From the
Equipe d'Accecil Signalisations
Moléculaires et Neurodégénéréscence,
Université Louis Pasteur, Strasbourg 67000, France, the
¶Department of Biological Sciences, Columbia
University, New York, New York 10027, the
||Institut National de la Santé et de la
Recherche Médicale, Unité 381, Strasbourg 67000, France, and the
**National Cancer Center, Tokyo 1004-0045, Japan
Received for publication, January 9, 2003 , and in revised form, March 31, 2003.
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ABSTRACT |
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INTRODUCTION |
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The precise functions of p73 proteins in the organism and the signaling
pathways that regulate their activity are still not well established. However,
various in vitro and in vivo experiments have shown or
suggested that, like p53, p73 proteins may play a role both in developmental
processes and DNA damage response. Several lines of evidence show that p73 may
play a role in nervous system and immune system development. Indeed,
p73/ mice harbor defects in the development of the hippocampus
and in the inflammatory response
(11). In lymphoid cells, p73
is involved in T cell receptor-induced apoptosis
(12). Supporting role(s) of
p73 in neurons, in vitro experiments have shown that p73 protein
levels are up-regulated during neuronal differentiation and that p73
overexpression is sufficient to induce neuronal differentiation
(13). Furthermore, N
isoforms of p73 can protect neurons from apoptosis
(14).
Several reports have attributed to p73 a role in the response to cellular
stress, as has been well described for p53. p73 overexpression can induce
apoptosis (1,
3,
6,
10). Moreover, up-regulation
of p73 protein levels and c-Abl-dependent activation of p73 in response to
radiation or cisplatin treatment leads to apoptosis
(1518).
c-Abl can also activate p73 activity through the p38 kinase that
phosphorylates p73 at a still unidentified threonine phosphorylation site
adjacent to a proline (TP
site)1
(19). Other oncogenes can also
increase p73 protein levels
(20). An interesting recent
report shows that p73 is required for p53-dependent apoptosis induced by DNA
damage (21). These
observations and the fact that p73 expression is affected in certain tumors
suggest that p73 may function as a tumor suppressor gene. However,
p73/ mice are not particularly prone to cancer
(11), and only rarely have
mutation or inactivation of p73 expression been found in human tumors.
Further, contradictory results have been reported concerning either
overexpression or repression of the p73 gene in some cancers.
Various proteins have been described to interact or regulate p73 protein activity. Among negative regulators, Hdm2 inhibits p73 but does not affect its stability (2225) and a subset of p53 tumor-derived mutants down-regulate p73 functions (9, 2729). WW proteins were reported to interact and stimulate p73 as well as Ik3-1/Cable (30, 31). However, the physiological relevance of these interactions and regulation have not yet been clearly established.
Irvin et al. reported that p73 protein levels are regulated through the cell cycle in experiments showing that cells released from serum starvation produce increasing levels of p73 starting at the end of G1 (32). p73 expression in this context is mediated by E2F protein(s) that bind to the p73 promoter. In fact, E2F proteins are transcriptional regulators of p73 expression, and it has been shown that p73 is involved in E2F-dependent apoptosis (12, 33). Because p73 expression is regulated through the cell cycle, we hypothesized that p73 activity might be modulated by cyclin-dependent kinases at a post-translational level.
In this study, we analyzed the physical and functional interactions between p73 proteins and cyclin-dependent kinases (CDK). We found that p73 can interact physically with cyclins and can be phosphorylated by S/G2/M CDK complexes. We identified Thr86 as a phosphorylation site for CDK complexes and showed that Thr86 is phosphorylated in a cell cycle-dependent manner in vivo. Finally, we found that mutation of Thr86 significantly affects p73 transcriptional activity, suggesting a regulatory role for the CDK complexes through this site.
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EXPERIMENTAL PROCEDURES |
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Expression VectorsExpression of human
HA-p73, HA-p73
, and HA-p73
cDNA
from the CMV promoter are as described
(7). pCMV cyclin A, pCMV cyclin
B, and pCMV p16 expression vector were gifts from Dr. K. Okamoto (Cancer
Center, Tokyo, Japan). PCDNA constructs encoding CDK1 or CDK2 proteins are a
gift from Dr. S. van den Heuvel. pBacp73
were obtained by subcloning a
HindIII/XhoI fragment of the CMVp73 expression constructs
into the FastBac vector (Invitrogen).
Expression and Purification of p73 Proteins and CDK Complexes Infection, expression, and purification of CDK complexes from insect cells were done as previously described (34). Generation and amplification of the p73 baculoviruses using the FastBac system were done as indicated by the manufacturer (Invitrogen). Purification of p73 proteins was performed as described previously (27). Briefly, Sf9 cells freshly seeded (1 h) at 90% confluence in 20-cm plates (2040 plates) were infected for 1 h with 500 µl of virus diluted in 2 ml of medium for each plate. After 48 h, the cells were removed from the plates in their medium and washed twice in phosphate-buffered saline. The cells were then lysed for 30 min in buffer A (50 mM Tris-HCl, pH 8, 0.5% Nonidet P-40, 150 mM NaCl, 1 mM DTT, 10% glycerol, 0.5 mM PMSF, and protease inhibitors, 0,1% aprotinin). The soluble fraction was isolated by centrifugation at 4 °C for 30 min at 20,000 rpm and added to protein A-Sepharose beads (12 ml) cross-linked to HA monoclonal antibody (12CA5) that were rocked overnight at 4 °C. The beads were then washed two times in buffer B (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.5% Nonidet P-40, 100 mM NaCl, 1 mM DTT, 10% glycerol, 0.5 mM PMSF) and two times in buffer C (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 100 mM NaCl, 1 mM DTT, 10% glycerol, 0.5 mM PMSF) and then poured into a 5-ml syringe. After washing with three column volumes of buffer D (20 mM Tris-HCl, 1 mM EDTA, 10% glycerol, 250 mM NaCl, 1 mM DTT), p73 proteins were eluted with a HA peptide (final concentration, 10 µg/ml) in a buffer containing 20 mM Tris-HCl, 1 mM EDTA, 10% glycerol, 500 mM NaCl, and 1 mM DTT. Fractions containing p73 were assessed by SDS-PAGE and pooled according to their similarity in concentration and purity.
Reporter Vectorsp21 min luc contains a duplex oligonucleotide encoding the p53-responsive cis-acting element from p21, cloned upstream of the minimal c-fos promoter (53 to +42) in pGL3-OFLUC (kindly provided by N. Clarke). The synthesized oligonucleotides (Operon Technologies, Inc.) encoding the p21 cis-acting p53-responsive element were as follows: 5'-GATCCTCGAGGAACATGTCCCAACATGTTGCTCGAG-3' and 5'-GATCCTCGAGCAACATGTTGGGACATGTTCCTCGAG-3'. The resulting plasmid was sequenced to determine the orientation and number of inserts of the oligoduplex.
Transfection and Luciferase AssaysH1299 and Cos1 cells (American Type Culture Collection) were maintained in DMEM supplemented with 10% FBS in 5% CO2 at 37 °C. The cells were transfected by a lipopolyamine-based (TransfectamTM) protocol as described previously (35). Briefly, cells were grown in DMEM, 10% FBS and transfected with various amounts of DNA. The precipitate was left on the cells for 6 h, after which fresh DMEM, 10% FBS was added. For luciferase assays, the cells were seeded in 12-well, 3.8-cm2 plates and transfected at 90% confluency with one of the expression vectors (200 ng of each) and two different reporter constructs (250 ng of each), a CMV-expressed luciferase cDNA from Renilla, and a p53-responsive luciferase cDNA from firefly. Luciferase activity was measured in each well 24 h later by a dual luciferase reporter gene assay (Promega).
Preparation of Whole Cell Extracts, Immunoblotting, and
Immunoprecipitation AnalysisH1299 and Cos1 cells in 6-cm plates
were transfected with the indicated plasmids (8 µg) and harvested 48 h
later. Sf9 cells were infected with 50 µl of virus in six-well plates. The
cells were lysed in 300 µl of TEGN buffer (20 mM Tris-HCl, pH 8,
1 mM EDTA, 0.5% Nonidet P-40, 150 mM NaCl, 1
mM DTT, 10% glycerol, 5 mM PMSF, 100 µM
benxamidine, 300 µg/ml leupeptine, 10 µg/ml bacitracine, 100 µg/ml,
-macroglobin and phosphatase inhibitors) (Cocktails I and II; Sigma),
and the extracts were centrifuged at 13,000 rpm for 12 min to remove cell
debris. The protein concentrations were determined using a colorimetric assay
(Bio-Rad). p73 proteins (400750 µg of whole cell extract) were
incubated with 30 µl of 50% slurry protein A-Sepharose beads cross-linked
to an anti-HA antibody (12CA5) followed by rocking at 4 °C for 3 h. After
incubation, the samples were washed four times with 1 ml of wash buffer (20
mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM EDTA,
0.5% Nonidet P-40, 1 mM DTT, 10% glycerol). Excess liquid was
aspirated, 35 µl of sample buffer
(36) was added, and samples
were heated to 95 °C for 3 min and centrifuged for 3 min at 13,000 rpm,
followed by electrophoresis through a 10% SDS-polyacrylamide gel followed by
transfer to nitrocellulose membranes (Schleicher & Schuell). For HA
detection, the 16B12 monoclonal HA antibody (Babco; 1 mg/ml) was used at
1:1000. TP phosphorylation was observed using a phospho-specific TP antibody
(New England Biolabs; 1:5000). Thr86 phosphorylation was followed
with a Thr(P)86 antibody, antigen-purified, and used at a dilution
of 1:1000. To assess p73-regulated p21 expression, the cells were transfected
in 6-well plates with 1 µg of p73 expression vector (or control vector), 3
µg of dnCDK1 (or control vector), and 0.2 µg of EGFP expressing vector.
The cells were lysed 20 h after transfection, and the proteins were separated
on a 10% SDS gel. For each point, two wells were transfected, and lysates were
mixed. p21 protein was detected using a monoclonal p21 antibody (Pharmingen,
clone SX118, 1/500). Cyclin immunoprecipitation and immunoblotting were
performed with a monoclonal anti-cyclin A antibody (1:50 and 1:1000,
respectively; Sigma). The proteins were visualized with an enhanced
chemiluminescence detection system (Amersham Biosciences).
GST Pull DownInteractions with purified proteins were
assessed in a buffer containing 2 µg/µl bovine serum albumin, 20
mM Tris-HCl, pH 8, 1 mM EDTA, 0.5% Nonidet P-40, 250
mM NaCl, 1 mM DTT, 10% glycerol, and protease inhibitor.
Immunopurified GST-cyclin A (300 ng) was incubated for 30 min with anti-GST
beads (glutathione-Sepharose 4B; Pharmacia Corp.; 20 µl). After two washes,
HA-p73 proteins (100 ng) were added and incubated for another 30 min.
Then beads were washed five times with a TEGN buffer containing 250
mM NaCl. The samples were heated to 95 °C for 5 min,
centrifuged for 3 min at 13,000 x rpm, electrophoresed through a 10%
SDS-polyacrylamide gel, and transferred to nitrocellulose for immunoblotting.
GST and GST-cyclin A were detected with a GST antibody (Ab3; Oncogene Research
Products; 1:1000).
In Vitro Phosphorylation AssaysImmunopurified p73 or
RB proteins (100 ng) were incubated in the presence of immunopurified
cyclin/CDK complexes (100 ng) in 20 µl of a kinase buffer (20 mM
Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM EDTA, 1
mM DTT, 100 µM ATP, 15 µCi of
[
-32P]ATP) for 30 min at 37 °C. After the addition of 10
µlof3x sample buffer, the proteins were separated on a 10% SDS-PAGE
gel. The gels were dried and analyzed by autoradiography. The purity and the
quantity of the proteins used in the assay were verified simultaneously by
SDS-PAGE gel separation and silver staining.
FACS AnalysisFACS analysis was performed as described by Lamm et al. (37). Briefly, the cells were fixed in 2% paraformaldehyde in a fixative buffer (100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA, 10 mM PIPES, pH 6.8) for 20 min and then in 95% methanol for 1 h. The fixed cells were washed three times with phosphate-buffered saline and exposed to propidium iodide (60 µg/ml), and RNase A (50 µg/ml) for 30 min before analysis (FACScalibur; Becton Dickinson) for DNA content (propidium iodyide) with a 610-nm long pass filter. An excitation wavelength of 488 nm was used for propidium iodyide. The data were analyzed using CELLQuest software (Becton Dickinson).
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RESULTS |
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To further investigate whether p73 is a direct target of CDK complexes, we performed in vitro protein kinase assays using immunopurified proteins. Rb, which is a well known substrate for all of the common CDK complexes, was used for comparison in these experiments (38). Similar amounts of purified p73 and Rb proteins were used as substrates for purified cyclin A/CDK2, cyclin D/CDK4, and cyclin E/CDK2 (Fig. 1C, right panel, silver-stained gel). As expected, all three CDK complexes phosphorylated Rb protein in vitro (Fig. 1C, left panel). In the same experimental conditions, p73 was phosphorylated far more efficiently by cyclin A/CDK2 than by cyclin E/CDK2. No phosphorylation of p73 by cyclin D/CDK4 was observed under conditions that allowed Rb phosphorylation by this kinase. Thus, p73 proteins can serve as substrates for cyclin A/CDK2 and cyclin E/CDK2 but not for cyclin D/CDK4.
Because the p73 gene encodes multiple isoforms varying in their C or N
termini, we compared the ability of the different isoforms to be
phosphorylated by the CDK using the same approach. Upon co-infection of insect
cells with cyclin A and CDK2 expressing baculoviruses, we found that at least
three isoforms (p73, p73
, and p73
) displayed a mobility
shift (Fig. 1D). This
suggests that the CDK phosphorylation site(s) is located in the common portion
of the various p73 isoforms.
Our experiments indicate that p73 proteins are direct targets for a subset of CDK complexes. The fact that they phosphorylate p73 in vitro with a similar efficiency to Rb, leading to a gel mobility shift in p73 migration, suggests that p73 is a good substrate for S/G2/M CDK complexes.
p73 Proteins Interact with CyclinsIn some cases,
phosphorylation by CDK complexes is associated with a direct interaction
between the cyclin and the substrate through a cyclin recognition motif (CRM):
RXL or KXL. The p73 protein sequence exhibits two potential
CRM sites (position 149, KKL; position 515, RAL), suggesting that p73 may
interact with cyclins. The first CRM is located in the core domain of the
protein (position 149, KKL) and is common to all p73 isoforms (see
Fig. 3A). The second
CRM is located in the extreme C-terminal part of the p73 isoform at
position 515 (RAL; Fig.
3A). We first examined the interaction between p73 and
cyclins in insect cells co-infected with baculoviruses expressing HA-tagged
p73 and GST-tagged cyclin A, cyclin B, cyclin E, or cyclin D
(Fig. 2A). Infected
cells were lysed 48 h after infection, GST-tagged cyclins were pulled down
with GST beads, and HA-p73 proteins were detected by immunoblotting using an
anti-HA antibody. As shown in Fig.
2A, p73
was present in the complex with cyclin A,
cyclin B, cyclin E, and cyclin D. Co-expression of cyclin A and cyclin B with
CDK2 and CDK1, respectively, did not significantly interfere with the ability
of p73 to co-immunoprecipitate with these cyclins. Interestingly,
co-expression of cyclin E and cyclin D with CDK2 and CDK4 reduced the amount
of p73 proteins in the complex. Moreover, when we compared the ability of
p73
and p73
to co-precipitate with cyclins, we observed better
co-precipitation between p73
and cyclin A, although p73
is
expressed at higher levels than p73
(Fig. 2B). This
suggests that both CRM sequences participate in the interaction between
cyclins and p73
. To confirm that the physical interaction between p73
and the cyclins is direct, co-immunoprecipitation experiments using
immunopurified proteins were performed
(Fig. 2C). Purified
p73
proteins were pulled down with GST-tagged cyclin A protein but not
with GST alone. We estimate that
5% of each protein is associated with
each other (Input = 10% of the purified protein used for the
immunoprecipitation; Fig.
2C, left panel).
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To examine whether an interaction between p73 and cyclin A could be
demonstrated in mammalian cells, H1299 cells were co-transfected with
HA-p73 and cyclin A expression vectors. After immunoprecipitation of
HA-p73 proteins with HA beads, we observed the presence of cyclin A proteins
in the complex (Fig.
2D). In absence of p73 protein, only a very small amount
of cyclin A was detected after immunoprecipitation, even though cyclin A
protein was expressed at a similar level
(Fig. 2D, left
panel). Thus, p73 proteins can interact physically and directly in
vivo with at least one cyclin (cyclin A).
Expression of CDKs Leads to Phosphorylation of p73 on a TP Site in VivoAs mentioned above, Thr86 is a potential CDK phosphorylation site ((S/T)PB, where B indicates a basic residue) in the N terminus of p73 (Fig. 3A; T*PEH). To test the possibility that this site can be phosphorylated by CDK, we first used a commercially available antibody that recognizes phosphorylated TP sites (phospho-TP). Insect cells were co-infected with baculoviruses expressing various CDK complexes. After immunoprecipitation of p73 proteins, the proteins were separated by SDS-PAGE, transferred to nitrocellulose, and probed with the phospho-TP antibody (Fig. 3B). When p73 was co-expressed with cyclin A/CDK1, cyclin A/CDK2, cyclin B/CDK1, cyclin B/CDK2, or cyclin E/CDK2, the phospho-TP antibody was able to detect p73 proteins (Fig. 3B, upper panel). Consistent with the results shown in Fig. 1, the antibody did not recognize p73 when cyclin D/CDK4 (Fig. 3B) or cyclin D/CDK2 (data not shown) was co-expressed. The lower panel in Fig. 3B indicates that similar amounts of p73 protein were immunoprecipitated in the presence or absence of CDK. To support the possibility that CDKs can phosphorylate p73 protein in human cells, H1299 cells were transfected with constructs expressing p73 with or without cyclin A. p73 proteins were detected with the phospho-TP antibody even without overexpression of cyclin A (Fig. 3C, upper panel). However, upon cyclin A overexpression, p73 phosphorylation was increased as detected by the phospho-TP antibody. Conversely, when the cells were treated with an inhibitor of CDKs, roscovitine for 3 h, p73 phosphorylation on TP was strongly reduced (Fig. 3C, lower panel). Further evidence that reduction of p73 phosphorylation had occurred was obtained using an anti-p73 antibody, because the upper band of the doublet disappeared under roscovitine treatment (Fig. 3C, lower panel). Thus, CDK complexes phosphorylate p73 proteins on one or more TP sites, and this phosphorylation occurs in mammalian as well as insect cells.
p73 Threonine 86 Is Phosphorylated in Vitro and in VivoTo
confirm that Thr86 is a target for CDK, an antibody was raised that
recognizes p73 when Thr86 is phosphorylated. To validate the
anti-Thr(P)86 p73 antibody, a mutant form of p73 was
generated in which threonine 86 was changed to alanine. Constructs expressing
p73
wild type and p73
T86A proteins were transfected into H1299
cells and then immunoprecipitated from H1299 cell extracts with HA antibody
cross-linked to protein A-Sepharose beads. After washes, the pellets of p73
proteins and beads were resuspended in a kinase buffer containing 100 ng of
purified cyclin A/CDK2 complex or no kinase, and [
-32P]ATP
(Fig. 4A). Half of the
proteins were run on an SDS-PAGE gel that was subsequently dried and
autoradiographed (Fig.
4A, upper panels). The other half was also
resolved on an SDS-PAGE gel and then transferred to nitrocellulose for
immunoblotting using the p73 anti-Thr(P)86 antibody
(Fig. 4A, middle
panels). In the absence of cyclin A/CDK2 complex, we did not observe any
incorporation of radioactivity (upper left panel). The addition of
purified cyclin A/CDK2 led to readily detectable phosphorylation of wild type
p73 but not p73
T86A (only barely detectable upon long exposure). This
observation suggested that Thr86 is the main phosphorylation site
for cyclin A/CDK2 phosphorylation on p73. The Thr86
phospho-specific antibody weakly recognized wild type p73
but not
p73
T86A, and after incubation with cyclin A/CDK2, recognition of p73
wild type by the anti-phospho-Thr86 antibody was markedly
increased. Thus, the Thr86 phospho-specific antibody specifically
recognizes the Thr86 site of p73 when phosphorylated. The weak
reactivity observed in the absence of phosphorylation by the CDK complex
suggests that some p73 is normally phosphorylated in H1299 cells at
Thr86.
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To confirm that Thr86 can be phosphorylated by CDK complexes in vivo, insect cells were co-infected with p73 and CDK expressing baculoviruses as described in the legend to Fig. 1. Immunoprecipitated p73 was subsequently probed with the Thr86 phospho-specific antibody. Cyclin A/CDK2 and cyclin B/CDK2 induced phosphorylation of p73 at Thr86, but cyclin D/CDK2 or cyclin D/CDK4 complexes failed to do so (Fig. 4B).
These observations were then extended to examine H1299 cells in which
p73 was co-expressed with cyclin A and CDK2. p73 proteins were
immunoprecipitated, and the phosphorylation of p73 was detected by
immunoblotting using the Thr86 phospho-specific antibody. As we had
observed in vitro, Thr86 phosphorylation was detected even
in the absence of overexpressed cyclin A/CDK2 complex. However, cyclin A/CDK2
co-expression significantly increased phosphorylation at this site
(Fig. 4C, left
panel). As expected, p73
T86A was not detected by the
anti-phospho-antibody (Fig.
4C, right panel).
The p73 Cyclin Recognition Motif Facilitates Thr86
Phosphorylation by Cyclin-dependent KinasesAs shown above, p73
proteins possess at least one CRM sequence and can interact with cyclins. We
wished to determine the importance of the CRM in p73 for both p73
phosphorylation and p73 activity. To this end, a variant of p73 lacking
the three amino acids KKL (position 149) was generated (p73
CRM). We
tested the ability of cyclin A/CDK2 to phosphorylate and interact with this
mutant. Wild type p73 and p73
CRM proteins were expressed in H1299
cells, and endogenous cyclin A was immunoprecipitated from cell extracts.
After four washes, the proteins were separated on a 10% SDS gel and
immunoblotted, and p73 proteins were detected with HA antibody
(Fig. 5A). Although
interactions with both wild type p73 and p73
CRM were observed, the
CRM-deleted form of p73 interacted less efficiently than wild type p73. Next,
we examined phosphorylation of p73
CRM using the
Thr(P)86 antibody (Fig.
5B). Interestingly, p73
CRM was much less
efficiently phosphorylated by cyclin A/CDK2 than was wild type p73
,
suggesting that the CRM is important for Thr86 CDK-dependent
phosphorylation (Fig.
5B). Finally, we tested the activity of
p73
CRM on the p21 min luc reporter gene, and we observed that
this mutant was not able or barely able to induce p21 min luc expression (data
not shown). Because the CRM lies within the DNA-binding region of p73, this
result was not unexpected. Nevertheless, our results identify this region as
being important for facilitating phosphorylation of p73 at
Thr86.
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Phosphorylation of p73 at Thr86 Is Regulated through the
Cell CycleBecause we showed that several CDK complexes can
phosphorylate p73 on Thr86 in vitro and p73 can be
phosphorylated at Thr86 in vivo, it was of interest to
determine whether phosphorylation at this site is regulated through the cell
cycle in human cells. In the first set of experiments, HA-p73 was
overexpressed in Cos-1 cells, and the state of the cells or the activity of
the CDK were modulated experimentally. Either the cells were treated with
roscovitine (Fig. 6A,
rosc.) or serum-starved (serum) or p73 was
co-expressed with p16 an inhibitor of CDK in G1 phase
(Fig. 6B). The
phosphorylation status of Thr86 was then monitored by
immunoblotting with the Thr86 phospho-specific antibody. We
observed that all treatments known to reduce CDK activity and cell cycle
progression (p16, roscovitine, serum starvation) significantly reduced
Thr86 phosphorylation (Fig.
6). Note that in all conditions, the variation in Thr86
phosphorylation was not due to a decrease in the level of immunoprecipitated
p73 proteins as shown by reblotting with an anti-HA antibody
(Fig. 6, lower panels
in both A and B).
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We next examined phosphorylation of p73 at Thr86 upon cell cycle
re-entry. The cells were first depleted of serum for 48 h and then were
transfected with the p73 expression vector for 14 h before induction with
serum (Fig. 7A). The
cells were then harvested at various times after serum treatment, and both
Thr86 phosphorylation and cyclin A expression (an indicator of the
progression of the cell cycle) were monitored. Serum starvation abolished
almost all Thr86 phosphorylation (seen with a longer exposition).
In contrast, serum treatment led to phosphorylation of p73 at
Thr86. Remarkably, this phosphorylation correlated closely with
cyclin A expression levels (Fig.
7A, top and bottom panels). In all
conditions, similar levels of p73 were immunoprecipitated
(Fig. 7A, middle
panel).
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To analyze in more detail the phosphorylation status of endogenously expressed p73 at Thr86 through the cell cycle, we used T98G cells in which it was previously shown that p73 expression is regulated through the cell cycle (32). T98G cells were induced with serum after starvation, and progression of their cell cycle was monitored by FACS analysis (not shown) and expression of specific markers (cyclin A, cyclin B, and E2F; Fig. 7B). Endogenous p73 protein was immunoprecipitated with a p73 antibody and after SDS-PAGE was immunoblotted with the anti-Thr(P)86 antibody. We normalized immunoprecipitation conditions to have comparable levels of p73 proteins on the blot. When cells were starved for 48 h, only weak phosphorylation of p73 was observed (Fig. 7B, ST). In this condition, no markers of the cell cycle were significantly expressed, and the cell cycle profile obtained by FACS analysis indicated that 82% of the cells were in G1/GO. Six hours after serum treatment, the majority of the cells were in S phase, and E2F, as well as cyclin A, was detected. Thr86 phosphorylation was clearly increased at this stage and continued to rise as the cells progressed through the cell cycle (Fig. 7B). Twelve hours after serum treatment, the cells were mainly in G2/M phase (69%), and expression of both cyclin A and cyclin B was observed. It was also at this stage that p73 phosphorylation on Thr86 was maximal. These results strongly support the physiological relevance of our observations that CDK complexes containing cyclins A or B preferentially phosphorylate p73.
Cyclin A/CDK2 or Cyclin B/CDK1 Overexpression
Regulates p73 Transcriptional ActivityTo evaluate the impact of
phosphorylation by CDK on p73 transcriptional activity, a luciferase reporter
gene containing p53-binding sites from the p21 promoter upstream a minimal
c-fos promoter was transfected into H1299 cells
(39) (p21 min luc;
Fig. 8A). H1299 cells
do not express endogenous p53 but express low levels of p73. As controls for
the experiment, we also overexpressed c-Abl and HDM2, which were previously
shown to be positive and negative regulators of p73, respectively
(1518,
2225).
As expected, we observed that c-Abl expression increased the activity of p73
on the p21 min luc reporter gene (Fig.
8A). Note that results are shown as fold induction
relative to the control in the absence of Abl, cyclin A, or MDM2
overexpression. Overexpression of p73 alone induced the reporter gene activity
by a factor of 18 (data not shown). In contrast, HDM2 expression decreased the
activity of p73 by 50%. When cyclin A was overexpressed in similar
conditions, p21 min luc reporter activity was decreased to a similar extent as
observed with HDM2 (Fig.
8A, upper panel). In all of the conditions
described, p73 protein levels remained the same
(Fig. 8A, lower
panel). We also used the parental reporter gene p min c-fos luc
as a control, and no change in its activity was observed in any conditions,
indicating the specificity of the effects obtained
(Fig. 8B).
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To extend our analysis of the regulation of p73 activity by CDKs, we co-expressed p73 proteins along with cyclin A/CDK2 or cyclin B/CDK1 complexes. Both CDK complexes reduced p73 transcriptional activity as was observed when cyclin A was expressed alone (Fig. 8C). Strikingly, when we co-expressed p73 with dominant negative forms of CDK1 (dnCDK1) or CDK2 (dnCDK2), p73 activity was markedly increased (Fig. 8C).
We then tested whether phosphorylation at Thr86 is important for
the functions of p73 by comparing activity of wild type p73 and
p73
T86A on the p21 min luc reporter gene in H1299 cells in the presence
or absence of CDK complexes (Fig.
9A). Both p73
wt and p73
T86A activated p21
min luc expression. However, CDK complexes were far less effective in reducing
p73
T86A transcriptional activity than wild type p73
(Fig. 9A). This series
of experiments suggests that phosphorylation of p73 by CDK negatively
regulates p73 transcriptional activity.
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To confirm that CDKs control p73 function in a more physiological context,
we tested the ability of p73 to regulate endogenous p21 protein expression in
H1299 cells in the presence or absence of the dominant negative form of CDK1
(dnCDK1). As with the p21 min luc reporter gene, p73 overexpression in
H1299 cells led to increased p21 protein levels, and the capacity of p73 to
induce p21 expression was significantly enhanced by dnCDK1
(Fig. 9B).
Importantly, although p73
T86A induced p21 to the same extent than wild
type p73
, it did not show similar stimulation by dnCDK2
(Fig. 9B, see Ct
lane in p73
lanes). Altogether our results
support the likelihood that p73 phosphorylation at Thr86 by CDK1
leads to decreased p73 transcriptional activity.
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DISCUSSION |
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p73 Is a Relevant Target for S/G2/M CDK ComplexesTo assess whether p73 proteins are targets of cyclin-dependent kinases, we tested various CDK complexes and found that S/G2/M CDK complexes are able to phosphorylate p73 proteins. The phosphorylation was observed both in vitro and in various cell lines (mammalian and insect), either with exogenously expressed protein or endogenous p73 proteins, indicating that this process occurs normally in vivo. Although the significance of phosphorylation of p73 by CDKs is not yet established, our results suggest that the proportion of p73 protein that is phosphorylated is fairly substantial. First, we observed that both p73 and pRb can be phosphorylated to approximately similar extents in vitro, and pRb is a well known target of CDK complexes (Fig. 1). Second, CDK phosphorylation of p73 results in a significant gel mobility shift of the p73 polypeptide, in some cases leading to its complete retardation. Importantly, we found that modulation of p73 phosphorylation at Thr86 occurs during the cell cycle, suggesting strongly that CDK complexes can affect p73 status at this site in a physiologically relevant manner. However, our data do not exclude the possibility that in mammalian cells Thr86 phosphorylation could also be regulated by other cell cycle-regulated kinases in addition to CDKs. It is also possible that under other physiological conditions, protein kinases whose activity is unrelated to the cell cycle might phosphorylate Thr86.
The fact that p73 is most highly phosphorylated at Thr86 in G2 and M (Fig. 5) is consistent with our observation that it is phosphorylated most efficiently by cyclin A and cyclin B containing complexes (Fig. 1). By contrast cyclin E containing CDK complex phosphorylates p73 quite inefficiently, and cyclin D containing CDK complexes appeared to be virtually inert in this regard. We cannot exclude the possibility that under specific conditions cyclin E or cyclin D complexes phosphorylate p73, but the cell cycle specificity of p73 phosphorylation suggests that this is not likely to be the case.
Thr86 Is an Important Site for CDK Phosphorylation in p73
ProteinCyclin-dependent kinases phosphorylate proteins at TP or SP
sites one residue from a basic residue on the C-terminal side of the site. In
p73 proteins, we identified Thr86 as the most likely candidate
(sequence: TPEH). Using a Thr86-specific phospho-antibody, we
confirmed that different CDK complexes indeed phosphorylate this site. We
cannot exclude that CDKs can phosphorylate p73 on other residues. Indeed, we
were still able to observe residual phosphorylation of the p73T86A
mutant by cyclin A/CDK2 (data not shown), and we found that transcriptional
activity of p73
T86A is still partially repressed by CDK overexpression
(Fig. 7). Nevertheless, both
phosphorylation and the repression by CDKs of p73
T86A were drastically
reduced when compared with wild type p73, strongly suggesting that
Thr86 is the principal site phosphorylated by CDK in p73.
Role of a CRM Sequence in p73 Phosphorylation by CDK
ComplexesPhosphorylation by CDK complexes is sometimes dependent
on the interaction between cyclins and the target through cyclin recognition
motifs (40). All p73 proteins
have a CRM located within the N-terminal portion of the DNA-binding domain
(KKL; position 149). We identified a second CRM specific to p73 located
at position 515 (RAL). From our studies, it appears that both sequences are
involved in the ability of cyclins to interact with p73 and to allow
phosphorylation of p73 by CDK. Although we showed that deletion of the CRM
located at position 149 reduced strongly the interaction with cyclin A or the
phosphorylation by cyclin A/CDK2, the interaction between cyclin A and
p73
is stronger than with p73
(Figs.
2 and
5). The exact role of CRMs in
the regulation of p73 activity and in particular whether the interaction with
the cyclin might regulate p73 function(s) without phosphorylation is difficult
to assess because one of CRM sequence is located in the DNA-binding domain of
p73. The fact that CRM(s) are present in p73 and can impact its ability to be
phosphorylated by CDKs further support the likelihood that CDKs, particularly
those containing S and G2 type cyclins, play a significant role in
the regulation of p73.
How Is the Function of p73 Regulated by CDKs?We observed
that overexpression of two CDK complexes (cyclin A/CDK2 and cyclin B/CDK1)
reduces p73 transcriptional activity in our conditions. Importantly, the
p73T86A mutant is markedly less sensitive to CDK repression. This
conclusion was based on transient co-transfection experiments with a
p53/p73-responsive reporter (Fig.
8) and perhaps more convincingly by examining directly the
expression levels of endogenous p21 protein in H1299 cells
(Fig. 9). Therefore, under our
conditions, CDK phosphorylation leads to the repression of at least some of
the functions of p73. At this stage, we do not know the mechanism involved.
When the effect of in vitro phosphorylation by CDK on the ability of
purified p73 to bind DNA was tested, we observed a modest (2-fold) increase
under those conditions (data not shown). Although this is not consistent with
our observations, it should be noted there that when experiments were
performed with overly confluent cells, we observed that overexpression of CDKs
actually stimulated transcriptional activity of wild type p73 but not the
p73
T86A mutant (data not shown). This observation may be related to our
observation of modest stimulation of p73 DNA binding by CDKs in
vitro.
It is possible that the repression mechanism seen under the culture
conditions used in our experiments (i.e. use of subconfluent freshly
plated cells) reflects modulation of the interaction of p73 with other
molecules that would be affected by Thr86 phosphorylation. Indeed,
we observed that the mutant p73T86A is more strongly stabilized by MDM2
than wt p73
(data not shown). The physiological relevance of this
observation is still under investigation. It is also interesting to note that
the location of Thr86 within p73 is similar to threonine 81 in p53,
which is also adjacent to a proline. Recently, Thr81 in p53 was
shown to be required for interaction with and stimulation by the prolyl
isomerase PIN1 (41,
42). It is attractive to
speculate that CDK-dependent phosphorylation at Thr86 may also
control the activity of p73 by modulating the interaction of p73 with
PIN1.
What Is the Function of CDK-dependent Phosphorylation of p73 during the Cell Cycle?p73 phosphorylation of Thr86 increases during the cell cycle such that it is greatest at G2/M. Because p73 harbors pro-apoptotic and growth arrest properties, these functions require a tight control by CDKs to allow a cell cycle to be completed, which is consistent with our finding that CDKs down-regulate p73 activity. However, it is intriguing that the maximum of Thr86 phosphorylation is at G2/M where the mitotic checkpoint can take place when microtubules are disrupted. Interestingly, it has recently been shown that cyclin B/CDK1 is involved in this mitotic checkpoint by up-regulating the activity of Survivin (43), a gene that can be repressed by p53 (26). Although p53 is not involved in the mitotic checkpoint (43), it is possible that p73 and its phosphorylation by CDK might play a role at this key stage of the cell cycle.
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FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 33-3-90-24-30-86; Fax:
33-3-90-24-30-65; E-mail:
gaiddon{at}neurochem.u-strasbg.fr.
1 The abbreviations used are: TP site, threonine phosphorylation site
adjacent to a proline; CDK, cyclin-dependent kinase; DMEM, Dulbecco's modified
Eagle's medium; FBS, fetal bovine serum; DTT, dithiothreitol; PMSF,
phenylmethylsulfonyl fluoride; HA, hemagglutinin; GST, glutathione
S-transferase; FACS, fluorescence-activated cell sorter; PIPES,
1,4-piperazinediethanesulfonic acid; CRM, cyclin recognition motif;
phospho-TP, phosphorylated TP site; wt, wild type; dn, dominant negative; min
luc, minimal luciferase.
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ACKNOWLEDGMENTS |
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REFERENCES |
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