From the Laboratoire de Biologie Moléculaire et
Cellulaire de la Différenciation, INSERM U309, Institut Albert
Bonniot, Faculté de Médecine, Domaine de la Merci, 38706 La
Tronche Cedex, France,
Laboratoire de
Chimie des Proteines, ERIT-M0201, 38054 CEA-Grenoble, France, and
¶ Laboratoire de Biologie Moléculaire Eucaryote, UPR 9006 CNRS, 118, route de Narbonne, 31062 Toulouse, France
Received for publication, November 20, 2002, and in revised form, December 2, 2002
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ABSTRACT |
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Here we show that the phosphorylation of histone
acetyltransferase Tip60, a target of human immunodeficiency virus, type
1-encoded transactivator Tat, plays a crucial role in the
control of its catalytic activity. Baculovirus-based expression and
purification of Tip60 combined with mass spectrometry allowed
the identification of serines 86 and 90 as two major sites of
phosphorylation in vivo. The phosphorylation of Tip60 was
found to modulate its histone acetyltransferase activity. One of the
identified phosphorylated serines, Ser-90, was within a
consensus cyclin B/Cdc2 site. Ser-90 was specifically phosphorylated
in vitro by the cyclin B/Cdc2 complex. Accordingly, the
phosphorylation of Tip60 was enhanced after drug-induced arrest of
cells in G2/M. This G2/M-dependent phosphorylation of Tip60 was abolished by treating cells with a
specific inhibitor of the cyclin-dependent kinase,
roscovitin. All together, these results strongly suggest a
G2/M-dependent control of Tip60 activity.
Tip60 was first identified as a partner of human immunodeficiency
virus, type 1-encoded transactivator protein Tat (1). Later,
investigations clearly showed it to be a specific member of the
MYST family of nuclear histone acetyltransferases
(HATs)1 (2). Although the
targeting of Tip60 by Tat was found to interfere with its HAT activity
and to disturb the expression of at least one cellular gene (3), the
function of Tip60 remained elusive until recently. Indeed, the
identification of proteins associated with ectopically expressed Tip60
showed the presence of associated ATPase and DNA helicase activities.
Functional tests showed that Tip60 and associated proteins may play an
essential role in DNA repair and apoptosis (4). Moreover, considering
other recent reports, it appears that Tip60 is involved in a wide
variety of cellular functions. For instance, Tip60 was found to
interact with the androgen receptor and to enhance its transactivation in a ligand-dependent manner (5). Moreover, androgen
receptor was found to be a substrate for Tip60 (6). Other steroid
receptors such as estrogen and progesterone receptors have also shown
an enhanced activity in the presence of Tip60 and their ligands (5). Tip60 has also been involved in the NF The important function of Tip60 in cell signaling is probably the
reason for its targeting by the HIV-1-encoded transactivator Tat.
Indeed, it appears that Tat uses Tip60 to control cellular events for
the benefit of the virus (13). Here we evidenced a new property of
Tip60. First, Tip60 has been shown to be phosphorylated in
vivo, and second, two major sites of Tip60 phosphorylation have
been discovered. The sequence of one of these sites encompassing serine
90 perfectly matches a Cdc2 phosphorylation site. We showed that cyclin
B/Cdc2 complex can specifically phosphorylate Ser-90 in
vitro and in vivo and that the phosphorylation of Tip60
was enhanced in G2/M phase of the cell cycle. This specific
G2/M phosphorylation of Tip60 was inhibited when cells were
treated with a specific inhibitor of cyclin-dependent
kinase, roscovitin. Our data clearly show that the phosphorylation of
Tip60 controls its HAT activity and strongly suggest a role for Tip60
HAT activity in the control of G2/M-related events.
Production and Purification of Proteins in Baculovirus--
Wild
type or mutant Tip60 cDNA were cloned into pBacPAK9 transfer vector
(Clontech) in-frame with a histidine tag at the C terminus or at the N terminus of the coding sequence. Viral particles were generated using the BacPAK baculovirus expression system (Clontech) and Sf21 insect cells. 2-3 days
after infection, Tip60 proteins were purified from Sf21 cells by
nickel affinity column (NiTA-agarose, Qiagen), eluted with 250 mM imidazole, and finally dialyzed against 20 mM Tris, pH 7.5, 10% glycerol, and 1 mM
dithiothreitol. The purified proteins were kept at Plasmids--
The plasmid pcDNA-HA-Tip60 has been described
previously (14). The Tip60-(1-211) mutant was generated by PCR
and cloned in-frame with the HA tag in the pcDNA-HA vector. The
point mutations were generated by PCR, and the incorporation of all of
the mutations was confirmed by DNA sequencing. In the Gly-380 mutant,
glycine 380 was replaced by an alanine. In the Ser-86, Ser-90, and
Ser-86/Ser-90 mutants, the serines were replaced by alanines. In the
Leu-254/Leu-257 mutant, the two indicated leucines were replaced by alanines.
Phosphatase Treatment--
Five hundred nanograms of His-Tip60
proteins produced in baculovirus were incubated with 10 units of calf
intestine phosphatase (CIP) (New England BioLabs) in the presence or
absence of phosphatase inhibitor (5 mM NaF) for 30 min at
37 °C (or 1 h on ice when the dephosphorylation was followed by
a HAT assay). Tip60 was then removed by incubation of the reaction
mixture with NiTA-agarose beads and eluted with Laemmli sample buffer
and subsequently analyzed by SDS-PAGE and Western blotting. Phosphatase
treatment of endogenous Tip60 was performed as follows. HeLa cell
nuclei isolated from 107 cells were incubated with 50,000 units of CIP for 30 min at 37 °C. Nuclei were washed three times in
the lysis buffer (15 mM NaCl, 60 mM KCl, 12%
sucrose, 2 mM EDTA, 0.5 mM EGTA, 0.65 mM spermidine, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 0.5% Triton X-100) and
directly lysed in protein loading buffer. Tip60 was detected using an
anti-Tip60 antibody described by Legube et. al. (14).
Histone Acetyltransferase Assays--
HAT assays were performed
using 4 µg of free core histones or 5 µg of oligonucleosomes, wild
type, or mutant Tip60 proteins (100-200 ng) and 0.15 µCi of
[14C]acetyl-CoA (65 mCi/mmol) (ICN) in HAT buffer (25 mM Tris, pH 8.0, 10% glycerol, 100 mM NaCl, 1 mM dithiothreitol, 0.2 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 5 mM sodium
butyrate) for 30 min at 30 °C. 50% of the reactions was loaded on a
18% SDS-polyacrylamide gel followed by Coomassie Blue staining to
ensure equivalent loading of histones in each lane, and fluorography
was performed after incubation of the gel in the Amplify solution
(Amersham Biosciences). In some assays, the HAT activity was analyzed
with Tip60 immobilized on NiTA-agarose beads after alkaline phosphatase treatment.
MALDI-MS Analysis--
Mass spectra of the tryptic digests were
acquired on a Biflex (Bruker-Franzen Analytik, Bremen, Germany)
MALDI-TOF mass spectrometer equipped with a gridless delayed
extraction. The instrument was operated in linear mode. 0.5 µl of the
digest solution (in 25 mM NH4HCO3)
was deposited directly onto the sample probe on a dry thin layer of
matrix made of Peptide Dephosphorylation--
Phosphorylated tryptic peptides
were identified on the peptide mass fingerprint by a 80-Da (or
multiples of 80 Da) mass shifts after the dephosphorylation step. The
dephosphorylation reaction was done by treating the tryptic peptide
mixture with bovine alkaline phosphatase in which the peptide digest (5 µl) was mixed with 5 µl of calf intestinal alkaline phosphatase
immobilized on agarose beads (P0762, Sigma) in 25 mM
NH4HCO3 buffer. After 30 min (37 °C), the
peptide digest was directly deposited on the MALDI sample probe for analysis.
Phosphopeptide Microcropurification--
Phosphopeptides were
purified from the crude tryptic digest using immobilized metal ion
affinity chromatography essentially as previously described (15). The
tryptic digest (2 µl in 10 mM
NH4HCO3, 5% acetic acid, 30% acetonitrile)
was loaded on a ZipTipMC (ZT0MCS, Millipore,) that had been
previously equilibrated following the recommendations of the
manufacturer. The ZipTipMC column was then washed with
0.1% acetic acid followed by 0.1% acetic acid, 30% acetonitrile, and
0.1% acetic acid and finally eluted in 2% ammonium hydroxide (2 µl). Eluted peptides were then mixed with 50 mM
NH4HCO3 (v/v) and analyzed by MALDI-MS.
Nanoelectrospray-MS/MS Analysis--
The digest
solution was dried in a vacuum centrifuge and desalted with ZipTip C18
(Millipore, Bedford, MA) before the nanospray MS/MS analysis. A Q-TOF
instrument (Micromass, Manchester, United Kingdom) was used with a
Z-spray ion-source working in the nanospray mode. Approximately 3-5
µl of the desalted sample was introduced into a needle (medium sample
needle, PROTANA Inc. Odense, Denmark) to run MS and MS/MS experiments.
The capillary voltage was set to an average voltage of 1000 V, and the
sample cone was set to 50 V. Glufibrinopeptide was used to calibrate
the instrument in the MS/MS mode. MS/MS spectra were transformed using
MaxEnt3 (MassLynx), and amino acid sequences were analyzed using PepSeq (BioLynx).
Kinase Assays--
Wild type or mutant Tip60 peptides (mutant
Ser-90 and mutant Ser-86/Ser-90) encompassing serines at positions 86 and 90 were synthesized, purified (Sigma), and reconstituted in
distilled water at 5 mM. For the phosphorylation assays, 15 nmol of wild type or mutant peptides at a final concentration of 500 µM were incubated in Cdc2 kinase buffer supplemented with
100 µM cold ATP, 100 µM sodium vanadate, 10 µCi of [ Cell Culture, Transfection, Drug Treatments, and Western
Blot--
HeLa cells were grown as described previously (14) and
transfected using Exgen (Euromedex) or FuGENE 6 (Roche Molecular Biochemicals) as indicated by the suppliers. 24-48 h later, cells extracts were prepared by lysing cells directly in Laemmli sample buffer and sonicated. Stably Tip60 expressing HeLa cells (a gift of Dr.
V. Ogrysko) were grown in Dulbecco's modified Eagle's medium supplemented with fetal calf serum (10%) and antibiotics.
Phosphorylation of Tip60 in Vivo--
To investigate the
biochemical property of Tip60, we have expressed a histidine-tagged
human Tip60 in insect cells using a baculovirus-based expression
system. The recombinant protein was found to be an active HAT on
purified histones as well as on nucleosomes (Fig.
1A). Interestingly, whereas
Tip60 efficiently acetylated free histone H4 and H3, in nucleosomes, it
essentially acetylated histone H4. However, the acetylation of
nucleomosal H4 was much less efficient than that of free histones.
Indeed, the gel containing nucleosomal histones had to be exposed for a
longer period of time to obtain a comparable signal for the acetylation
of free and nucleosomal H4. We have also noticed that the purified
recombinant Tip60 migrated as a doublet on a 8% SDS-PAGE, and the
treatment with the protein calf intestinal alkaline phosphatase
prior to electrophoresis caused a shift of Tip60 to a faster migrating band (data not shown). This observation showed that protein
phosphorylation was responsible for the observed shift in the
electrophoretic mobility of Tip60. To determine the region of Tip60
concerned by phosphorylation, two fragments of Tip60 were expressed in
insect cells, purified, and analyzed on a 15% SDS-PAGE, one containing the N-terminal chromodomain of Tip60 encompassing the first 211 amino
acids and the other corresponding to the rest of the protein (Fig.
1B, schemes). Fig. 1B shows that the
N-terminal fragment migrated as a doublet and that the CIP treatment
caused a shift of the majority of the protein into a single fast
migrating band (compare lane 1 with
2). The 212-513 region of Tip60 migrated as a single band,
and the CIP treatment did not affect its electrophoretic mobility (Fig
1B, lanes 3 and 4).
Two Major Sites of Tip60 Phosphorylation in Vivo--
Data
presented above showed that the major sites of Tip60 phosphorylation
are located in the first 211 amino acids of the protein. To determine
precisely the sites of Tip60 phosphorylation, we chose a mass
spectrometry-based approach. First, the mass spectra of the tryptic
fragments of the 1-211 region of Tip60 were compared with that of the
tryptic fragments of the CIP-treated protein. The MALDI-TOF analysis
showed that peptide-(81-93) bears two phosphate groups. The Q-TOF
sequencing of peptide-(81-93) from phosphorylated and CIP-treated
proteins confirmed the above conclusion and showed that serines 86 and
90 are phosphorylated. Finally, tryptic fragments of Tip60-(1-211)
were loaded on a ZipTip-MC column capable of retaining
phospho-peptides, and the MALDI-TOF analysis of these peptides
confirmed again the presence of two phosphate groups on
peptide-(81-93). This analysis also showed the possible presence of
phosphate groups on 190-211 and 204-211 tryptic fragments (data not
shown). To evaluate the respective participation of serines 86 and 90 in Tip60 phosphorylation, we mutated either serine 86 or serine 90 or
both to alanine and monitored their migration on 8%
SDS-PAGE after an ectopic expression in HeLa cells. As controls, we
also expressed either a Tip60 mutant in which glycine 380 was replaced
by an alanine or Tip60 Leu-254/Leu-257 mutant in which the two
indicated leucines potentially involved in a nuclear export signal were
also changed to alanines. Fig.
2B shows that only mutations
affecting serines 86 and 90 affect the mobility of Tip60, which
strongly suggests that these sites are majors sites of Tip60 phosphorylation in mammalian cells. This hypothesis was further confirmed by showing that CIP-treated Tip60-(1-211) fragment expressed in HeLa cells migrated at the same position as a Tip60-(1-211) fragment containing the Ser-86/Ser-90 mutation (Fig. 2C).
All of these results showed that Tip60 can be phosphorylated both in
insect and mammalian cells, and serines 86 and 90 represent two major
sites of phosphorylation in vivo. Finally, we showed that
endogenous Tip60 detected in HeLa cell nuclear extracts migrated as a
doublet and that the CIP treatment of the nuclei before the extract
preparation led to the migration of Tip60 as a single band (Fig.
2D).
Phosphorylation of Tip60 Modulates Its HAT Activity--
To
evaluate the role of phosphorylation in the activity of Tip60, the HAT
activity of purified phosphorylated Tip60 was compared with that of the
CIP-treated protein. Fig. 3A
shows that the pretreatment of Tip60 with increasing amounts of CIP
considerably reduced the HAT activity of the protein (compare
lanes 2 and 3 with 1). In the same
experiment when CIP was added in the presence of its inhibitor NaF
(Fig. 3A, lanes 4-6) or heat-inactivated prior
to its addition (Fig. 3B, lane 9), no detectable
modification of Tip60 HAT activity was observed compared with the
control. These experiments show that the inhibition of Tip60 HAT
activity is not because of the presence of CIP itself in the reaction
medium but because of its ability to dephosphorylate Tip60. Moreover, the activity of the CIP-treated Tip60 was comparable with that of a
mutated Tip60, Tip60Gly-380, containing a mutation
severely affecting its HAT activity (Fig. 3B, lane
11). In this mutant, a glycine critical for acetyl-CoA binding in
all MYST members of HATs was replaced by an alanine. This mutation had
been previously shown to inactivate the Drosophila Tip60 homologue
MOF (16) and was also one of the two mutations introduced by
Ikura et al. (4) to inactivate Tip60. The above experiment
clearly showed that dephosphorylated Tip60 has a poor catalytic
activity. To show the specific involvement of the serines 86 and 90 in
the control of the HAT activity of Tip60, baculovirus-based expression was used to express and purify a mutated form of Tip60 (Tip60Ser-86/Ser-90) in which these two serines were
replaced by alanines. A HAT assay was set up to compare the HAT
activity of the wild type, phosphorylated Tip60 with that of
Tip60Ser-86/Ser-90. Fig. 3C shows that, as in
mammalian cells, the double mutant Tip60 migrated faster than the wild
type protein (upper panel). Moreover, this experiment also
showed that Tip60Ser-86/Ser-90 was not as efficient as
wild-type Tip60 at acetylating histones.
Kinases Involved in the Phosphorylation of Tip60--
The data
presented thus far showed the critical role of Tip60 phosphorylation in
the control of its catalytic activity. Therefore, it appeared very
important to identify the kinases involved to better understand the
functional significance of this regulation of Tip60 activity by its
phosphorylation. The analysis of the sequence encompassing Ser-86 and
Ser-90 showed that Ser-90 corresponded to a potential p34 Cdc2
phosphorylation site XSPX(R/K) (17). To
test the ability of cyclin B/Cdc2 to phosphorylate Ser-90, we have
synthesized three peptides encompassing the amino acid 82-96 region of
Tip60. One peptide corresponded to the wild type sequence, and the two
others contained serine replacement (Ser to Ala) of either Ser-90 or
both Ser-86 and Ser-90 (Fig.
4A). The peptides were
incubated with purified cyclin B/Cdc2 complex in the presence of
32P-labeled ATP, and peptide phosphorylation was monitored.
Fig. 4B shows that only the wild type peptide was
phosphorylated by the purified cyclin B/Cdc2 complex. The
phosphorylation of a peptide containing the "Ser-90 to Ala"
mutation was comparable with that of a peptide containing replacements
of both serines, Ser-86 and Ser-90, to alanines or that of the wild
type peptide incubated with [32P]ATP in the absence of
the enzyme. This experiment shows that cyclin B/Cdc2 specifically
phosphorylates Ser-90 of Tip60. To show the participation of
cyclin-dependent kinases (CDKs) in the phosphorylation of
Tip60, HeLa cells were treated with nocodazole, inducing an arrest at
G2/M phase of the cell cycle because of its ability to
depolymerize microtubules. Nocodazole-treated cells were lysed, and
Tip60 was detected using an anti-Tip60 antibody (14) when ~90% of
cells were in the G2/M phase of the cell cycle (data not
shown). Fig. 5A shows that, as
expected, in cycling cells, Tip60 appeared as a doublet. Interestingly,
after the treatment of cells with nocodazole, almost only the
phosphorylated form of Tip60 was visible. However, in our hands for
unknown reasons, the endogenous Tip60 could not be detected with a
reasonable sensitivity in a reproducible manner. Therefore, to better
investigate the involvement of the CDKs in the phosphorylation of
Tip60, we used a characterized HeLa cell line stably expressing
HA-tagged Tip60 (4) to evaluate Tip60 phosphorylation in response to
specific drugs. In cycling HeLa cells, the HA-Tip60 appeared also as a doublet. After the treatment of cells with nocodazole, the HA-Tip60 behaved like the endogenous protein because the amount of the phosphorylated form of the protein increased (Fig. 5B,
lane 3). This nocodazole-induced accumulation of
phosphorylated HA-Tip60 was severely reduced when the
nocodazole-treated cells were treated with roscovitin, a specific
inhibitor of CDKs (18), for 7 h before the cell harvest (Fig.
5B, lane 4). All together, these data show the
participation of CDKs in the phosphorylation of Tip60 and strongly
suggest a role for Tip60 in the control of cell cycle-related
events.
The MYST family of histone acetyltransferases are evolutionary
conserved enzymes from yeast to humans. The specific function of
several members of this family is now emerging in different species
(19). For instance, in yeast, two HATs of the MYST family, SAS2 and
SAS3, play a role in silencing. The HAT activity of SAS3 has been shown
in vitro (20), and moreover, it has been found within the
NuA3 complex, which specifically acetylates histone H3 in nucleosomes
(21). The third member of MYST HATs in yeast, Esa1, appears to be
responsible for cell cycle progression (22). This HAT is also present
in a complex known as NuA4 (23) and is required for DNA double-stranded
break repair (24). In Drosophila, MOF, a MYST HAT, has been
clearly shown to be involved in the male X chromosome hyperactivation
by specifically acetylating histone H4 lysine 16 (16). Interestingly,
as Tip60, MOF has a canonic chromodomain, which possesses the
ability to bind to RNA in vitro and to the X
chromosome-associated RNA in vivo (25). The role of other
vertebrate MYST members, HBO1, MORF, and MOZ, has not yet been
defined (19). Therefore, it appears that the HATs of the MYST family
are involved in the control of a variety of critical cellular events.
This conclusion implies that the activity and the expression of these
enzymes should be tightly regulated. In the literature, there is almost
no hint regarding this issue. Our data provide the first indication of
the regulated expression and activity of a member of the MYST
acetyltransferases, Tip60. Indeed, we have previously shown that the
cellular concentration of Tip60 is controlled by Mdm2-mediated
ubiquitination and proteasome-dependent degradation of the
protein (14). Here we show that the phosphorylation of Tip60 is a
controlled phenomenon and modulates the activity of the protein. Tip60
is not the only HAT whose activity is controlled by phosphorylation.
Indeed, it has been shown that the transcription factor ATF-2 possesses
an intrinsic HAT activity and that its phosphorylation considerably
stimulates its HAT activity (26). Another example is CREB-binding
protein, which is phosphorylated at the G1/S boundary, and
this phosphorylation was also shown to stimulate its HAT activity (27).
This phosphorylation-dependent control of CREB-binding
protein activity allows this HAT to be responsive to distinct signaling
pathways. Indeed, kinases as different as p44 MAPK/ERK1 (28), cell
cycle-dependent kinases such as cyclin E/Cdk2 (27), as well
as MEKK1 (29) are capable of phosphorylating and, hence, activating the
CREB-binding protein catalytic activity. In the case of Tip60, Ser-90
is located within a canonic cyclin B/Cdc2 site and the enzyme was found
here to specifically phosphorylate this specific serine. Interestingly, Ser-90 is highly conserved among Tip60 homologues in different species
(data not shown), suggesting that this mode of regulation is also
conserved during evolution. Furthermore, our data provide a new basis
to reconsider the many functions of Tip60. Indeed, Tip60 appears as an
important intermediate in several unrelated receptor-mediated signaling
processes (5, 6, 8, 9, 11). Therefore, our data suggest that an
additional control level, that of Tip60 phosphorylation, needs to be
coordinated with signals induced by steroids (5, 6), IL-1 In summary, this work has allowed to establish a link between CDKs and
the activity of Tip60. However, other kinases may also phosphorylate
Tip60, establishing a specific linkage between Tip60 activity and one
or several of the signaling pathways discussed above.
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B response system, because it
was found to interact directly with BCL-3, a member of I
B family.
BCL-3 is thought to serve as an adaptor-bridging Tip60 to the NF
B
p50/p52 and to participate in the gene activation function of this
transcription factor (7). Moreover, it has recently been shown that
interleukin-1
induces the activation of a specific group of
NF
B-responsive genes including KAl1 in relation to the
selective recruitment of Tip60 to the promoter of this gene (8). Other
studies (9) showed that Tip60 can interact with interleukin-9 (IL-9)
receptor, suggesting its possible role in IL-9 signaling. Tip60 was
also found to interact directly with cAMP response element-binding
protein (CREB) and interfere with its activity, implicating this HAT in
the cAMP-dependent signaling process (10). Moreover, Tip60
was also shown to interact with one of the endothelin receptors, ETA
(11). All of these examples point to an important involvement of Tip60
in various receptor-mediated signaling processes. In agreement with
this hypothesis, it has recently been shown that Tip60 is an essential component linking the proteolytic cleavage of Amyloid-
precursor protein to transcriptional activation (8, 12).
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20 °C and used
in phosphatase treatment experiment, mass spectrometry analysis, and
HAT assay (see below).
-cyano-4-hydroxy-trans-cinnamic acid mixed
with nitrocellulose (mixture (4:3, v/v) of a saturated solution of
-cyano-4-hydroxy-trans-cinnamic acid in acetone and a
solution consisting of 10 mg of nitrocellulose dissolved in 1 ml of
isopropyl alcohol/acetone (1:1, v/v)). The deposits were washed
with 5 µl of 0.1% trifluoroacetic acid before the analysis. A
mass list of peptides was obtained for each protein digest.
-32P]ATP (3000 Ci/mmol, ICN), and 5 units of
Cdc2 kinase/cyclin B (New England BioLabs) for 30 min at 30 °C
(final volume of 30 µl). In control reactions, the kinase or the
peptide was omitted. Two volumes of 10% trichloroacetic acid and 20 µg of carrier bovine serum albumin were added at the end of the
reaction, and the samples were spotted on P81 cellulose phosphate
paper. Filters were then washed in 0.5% phosphoric acid (4 times, 10 min), rinsed in acetone, and finally used to measure 32P
incorporation in a scintillation counter.
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Fig. 1.
The enzymatically active Tip60 expressed in
insect cells is phosphorylated. Baculovirus-based expression
system was used to produce His-tagged human Tip60 (scheme)
in insect cells. A, the purified enzyme is capable of
efficiently acetylating free histones (left panel) and
nucleosomal histone H4 (right panel). B, the two
indicated His-tagged fragments of Tip60 (scheme) were
produced in insect cells, treated or not with CIP (+ and ,
respectively) and analyzed on 15% SDS-PAGE stained with Coomassie
Blue.
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Fig. 2.
Serines 86 and 90 are two major sites of
Tip60 phosphorylation. A, amino acid 1-211 region of
Tip60 expressed and purified from insect cells was digested with
trypsin, and the CIP-treated and untreated tryptic fragments were
analyzed by mass spectrometry. The CIP treatment led to the complete
displacement of a peak at 1483.30 kDa and the appearance of a
new one at 1323.30. The loss of mass attributed to the CIP treatment
(160 Da) shows the removal of two phosphate groups by CIP. The Q-TOF
sequencing of this peptide shows that it corresponds to the amino acid
81-93 region of Tip60 containing only two phosphorylable residues,
serine 86 and serine 90 (gray box). B, serines 86 (S86) and 90 (S90) are sites of phosphorylation
in mammalian cells. Site-directed mutagenesis was used to replace
Ser-86, Ser-90, or both by alanines. As a control, other irrelevant
sites of Tip60 have been also mutated to alanines. These sites are
glycine 380 (G380) and leucines 254 (L254) and
257. These proteins were expressed in HeLa cells, and their mobility
was monitored after a Western blot and immunodetection with an anti-HA
antibody. C, Tip60-(1-211) fragment containing the wild
type sequence or mutated on Ser-86 and Ser-90 (Tip1-211
S86/90) was also expressed in HeLa cells, and the mobility
of the CIP-treated wild type peptide was compared with that of the
Ser-86/Ser-90 mutant. D, purified HeLa cell nuclei were
isolated, and a fraction was incubated with CIP before extract
preparation (+). The extracts were then analyzed by Western blotting
using an anti -Tip60 antibody (14).
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Fig. 3.
Tip60 HAT activity is controlled by
phosphorylation. A, purified Tip60 expressed in insect
cells was treated with increasing amounts of CIP in the absence
(lanes 2 and 3) or presence of CIP inhibitor NaF
(lanes 4-6). Lanes 1 and 4 show the
activity of untreated Tip60. Treated and untreated Tip60 proteins were
incubated with purified histone and [14C]acetyl-CoA. The
reaction was stopped, loaded on a 15% SDS-PAGE, and analyzed by
autoradiography. B, purified Tip60 was left untreated
(lane 7) or treated with CIP or heat-inactivated CIP
(lanes 8 and 9, respectively). The activity of
wild type Tip60 was also compared with a protein harboring a G to A
mutation at position 380 (lanes 10 and 11,
respectively). C, the Ser-86/Ser-90 Tip60 mutant shows a
reduced HAT activity. Wild type Tip60 or a mutated version of the
protein harboring double S to A mutations at positions 86 and 90 was
produced in insect cells and purified, and their HAT activity was
tested as noted above. After the reaction, a fraction was analyzed on a
silver-stained 8% SDS-PAGE to compare the amounts of enzyme used
(upper panel). The middle panel
(Autorad) shows the labeling of histone after the reaction
by wild type or mutated Tip60. The corresponding Coomassie Blue-stained
gel before autoradiography shows histones used in each reaction
(lower panel).
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Fig. 4.
Tip60 Serine 90 is phosphorylated by cyclin
B/Cdc2 in vitro. A, peptides
corresponding to the Tip60 amino acids 82-96 region, either wild type
(wt) or containing the indicated mutations, were synthesized
and used in a kinase reaction. B, the histogram shows that
only the wild type peptide is a substrate for Cdc2 kinase.
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Fig. 5.
The arrest of cells at the G2/M
phase of the cell cycle is associated with an enhancement of Tip60
phosphorylation. A, HeLa cells were treated with
nocodazole (100 ng/ml) for 16 h and lysed, and the migration of
the endogenous Tip60 was monitored by Western blotting using an
anti-Tip60 antibody. B, HeLa cells stably expressing
HA-Tip60 were treated with nocodazole for 23 h, and 7 h
before harvest, roscovitin (50 µM) was added where
indicated. Cells were lysed and analyzed as above using an anti-HA
antibody. -, non-treated cells.
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(8), IL-9 (9), or endothelin (11). Besides, the HAT activity of Tip60 has been
shown to be essential in the cellular response to DNA damage (4).
Therefore, the phosphorylation of Tip60 regulating its HAT activity
could well play an important role in this process and somehow link
Tip60 function to the replication checkpoint. Finally, the HAT activity
of Tip60 was found to mediate gene activation following the cleavage of
amyloid-
precursor (12). These data should also be reconsidered in
the light of a linkage between Tip60 catalytic activity and its phosphorylation.
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ACKNOWLEDGEMENTS |
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We thank Dr. Vasily Ogrysko for the generous gift of the stable Tip60-expressing HeLa cell line, Sandrine Curtet and Marie-Paule Brocard for technical assistance, and Dr. Sophie Rousseaux for the critical reading of the paper.
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FOOTNOTES |
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* This work was supported by a grant from "Sidaction - Ensemble Contre Le Sida" (to S. K.) and a grant from "La Ligue Nationale Contre le Cancer" as a "équipe labelisée" (to D. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a postdoctoral fellowship from "Sidaction-Ensemble, Contre Le Sida from 1999 to 2001.
Recipient of a postdoctoral fellowship from Ligue Nationale
Contre le Cancer.
** Supported by the INSERM "Delegation" program.
§§ To whom correspondence should be addressed. Tel.: 334-7654-9583; Fax: 334-7654-9595.
Published, JBC Papers in Press, December 4, 2002, DOI 10.1074/jbc.M211811200
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ABBREVIATIONS |
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The abbreviations used are: HAT, histone acetyltransferase; IL, interleukin; CIP, calf intestine phosphatase; CREB, cAMP response element-binding protein; CDK, cyclin-dependent kinase; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MS, mass spectrometry; HA, hemagglutinin; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEKK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase.
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