawa et al., 1997; Senatorov et al., 2003; Sirover, 2005; Park et al.,
2009). Serum depletion in NIH3T3 fibroblasts also induced GAPDH
import to the nucleus (Schmitz, 2001). In the nucleus, GAPDH plays
several roles being one of them the regulation of cell cycle progres-
sion. It binds to the cell cycle regulator p21
Cip1
and this interaction is
mediated by the chromatin remodelator SET (Carrascal et al., 2002;
Carujo et al., 2006). Interestingly, GAPDH also binds to cyclin B
and over-expression of GAPDH induces an acceleration of cell cycle
mediated by its effect on cyclin B-cdk1 activity (Carujo et al., 2006).
1357-2725/$ see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biocel.2010.06.014
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M. Ventura et al. / The International Journal of Biochemistry & Cell Biology 42 (2010) 1672–1680 1673
Likewise, GAPDH is a key component of the Oct-1 co-activator in
S-phase (OCA-S) complex that is essential for S-phase-dependent
histone H2B transcription (Zheng et al., 2003; Dai et al., 2008).
A number of reports reveals that nuclear accumulation of
GAPDH precedes apoptosis (Colell et al., 2009). However, the mech-
anisms involved in the nuclear translocation of GAPDH are still a
controversial matter. It has been reported that GAPDH is a sensor of
nitric oxide (NO) stress (Hara et al., 2006). NO causes S-nitrosylation
of GAPDH at its active site (Cys 152 in human GAPDH) increas-
ing the binding to the E3-ubiquitin ligase Siah1 (Hara et al., 2005;
Hara and Snyder, 2006). For this association it is important the
integrity of Lys 227. As GAPDH has not a NLS sequence, it needs the
association with Siah1 for its translocation. In the nucleus, GAPDH
stabilizes Siah1 enhancing its ubiquitin ligase activity. Moreover,
nuclear GAPDH/Siah1 complexes increases p300/CBP-associated
acetylation of nuclear proteins, including p53 and thus trigger-
ing degradation of nuclear proteins (Fiucci et al., 2004; Hara et
al., 2005; Sen et al., 2008). The Siah1 protein is also necessary for
high glucose-induced GAPDH nuclear accumulation and cell death
in Müller cells (Yego et al., 2009; Yego and Mohr, 2010). A recent
report describes that the cytosolic protein GOSPEL binds to GAPDH
and competes with Siah1 thus preventing its nuclear translocation
(Sen et al., 2009).
We have analyzed here the role of GAPDH acetylation on its
nuclear translocation. Our results indicate that GAPDH is acety-
lated by the acetyltransferase p300/CBP-associated factor (PCAF).
Moreover, we identified three Lys residues of GAPDH that are nec-
essary for its nuclear accumulation. Thus, we describe here for the
first time that GAPDH acetylation at specific Lys residues is a new
pathway that regulates its nuclear transport.
2. Materials and methods
2.1. Materials
Anti-acetyl-Lys (Ab193) was purchased from Abcam. Anti-
HA (H6908) and anti-PCAF (p7493) were obtained from Sigma
and anti-GAPDH (MAB374) was from Chemicon. Glutathione-
Sepharose beads and CNBr-activated-Sepharose 4B beads were
from Amersham Bioscience. Purified human erythrocyte GAPDH,
Trichostatin A (TSA) and chemicals for SDS-PAGE were purchased
from Sigma. GAPDH synthetic peptides for spot mapping analysis
were provided by Sigma–Genosys.
2.2. Cell culture and transfection
NIH3T3 cells were grown in Dulbecco’s minimum essential
medium (DMEM) supplemented with 10% donor calf serum.
HCT116 cells were grown in DMEM–HAM (F12) 1:1 medium sup-
plemented with 10% fetal calf serum. Cell transfections were made
with lipofectamine-2000 (Invitrogen). In several experiments, TSA
was added to the medium at a concentration of 100 nM at 24 h post-
transfection. For siRNA experiments NIH3T3 cells were transfected
with PCAF siRNA or control siRNA with lipofectamine-2000 for 24 h.
Then, cells were treated with 100 nM TSA for 20 h and subsequently
subjected to immunocytochemical analysis of GAPDH according to
the protocol described in this manuscript.
2.3. Plasmids
Human placenta GAPDH cDNA was a generous gift from Dr.
Sirover. This cDNA, was cloned into pGEX-5X-3, pGFP-C2 and
pEF-HA vectors. GAPDH fragments were generated by PCR. Site-
directed mutagenesis (Quick Change-Site-Directed Mutagenesis,
Stratagene Kit) was used to mutate Lys to Arg or Gln; The pCDNA3-
Flag-C-terminal-PCAF (aa352–832) and pCDNA3-Flag-PCAF-HAT
(aa352–658) constructs were a generous gift from Dr. Martínez-
Balbás; these PCAF fragments were cloned into pTRE vector for
tetracycline-dependent-induction experiments in NIH3T3 cells.
2.4. Protein expression and purification of GST-fusion proteins
GST–GAPDH and the different GST–GAPDH fragments were
expressed in Escherichia coli BL21 strain, under 0.5 mM IPTG
for 4 h at 37
C. These fusion proteins were purified with
glutathione–sepharose beads as previously described (Frangioni
and Neel, 1993).
2.5. Pull-down and immunoprecipitation
Pull-down experiments were performed as previously
described (Carujo et al., 2006). The bound and unbound frac-
tions were analysed by SDS-PAGE and Coomassie Blue staining or
by western blot (WB) using anti-PCAF. For immunoprecipitation
(IP) experiments, cells were lysed in buffer A (50 mM Tris–HCl, pH
7.4, 5 mM EDTA, 250 mM NaCl and 0.1% Triton X-100) containing
50 mM NaF, 0.1 mM Na
3
VO
4,
1 mM PMSF, 10 g/ml leupeptin,
1 g/ml aprotinin and 0.2 g/ml TSA, on ice for 30 min. Samples
were then clarified by centrifugation at 10,000 × g for 10 min at
4
C. Supernatants (0.5–1 mg of protein) were incubated overnight
with specific antibodies (1–4 g), followed by incubation with
Protein A- (Pierce), Protein G- (Sigma) or Protein A/G- (Pierce)
agarose beads for 1 h at 4
C. After washing three times with buffer
B, the immunoprecipitates were then analysed by SDS-PAGE and
WB. The blots were visualized using the ECL system (Amersham,
Pharmacia Biotech).
2.6. In vitro acetylation
Acetylase assays were performed as described (Martinez-Balbas
et al., 2000). For spot mapping experiments, 23 peptides were
bound to a cellulose--Ala membrane. Then, the membrane was
incubated in 3 ml of HAT modified buffer (50 mM Tris–HCl, pH 8,
500 mM NaCl, 0.1 mM EDTA, 5% glycerol, and 0.1% NP-40) in the
presence of GST–HAT and (
14
C)-acetyl CoA, for 30 min at 30
C.
Finally, acetylation was visualized by autoradiography that was
performed at 80
C for 24–48 h.
2.7. Immunocytochemistry
Cells were seeded in culture dishes containing glass cover-slips
and allowed to grow for 24 h. Cells were fixed in 4% paraformalde-
hyde for 30 min. Cells were permeabilized with methanol or with
0.1% Triton X-100. Then, they were incubated with primary anti-
bodies for 1 h at room temperature or 37
C. After washing in PBS,
cells were incubated with secondary antibodies conjugated with
FITC or TRITC, for 45 min at room temperature. After washing twice
in PBS, cover-slips were mounted on slides with Mowiol
®
. Confocal
images were acquired using a Leica TCS SL laser scanning confo-
cal spectral microscope (Leica Microsystems) equipped with Argon
and HeNe lasers and a Leica DMIRE2 inverted microscope. Alexa
594 emissions were acquired with a triple dichroic beam-splitter
(TD 488/543/633 nm) and emission detection ranges: 555–700 nm.
All images were obtained using 63× oil immersion objective lens
(NA 1.32) and the confocal pinhole set at 1 Airy unit. Due to small
size of cells, electronic zoom (2) was necessary for stronger magnifi-
cation and better image resolution. Image assembly and treatment
were performed using the Image J software. To analyze mean inten-
sities, ROIs of equal size were located in cytoplasm and nucleus. In
some cases, the ratio of mean intensities of nucleus versus cyto-
plasm was calculated. To count the number of cells showing major
staining in cytoplasm or nucleus, the cell counter plug-in was used
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1674 M. Ventura et al. / The International Journal of Biochemistry & Cell Biology 42 (2010) 1672–1680
Fig. 1. The acetyltransferase PCAF acetylates GAPDH. (A) Purified GST-PCAF, the catalytic domain of PCAF (GST-HAT) or GST were pulled-down with GAPDH-Sepharose 4B
or Sepharose 4B beads (used as a control). Bound (B) and unbound (NB) proteins were analysed by SDS-PAGE and coomassie blue staining. (B) Lysates from HCT116 cells
transfected with PCAF-Ct-WT were immunoprecipitated with anti-GAPDH or NMS as a control. The presence of GAPDH and PCAF in the immunoprecipitates was analysed
by WB. (C) GAPDH (3 M) was subjected to in vitro acetylation assays using GST–HAT in the presence of (
14
C)-Acetyl-CoA. Acetylation was visualized by autoradiography
(ARG) and GAPDH by WB. (D) HCT116 cell extracts were immunoprecipitated with anti-GAPDH or NMS, used as a control. The endogenous acetylated GAPDH was visualized
in the immunoprecipitates by WB using an anti-acetyl Lys antibody (AcLys).
(Kurt De Vos, University of Sheffield, Academic Neurology). In all
experiments quantified, at least 300 cells from two independent
experiments were analyzed.
When immunocytochemistry was developed with diaminoben-
zidine (DAB, Sigma) the protocol was: 7 min in 0.3% H
2
O
2
in
methanol, 2 washes of 15 min each in PBS, overnight in primary anti
anti-GAPDH (1:400), 2 washes of 15 min each in PBS, 1 h in biotiny-
lated goat anti-rabbit antibody (1:200, Vector), 2 washes of 15 min
each in PBS, 1 h in ExtrAvidin-HRP (1:500, Sigma), 5–10 min in DAB
(1 mg/ml), 0.15% H
2
O
2
in PBS. All steps were performed at room
temperature except primary antibody incubation (4
C). All anti-
bodies were diluted in PBS containing 7% normal goat serum. All
samples were photographed on a Nikon fluorescence microscope
Eclipse E1000 equipped with a digital camera Color View 12.
2.8. Quantification of nuclear average intensity
The average of intensity (gray level) of nuclear staining in each
sample after apoptotic stimulus was measured using Image J Soft-
ware. The levels of nuclear staining in each sample were measured
in the same manner using the average intensity of time 0 to nor-
malize the nuclear staining intensity in each experiment. For each
experimental situation the intensity of at least 600 nuclear profiles
was estimated. Results are expressed as relative units (mean ± SD
of the mean for each experimental group) of 3–4 replicate samples
per treatment. Each experiment was carried out at least 3 times.
Statistical analysis of each time point vs. time 0 h was performed
using a Student’s t-test.
3. Results
3.1. PCAF acetylates GAPDH
By pull-down experiments, using GAPDH bound to Sepharose
4B beads and purified GST–PCAF, we observed that PCAF associ-
ated with GAPDH in vitro (Fig. 1A). Similar experiments indicated
that the catalytic domain of PCAF (GST–HAT) also interacted with
GAPDH (Fig. 1A). GAPDH and PCAF also interact in the cells as
observed by IP of lysates from HCT116 cells transfected with an
active fragment of PCAF (PCAF-Ct-WT) using anti-GAPDH or nor-
mal mouse serum (NMS) that was used as a control (Fig. 1B).
Because GAPDH interacts with the catalytic domain of PCAF we
aimed to study whether PCAF could acetylate GAPDH. To check
this possibility, in vitro acetylation assays were performed using
purified GST–HAT as enzyme and GAPDH as a substrate. As shown
in Fig. 1C, GAPDH was directly acetylated by PCAF. Subsequent IP
experiments with anti-GAPDH followed by WB using anti-acetyl-
Lys antibodies revealed that the endogenous GAPDH was acetylated
in the cells (Fig. 1D).
3.2. Identification of the GAPDH acetylation sites
To identify the Lys residues of human GAPDH (Fig. 2A) suscep-
tible to be acetylated by PCAF, four fragments of this protein were
generated: F1 (aa 1–82), F2 (aa 1–162), F3 (aa 162–249) and F4
(aa 249–335) (Fig. 2B). These fragments were then subjected to
in vitro acetylation assays. Results revealed that fragments F2, F3
and F4 were acetylated (Fig. 2C). These data indicate that differ-
ent Lys distributed along a GAPDH region including aa 82–335 can
be acetylated by PCAF. To identify these Lys residues spot map-
ping analysis were performed. Thus, 22 peptides that covered the
whole extension of this GAPDH region were synthesized (Fig. 2D,
bottom panel), subsequently spotted on a membrane and finally
subjected to an in vitro acetylation assay. An acetylatable peptide
from histone H3 was added as a positive acetylation control (pep-
tide 23). Results revealed that peptides 5 and 14 containing Lys 117
and 251, respectively, were acetylated (Fig. 2D, upper panel). Lys
117 belongs to GAPDH fragment F2 and Lys 251 to fragment F4.
By this experimental approach we failed to identify the putative
acetylation site/s in fragment F3.
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M. Ventura et al. / The International Journal of Biochemistry & Cell Biology 42 (2010) 1672–1680 1675
Fig. 2. Identification of the Lys residues of GAPDH acetylated by PCAF. (A) Human sequence of GAPDH. (B) Schematic representation of GAPDH fragments used in the in vitro
acetylation experiments. (C) Four fragments of GAPDH and purified GST, used as a control, were subjected to in vitro acetylation assays using purified GST–HAT as acetylase
in the presence of (
14
C)-Acetyl-CoA. The acetylated fragments were visualized by autoradiography (ARG) (upper panel). The loading control gel was stained with coomassie
blue (CB) (bottom panel). (*) Indicates a degradation fragment of F3. (D) Twenty two peptides from the GAPDH region including aa 82–335 were synthesized (sequences are
shown in the bottom panel). Each peptide included one or two very closed Lys. A peptide from histone H3 was used as a positive acetylation control (peptide 23). All peptides
were spotted in a membrane and the membrane subjected to in vitro acetylation assays using GST–HAT in the presence of (
14
C)-Acetyl-CoA. The acetylated peptides were
visualized by autoradiography (ARG) (upper panel).
3.3. Over-expression of PCAF induced the nuclear translocation of
GAPDH
To examine whether acetylation of GAPDH could be involved in
its nuclear translocation, we first developed NIH3T3 cell lines that
expressed PCAF in an inducible manner on depending of the tetra-
cycline concentration in the culture medium (Tet-Off system). Thus,
we generated two cell lines, one expressing an active fragment of
PCAF (PCAF-Ct-WT) and the other one that expressed an inactive
fragment (PCAF-Ct-HAT). We derived a number of clones from
these two cell lines that showed different levels of PCAF. Neverthe-
less, all of them showed significant levels of this protein at 24 h after
elimination of tetracycline from the medium (Fig. 3A). Immunocy-
tochemical analysis performed in these clones revealed that when
PCAF was not expressed (tetracycline+), GAPDH was mainly located
in the cytoplasm (Fig. 3B and C). In contrast, when active PCAF was
expressed (tetracycline), GAPDH was preferentially located in the
nucleus (Fig. 3B and C). Interestingly, in the clones expressing the
inactive fragment of PCAF, GAPDH was unable to translocate to the
nucleus (Fig. 3B and C). All these results indicate that PCAF induces
the translocation of endogenous GAPDH from cytoplasm into the
nucleus and that for this translocation the acetylase activity of PCAF
is needed.
To further confirm this possibility we analyzed the effect of
knocking down PCAF on the nuclear translocation of GAPDH, in
NIH3T3 cells subjected to an apoptotic stimulus. First of all, we ana-
lyzed the interaction between endogenous PCAF and endogenous
GAPDH in these cells. As shown in Fig. 4A, IP experiments using anti-
GAPDH revealed that PCAF co-immunoprecipitates with GAPDH
in these cells. Then, we subjected NIH3T3 cells to an apoptotic
stimulus (100 nM TSA) and determined the nuclear translocation
of GAPDH at different times after TSA treatment. Results indi-
cate that endogenous GAPDH translocates from cytoplasm into
the nucleus at 16 h after TSA treatment remaining in the nucleus
at least until 30 h (Fig. 4B). Similar experiments were performed
in cells transfected with siRNA for PCAF versus transfected with
an unrelated siRNA. As shown in Fig. 4C, PCAF siRNA transfection
induced a decrease of 70% of the amount of endogenous PCAF. Then,
PCAF knocked down cells were subjected to an apoptotic stimulus
(100 nM TSA for 24 h) and the GAPDH nuclear intensity was deter-
mined. As shown in Fig. 4D the nuclear intensity of GAPDH was
significantly reduced in cells knocked down for PCAF, further con-
firming that this acetylase participates in the regulation of nuclear
translocation of GAPDH.
3.4. Acetylation of Lys 117 and 251 of GAPDH is necessary for its
nuclear translocation
With the aim to study whether the nuclear translocation of
GAPDH stimulated by PCAF was depending on the acetylation of Lys
117 and 251, we first generated two HA-tagged GAPDH mutants:
the HA-GAPDH-2R mutant in which both Lys were substituted by
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1676 M. Ventura et al. / The International Journal of Biochemistry & Cell Biology 42 (2010) 1672–1680
Fig. 3. Over-expression of active PCAF induces GAPDH nuclear translocation. (A) The levels of PCAF in different clones expressing active (Ct-WT) (clones 1, 2, 6 and 7) or
inactive PCAF (Ct-HAT) (clon 4) were determined by WB with anti-PCAF at different times after eliminating tetracycline from the media. (B) These different clones were
subjected to immunocytochemical studies using anti-GAPDH, in the presence or in the absence of tetracycline in the medium (24 h). (C) Immunocytochemistry was quantified
and the percentage of cells showing nuclear staining in the presence (white bars) or absence (black bars) of tetracycline was represented in the graph. Results are the mean
value ± SD of three independent experiments. Bar: 10 m.
Arg and the pseudoacetylated form, in which these two Lys were
substituted by Gln (HA-GAPDH-2Q). Then, we analyzed the intra-
cellular distribution of these mutants in the presence or in the
absence of an apoptotic stimulus (100 nM TSA). All these ectopic
forms were mostly cytoplasmatic in the absence of TSA treatment.
Specifically, the percentages of cells showing nuclear staining were
5% in HA-GAPDH-WT transfected cells, 9% for HA-GAPDH-2R and
10% for HA-GAPDH-2Q (Fig. 5A). After TSA treatment the percent-
age of cells showing nuclear staining was strongly increased in
HA-GAPDH-WT (77%) and HA-GAPDH-2Q (85%) transfected cells
whereas this increase was much lower in the case of the mutant HA-
GAPDH-2R (55% of cells with nuclear staining). These data indicate
that acetylation of Lys 117 and 251 of human GAPDH plays a role
in its translocation into the nucleus. Moreover, these results also
revealed that the acetylation of both Lys residues is not sufficient
to induce its translocation. Likely, other modifications induced by
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M. Ventura et al. / The International Journal of Biochemistry & Cell Biology 42 (2010) 1672–1680 1677
Fig. 4. Knocking down PCAF in NIH3T3 cells blocks GAPDH nuclear translocation (A) NIH3T3 cell extracts were subjected to IP with anti-GAPDH. The presence of PCAF in
the immunoprecipitates was analyzed by WB. (B) Intracellular localization of endogenous GAPDH at different times after treatment of NIH3T3 cells with 100 nM of TSA.
Cells were immunostained with anti-GAPDH. The graph represents the nuclear average intensity of staining at different times of treatment using arbitrary units. Results
are expressed as the mean value ± SD of three independent experiments. Statistical analysis of each time point vs time 0 h was performed using the Student’s t-test. (***)
Indicates a p < 0.001. (C) The levels of PCAF were determined by WB in NIH3T3 cells treated with a siRNA for PCAF or an unrelated siRNA used as a control. (D) Intracellular
localization of endogenous GAPDH at 24 h after treatment of NIH3T3 cells with 100 nM of TSA. Cells were transfected with siRNA for PCAF or with an unrelated siRNA, used
as a control. Results are expressed as the mean value ±SD of three independent experiments. Statistical analysis of the different values vs that obtained in the experiment
with control siRNA in the presence of TSA was performed using the Student’s t-test. (***) Indicates a p < 0.001.
PCAF and by the apoptotic stimulation (probably acetylation of
other specific sites) are necessary for the nuclear translocation of
GAPDH.
3.5. Acetylation of Lys 117, 251 and 227 signals the nuclear
translocation of GAPDH
As mentioned above, fragment F3 of GAPDH (aa 162–249) was
acetylated in vitro by PCAF (Fig. 2C). However, by spot mapping
analysis we could not identify which Lys residue/s in this fragment
was/were the acetylation site/s. This GAPDH fragment contains
six Lys residues at positions K162, K186, K194, K215, K219 and
K227. It has been reported that K227 is important for the inter-
action of GAPDH with the Siah1 protein (Jenkins and Tanner,
2006) and that the interaction GAPDH–Siah1 is important for the
translocation of GAPDH to the nucleus (Hara et al., 2005). Thus,
we analyzed the possibility that K227 could be a residue from
fragment F3 of GAPDH susceptible to be acetylated and that the
acetylation of this Lys could participate in the nuclear translo-
cation of GAPDH. To analyze this possibility, we generated triple
GAPDH mutants in which Lys 117, 227 and 251 were substi-
tuted by Arg (HA-GAPDH-3R) or by Gln (HA-GAPDH-3Q). Then,
the intracellular distribution of these mutants was analyzed by
immunocytochemistry. As shown in Fig. 5B in non-treated cells
HA-GAPDH-WT and HA-GAPDH-3R were mainly cytoplasmatic
whereas in contrast the pseudoacetylated mutant HA-GAPDH-3Q
was preferentially located in the nucleus. These results strongly
indicate that these three Lys residues when acetylated induce the
nuclear translocation of GAPDH. After TSA treatment, the pseu-
doacetylated mutant HA-GAPDH-3Q remains in the nucleus and
both HA-GAPDH-WT and HA-GAPDH-3R translocated from cyto-
plasm into the nucleus although the mutant HA-GAPDH-3R in a
less extension. Quantification data of these experiments are shown
in Fig. 5C. All these findings indicate that acetylation of Lys 117,
227 and 251 signals GAPDH for translocation from cytoplasm to
the nucleus. Moreover, results from the mutant HA-GAPDH-3R
also indicate that under apoptotic stimuli other mechanisms for
GAPDH translocation to the nucleus are activated in parallel to
acetylation.
4. Discussion
In addition to its glycolytic activity, GAPDH performs a number
of nuclear functions and for that reason it has to translocate from
the cytoplasm, where it is mostly located, into the nucleus. This
nuclear transport is mediated by different signals including the
O-linked-N-acetylglucosamine modification (O-GlcNAcylation) of
Thr 229 (Park et al., 2009) and the S-nitrosylation at Cys 152
(Hara et al., 2005; Hara and Snyder, 2006) in human GAPDH. The
O-GlcNAcylation has been reported to be related to the nuclear
translocation of other proteins (Majumdar et al., 2006; Andrali et al.,
2007). Specifically, this modification disrupts the GAPDH tetramer,
facilitating nuclear translocation. The relevance of this mechanism
is evidenced by the fact that mutation of Thr 229 to Ala, resulted
in an accumulation of GAPDH in the cytoplasm (Park et al., 2009).
The S-nitrosylation of GAPDH at Cys 152 activates the binding of
GAPDH to Siah1 that depends on the integrity of Lys 227, being the
GAPDH mutant K225A not able to interact with Siah1 (Fiucci et al.,
2004).
We report here that GAPDH directly interacts with the acetyl-
transferase PCAF and that as a consequence it becomes acetylated.
By in vitro acetylation assays and spot mapping analysis we iden-
tified Lys 117 and 251 as the putative PCAF dependent acetylation
sites. We report here that over-expression of an active fragment
of PCAF induces the nuclear translocation of GAPDH, being this
translocation dependent on the catalytic activity of the acety-
lase. These results indicated that acetylation is needed for GAPDH
translocation and that the acetyltransferase PCAF plays an impor-
tant role.
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1678 M. Ventura et al. / The International Journal of Biochemistry & Cell Biology 42 (2010) 1672–1680
Fig. 5. Nuclear translocation of GAPDH depends on its acetylation status. (A) Immunocytochemical studies of the intracellular localization of ectopic HA-GAPDH-WT, HA-
GAPDH-2R and HA-GAPDH-2Q in the absence of treatment (TSA) or at 24 h after addition of 100 nM TSA (TSA+). Cells were immunostained with anti-GAPDH antibody
and examined by confocal microscopy. Nuclei were stained with DAPI. (B) Intracellular localization of HA-GAPDH WT, HA-GAPDH-3R and HA-GAPDH-3Q in cells untreated
(TSA) or treated (TSA+) with 100 nM TSA. Immunocytochemistry was performed with anti-GAPDH antibody and examined by confocal microscopy. (C) The percentage of
cells showing nuclear or cytoplasmatic GAPDH staining is represented. Results are expressed as the mean value ±SD of three independent experiments. Bars: 10 m.
Both, the mutants GAPDH-2R (aa 117 and 251 non-acetylatable)
and GAPDH-2Q (aa 117 and 251 pseudoacetylated lysines) were
mainly located in the cytoplasm. However, under apoptotic stim-
uli both mutants translocate into the nucleus although GAPDH-2R
did it in a much less extension. These results indicate that acety-
lation of these two Lys in the GAPDH sequence is a mechanism
that participates in GAPDH nuclear translocation. These results
also indicated that acetylation of these two Lys residues was not
sufficient for GAPDH nuclear translocation. Thus we subsequently
examined the putative role of K227 acetylation in the GAPDH
entry into the nucleus. Interestingly, the mutant HA-GAPDH-3Q
was mostly nuclear even in the absence of apoptotic stimulus. In
contrast, HA-GAPDH-3R remained in the cytoplasm under these
conditions. These data revealed that acetylation of K117, K227 and
K251 is sufficient to move GAPDH from the cytoplasm into the
nucleus and also suggest that these acetylations might be necessary
in order to interact with Siah1. Altogether these results indicate
that the acetylation of specific Lys residues is a key mechanism for
nuclear transport of GAPDH.
Our results strongly suggest that nuclear transport of GAPDH
might be mediated by PCAF. We clearly have shown that K117
and K251 are acetylated by PCAF although we still do not know
whether K227 is also acetylated by this acetyltransferase. Never-
theless, the results showing that PCAF over-expression leads to the
nuclear translocation of GAPDH together with the evidence that
decreasing the cellular levels of PCAF blocks the nuclear transloca-
tion of GAPDH strongly suggest that K227 could also be acetylated
by PCAF. PCAF is a transcriptional co-activator that normally resides
in the nucleus. However, a recent report indicates that under apop-
totic conditions, PCAF moves from the nucleus to the cytoplasm
and this depends on its autoacetylation status (Blanco-Garcia et al.,
2009). Thus, likely, apoptotic stimuli induce PCAF re-localization
from nucleus to the cytoplasm, and there it can acetylate GAPDH
that then translocates into the nucleus. Altogether, these results
Page 7
M. Ventura et al. / The International Journal of Biochemistry & Cell Biology 42 (2010) 1672–1680 1679
suggest that the S-nitrosylation at Cys 152, the O-GlcNAcylation
at Thr 229 and acetylation at Lys 117, 227 and 251 can cooperate
in signaling GAPDH interaction with Siah 1 and subsequently in
nuclear translocation. Whether these different modifications have
to be produced simultaneously or are alternative pathways for
GAPDH transport still remains to be established. However, the evi-
dence reported here indicating that the pseudoacetylated mutant
HA-GAPDH-3Q mostly resides in the nucleus clearly indicates that
acetylation of Lys 117, 227 and 251 is sufficient for nuclear translo-
cation of GAPDH.
In the nucleus, GAPDH participates in the apoptotic process by
stabilizing the E3-ubiquitin ligase Siah1, involved in protein degra-
dation. It is worth to mention that PCAF also possesses an intrinsic
ubiquitin ligase activity that could participate in the apoptotic pro-
cess (Linares et al., 2007). Moreover, it has been shown that nuclear
GAPDH is acetylated at Lys 160 by the acetyltransferase p300/CREB
binding protein (CBP) through direct protein interaction, which in
turn stimulates the autoacetylation and the catalytic activity of
p300/CBP (Sen et al., 2008). Consequently, downstream targets of
p300/CBP, such as p53 (Lill et al., 1997) are activated and causes cell
death. Thus, acetylation plays an important role in the apoptotic
role of GAPDH and at least two different acetylases participate in
this process: PCAF in the cytoplasm helping GAPDH nuclear translo-
cation and p300/CBP in the nucleus inducing cell death.
As GAPDH participates in cell death, it may have interest as a
therapeutic target, especially in neurodegenerative diseases. It has
been shown that CGP 3466 and R-()-deprenyl, two anti-apoptotic
agents with neuro-protective activity interact with GAPDH and
prevent its nuclear transportation (Kragten et al., 1998). The
identification of acetylation as a mechanism involved in GAPDH
translocation raises the possibility that modulating GAPDH acety-
lation might be also of interest for therapy. For instance, blocking
PCAF activity and as a consequence GAPDH acetylation might
be used for neuroprotection whereas on the contrary promoting
GAPDH acetylation by using HDAC inhibitors might favour GAPDH
translocation and apoptosis that can be useful as oncological treat-
ments.
Acknowledgments
The confocal microscopy studies were performed at the Con-
focal microscopy facility, Serveis cientificotècnics (University of
Barcelona-IDIBAPS). We are grateful to S. Brun, A. Dominguez and
E. Esteve from the Department of Cell Biology, Immunology and
Neurosciences for helpful collaboration in different aspects of this
work. We are also grateful to Dr. M. Martinez-Balbas (Barcelona
Molecular Biology Institut) for her collaboration in the work. This
research was supported by grants SAF2006-05212 and SAF2009-
07769 from the Ministerio de Educación y Ciencia of Spain and
RETICS RD06/0020/0010 from the Instituto de Salud Carlos III.
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  • Source
    • "This result seemed to rule out the involvement of a redox modification of the catalytic cysteine for the nuclear translocation of GAPDH in Arabidopsis. Recently, a mechanism based on the acetylation of three lysine residues was alternatively proposed for the nuclear translocation of GAPDH after the apoptotic stimulus in animal cells (Ventura et al., 2010). Whether a mechanism of this type may apply to Cd-induced nuclear translocation of GAPC in plants is still an open question. "
    [Show abstract] [Hide abstract] ABSTRACT: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous enzyme involved in glycolysis and shown, particularly in animal cells, to play additional roles in several unrelated non-metabolic processes such as control of gene expression and apoptosis. This functional versatility is regulated, in part at least, by redox post-translational modifications that alter GAPDH catalytic activity and influence the subcellular localization of the enzyme. In spite of the well established moonlighting (multifunctional) properties of animal GAPDH, little is known about non-metabolic roles of GAPDH in plants. Plant cells contain several GAPDH isoforms with different catalytic and regulatory properties, located both in the cytoplasm and in plastids, and participating in glycolysis and the Calvin-Benson cycle. A general feature of all GAPDH proteins is the presence of an acidic catalytic cysteine in the active site that is overly sensitive to oxidative modifications, including glutathionylation and S-nitrosylation. In Arabidopsis, oxidatively modified cytoplasmic GAPDH has been successfully used as a tool to investigate the role of reduced glutathione, thioredoxins and glutaredoxins in the control of different types of redox post-translational modifications. Oxidative modifications inhibit GAPDH activity, but might enable additional functions in plant cells. Mounting evidence support the concept that plant cytoplasmic GAPDH may fulfill alternative, non-metabolic functions that are triggered by redox post-translational modifications of the protein under stress conditions. The aim of this review is to detail the molecular mechanisms underlying the redox regulation of plant cytoplasmic GAPDH in the light of its crystal structure, and to provide a brief inventory of the well known redox-dependent multi-facetted properties of animal GAPDH, together with the emerging roles of oxidatively modified GAPDH in stress signaling pathways in plants.
    Full-text · Article · Nov 2013 · Frontiers in Plant Science
  • Source
    • "In order to trigger cell cycle related events, it is possible that both GAPDH and FoxM1 translocate from the cytoplasm to the nucleus in cancer cells. Nuclear translocation of GAPDH may be regulated by acetylation [32]. FoxM1 is localized predominantly in the cytoplasm in late G1 and S phases. "
    [Show abstract] [Hide abstract] ABSTRACT: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is often used as a stable housekeeping marker for constant gene expression. However, the transcriptional levels of GAPDH may be highly up-regulated in some cancers, including non-small cell lung cancers (NSCLC). Using a publically available microarray database, we identified a group of genes whose expression levels in some cancers are highly correlated with GAPDH up-regulation. The majority of the identified genes are cell cycle-dependent (GAPDH Associated Cell Cycle, or GACC). The up-regulation pattern of GAPDH positively associated genes in NSCLC is similar to that observed in cultured fibroblasts grown under conditions that induce anti-senescence. Data analysis demonstrated that up-regulated GAPDH levels are correlated with aberrant gene expression related to both glycolysis and gluconeogenesis pathways. Down-regulation of fructose-1,6-bisphosphatase (FBP1) in gluconeogenesis in conjunction with up-regulation of most glycolytic genes is closely related to high expression of GAPDH in the tumors. The data presented demonstrate that up-regulation of GAPDH positively associated genes is proportional to the malignant stage of various tumors and is associated with an unfavourable prognosis. Thus, this work suggests that GACC genes represent a potential new signature for cancer stage identification and disease prognosis.
    Full-text · Article · Apr 2013 · PLoS ONE
  • Source
    • "Besides directly regulating the accessibility of substrate, acetylation can indirectly regulate substrate acces­ sibility by affecting subcellular localization of the metabolic enzyme. One such an example is acetylation­mediated nuclear translocation of glyceraldehyde­3­phosphate dehydrogenase (GAPDH; Ventura et al., 2010). GAPDH, long considered to be a housekeeping gene that is widely used as a protein loading control because of its relatively constant levels, catalyzes the NAD + ­dependent conversion of glyceraldehyde­3­phosphate (G3P) to 1,3 bisphosphoglycerate (1,3BPG). "
    [Show abstract] [Hide abstract] ABSTRACT: The activity of metabolic enzymes is controlled by three principle levels: the amount of enzyme, the catalytic activity, and the accessibility of substrates. Reversible lysine acetylation is emerging as a major regulatory mechanism in metabolism that is involved in all three levels of controlling metabolic enzymes and is altered frequently in human diseases. Acetylation rivals other common posttranslational modifications in cell regulation not only in the number of substrates it modifies, but also the variety of regulatory mechanisms it facilitates.
    Preview · Article · Jul 2012 · The Journal of Cell Biology
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