From the Institute for Research in Biomedicine, Via Vincenzo Vela 6, Bellinzona CH 6500, Switzerland
Received for publication, February 17, 2003 , and in revised form, April 24, 2003.
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ABSTRACT |
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INTRODUCTION |
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Class I PI 3-kinases activities were shown to be regulated by
phosphorylation
(2123).
Phosphorylation of the regulatory subunit p85 by the catalytic subunit
p110 of class I PI 3-kinase (p110/p85 heterodimer) down-regulates lipid
kinase activity of the complex
(21,
22). Phosphorylation of class
II PI 3-kinases was demonstrated; however, the physiological role of this
phosphorylation remained unclear. Increased phosphorylation of class II PI
3-kinase C2
was found to correlate with a moderately elevated enzyme
activity in insulin-stimulated cells
(24). In contrast, our data
demonstrated that the phosphorylation status neither changes the lipid kinase
activity of PI3K-C2
nor affects the substrate specificity, but
influences the intranuclear localization
(1).
In this study we investigated the phosphorylation of HsPI3K-C2
induced by genotoxic stress and during the cell cycle. We show that the kinase
becomes phosphorylated upon exposure of cells to UV irradiation and in
proliferating cells at the G2/M transition of cell cycle.
Stress-dependent and mitotic phosphorylation of HsPI3K-C2
occurs on the
same serine residue (Ser259) within a recognition motif
(serine-proline sequence) for proline-directed kinases, such as
mitogen-activated protein (MAP) kinases and cyclin-dependent protein kinases
(Cdk). By using different selective inhibitors of MAP kinases and Cdks in
in vitro and in vivo assays, we found that Cdc2 mediates
mitotic phosphorylation, whereas JNK/SAPK is responsible for stress-induced
phosphorylation of HsPI3K-C2
. In either case phosphorylation provides a
signal for proteasome-dependent degradation of the protein.
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EXPERIMENTAL PROCEDURES |
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PlasmidsThe cDNA encoding human HsPI3K-C2
(8) was a kindly provided by
Dr. J. Domin (London). Various HsPI3K-C2
cDNAs were amplified by the
PCR using gene-specific primers with incorporated restriction sites to
facilitate their cloning into appropriate vectors. For expression in bacterial
cells, GST-tagged fusion constructs were generated by cloning wild-type and
mutant HsPI3K-C2
cDNAs into the BamHI site of pGEX-2T
(Amersham Biosciences). For expression in mammalian cells the cDNAs were
cloned into the eukaryotic expression vector pEGFP-C1 or pEGFP-N1 (Clontech).
To generate pEGFP:
HsPI3K-C2
expressing
GFP-
HsPI3K-C2
, a PCR product corresponding to the amino acids
240275 of HsPI3K-C2
was inserted into
XhoI-BamHI sites of pEGFP-C1. HsPI3K-C2
point
mutations, S254A, S259A, S259D, S259E, S262A, and S264A were created by PCR
amplification from pEGFP-C1:
HsPI3K-C2
, using mutant sequence
oligonucleotides. pBK-CMV:myc-HsPI3K-C2
, which encodes full-length
HsPI3K-C2
tagged at the NH2 terminus with myc epitope was
constructed as follows. The SacI-BspEI fragment from
pBK-CMV-HsPI3K-C2
(8)
containing the 5'-untranslated region and the first 52 nucleotides of
the HsPI3K-C2
coding sequence was replaced by the
SacI-BspEI PCR fragment carrying a Kozak consensus sequence,
an ATG start codon, and the sequence of myc tag joined in-frame to the
HsPI3K-C2
coding sequence (452 bp). pBK-CMV:HA-HsPI3K-C2
,
which encodes full-length HsPI3K-C2
tagged at the NH2
terminus with HA epitope, was constructed using a similar approach. To
generate pEGFP-C1:GFP-HA-HsPI3K-C2
, which encodes complete
HsPI3K-C2
double tagged at the NH2 terminus with GFP and HA
epitope (GFP-HsPI3K-C2
), the SacI-EcoRI fragment from
pBK-CMV:HA-HsPI3K-C2
was cloned into SacI-EcoRI sites
of pEGFP-C1. To generate pEGFP-N1:myc-HsPI3K-C2
-GFP that encoded
full-length HsPI3K-C2
tagged at the NH2 terminus with myc
epitope and at the COOH terminus with GFP (HsPI3K-C2
-GFP), the
SacI-AccI fragment from pBK-CMV:myc-HsPI3K-C2
containing the myc-tagged HsPI3K-C2
coding sequence up to 4884 bp was
cloned into to pEGFP-N1:HsPI3K-C2
-
N, in which the COOH-terminal
fragment of HsPI3K-C2
(48845055 bp) was joined in-frame to GFP.
JNKK2 mammalian expression vector
(25) was a gift from G. Natoli
(Bellinzona).
Cell Culture, Synchronization, Transient and Stable Expressions HeLa (ATCC), MCF7, COS-7, and HEK-293 cells were cultured at 37 °C in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% fetal calf serum and antibiotics. The following protocols were used to obtain HeLa cells arrested at specific stages of the cell cycle. Cells, enriched in G1, were obtained by treatment with mimosine (Sigma) at 400 µM for 20 h. For early S phase arrest, subconfluent cultures were blocked by serum deprivation for 48 h followed by the addition of 5 µg/ml aphidicolin (Sigma) for 24 h (26). To obtain a cell population enriched in G2 phase, cells were presynchronized in S phase as described above and then released by transferring into fresh medium for 8 h. For M phase synchronization, cells were treated with nocodazole (Sigma) at 400 ng/ml for 16 h before collecting mitotic cells by shake off. Cell cycle distributions were confirmed by flow cytometry (fluorescence-activated cell sorter). For fluorescence-activated cell sorter analysis of DNA content, cells were washed twice in phosphate-buffered saline and fixed in 90% methanol at -20 °C for 15 min. After an additional wash in phosphate-buffered saline cells were resuspended in 4 mM sodium-citrate, 0.1% Triton X-100 and treated with 10 µg/ml RNase A in the presence of 50 µg/ml propidium iodide for 10 min at 37 °C. To inhibit proteasome activity, MG132 (Calbiochem) was added to cells at a concentration of 20 µM.
Transient and stable transfections were carried out using PolyFect reagent
(Qiagen) according to the manufacturer's instructions. For transient
expression of GFP-HsPI3K-C2 and the mutants (S259A and S259D) COS-7
cells were transfected with the corresponding plasmids:
pEGFP-C1:GFP-HA-HsPI3K-C2
, pEGFP-C1:GFP-HA-HsPI3K-C2
/S259A, and
pEGFP-C1:HA-HsPI3K-C2
/S259D. For generation of stable HEK-293 lines
expressing wild-type and mutant HsPI3K-C2
-GFP fusion proteins, HEK-293
cells were transfected with pEGFP-N1:myc-HsPI3K-C2
-GFP or
pEGFP-N1:myc-HsPI3K-C2
/S259A-GFP. Two days after transfection, cells
were replated in medium containing 1 mg/ml G418. G418-resistant colonies,
selected at 23 weeks after transfection, were subcloned and analyzed
for the expression of recombinant proteins by immunoblotting with anti-GFP
antibody.
UV and -IrradiationCells were exposed to
genotoxic agents and analyzed 1.5 h later. An UV dose of 300 J/m2
was delivered in a single pulse using a Stratalinker (Stratagene). Prior to
pulsing, the medium was removed, being replaced immediately after the
treatment. 100 Gy of
-irradiation was delivered using a Gammacell 1000
apparatus.
Gel Electrophoresis, Immunoprecipitation, and Western Blot AnalysisProteins were separated on 8 or 6% SDS-polyacrylamide gels prepared from the stock (33.5% acrylamide, 0.3% bisacrylamide) and blotted onto Immobilon-P (Millipore). Membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Triton X-100 and probed with specific antibodies. Immunoreactive bands were decorated with horseradish peroxidase-labeled secondary antibodies and visualized by enhanced chemiluminescence (Pierce).
For immunoprecipitation cells were washed twice in phosphate-buffered
saline and lysed in buffer (1% Nonidet P-40, 150 mM NaCl, 50
mM Tris-HCl (pH 7.5), 1.5 mM MgCl2, 1
mM EDTA), supplemented with phosphatase inhibitors (40
mM NaF, 0.5 mM sodium orthovanadate, 40 µM
-glycerophosphate, 5 mM sodium pyrophosphate) and protease
inhibitors (Complete, Roche). Cell homogenates were centrifuged at 13,000
x g for 10 min, and supernatants were precleared with
Gamma-Bind Plus-Sepharose (Amersham Biosciences) for 15 min.
Immunoprecipitation of HsPI3K-C2
with antibody AXXIII was carried out
at 4 °C for 12 h. Immune complexes were bound to GammaBind
Plus-Sepharose for 30 min, collected by centrifugation, and washed twice in
lysis buffer, once in 10 mM Tris-HCl (pH 8), 0.5 M NaCl,
0.5% Nonidet P-40, 0.5% deoxycholate, 0.05% SDS; then in 10 mM
Tris-HCl (pH 8), once in 10 mM Tris-HCl (pH 8), 150 mM
NaCl, 0.5% Nonidet P-40, 0.5% deoxycholate, 0.05% SDS, and finally in 10
mM Tris-HCl (pH 8), 0.05% SDS. For
-phosphatase treatment
immunoprecipitates were additionally washed twice in phosphatase buffer (50
mM Tris-HCl (pH 7.5), 2 mM MnCl2, 0.1
mM EDTA, 5 mM dithiothreitol, 0.01% Brij 35) and
resuspended in 50 µl of the same buffer. After warming up at 30 °C for
3 min, 50 units of
-phosphatase (New England Biolabs) was added, and
samples were incubated at 30 °C for 40 min.
Pulse-Chase ExperimentsSubconfluent cultures of HeLa cells
were labeled overnight with 50 µCi of [35S]methionine/cysteine
(Amersham Biosciences)/ml in methionine-free DMEM (Invitrogen) supplemented
with 10% dialyzed fetal calf serum. After labeling cells were washed in
phosphate-buffered saline, replated (1:3 dilution), and chased with complete
DMEM containing 10% fetal calf serum for 48 h. To obtain mitotic cells, 400
ng/ml nocodazole was added to the medium for the last 36 h of the chase.
Labeled mitotic cells were collected by shake off, washed three times in
prewarmed DMEM, and released into fresh complete medium for 3 h. For metabolic
labeling of cells at M/G1 transition of cell cycle,
nocodazole-treated mitotic HeLa cells were released into the labeling DMEM in
the presence of 50 µCi of [35S]methionine/cysteine for 1 or 3 h.
Cells were subsequently subjected to immunoprecipitation analysis with
anti-HsPI3K-C2 antibody (AXXIII) as described above. Immunoprecipitated
proteins were resolved by SDS-PAGE, transferred to polyvinylidene difluoride
membranes, and visualized by autoradiography and immunoblotting.
Kinase and Protease Inhibitor TreatmentsRoscovitine (Calbiochem) was used to inhibit Cdk activity. HeLa cells at late S phase (6 h after release from aphidicolin block) were treated or not with 30 µM roscovitine for 2 h. 400 ng/ml nocodazole was added and treatment continued for 15 h. Nonadhering mitotic and adhering G2 cells were collected by mechanical shock and trypsin treatment, respectively. Nocodazole-arrested mitotic HeLa cells were treated with 75 µM roscovitine for 15, 45, and 90 min. For okadaic acid treatment nocodazole-arrested mitotic HeLa cells were treated with 0.5 µM okadaic acid for 30 min, then 75 µM roscovitine was added, and treatment continued for 30 min.
SP600125 (Tocris) was used to inhibit JNK activation, and PD98059 and SB202190 (both from Alexis) were used to inhibit ERK and p38 activation, respectively. HeLa cells were preatreated with the inhibitors at concentrations indicated for 30 min before UV irradiation. Irradiated cells were cultured for 90 min in the presence of the inhibitors before harvesting.
The specific protease inhibitors MG132, ALLM, and lactacystin were from Calbiochem. HEK-293 cells were UV irradiated as described above. After 2 h of recovery in fresh medium, cells were treated with protease inhibitors (20 µM MG132, 100 µM ALLM, or 50 µM lactacystin) for the indicated times prior to Western blot analysis.
In Vitro Kinase AssayHsPI3K-C2 was
immunoprecipitated from mimosine-treated HeLa cells using affinity-purified
anti-HsPI3K-C2
antibody AXIX
(1). The antibody-antigen
complexes were collected with GammaBind Plus-Sepharose and used as substrate
for in vitro phosphorylation by cellular extracts. To obtain cellular
extracts, pellets of interphase or mitotic HeLa cells were resuspended in 2
pellet volumes of ice-cold hypotonic buffer (50 mM Tris-HCl (pH
7.5), 1 mM dithiothreitol, 40 mM NaF, 0.5 mM
sodium orthovanadate, 40 mM
-glycerophosphate, 5
mM sodium pyrophosphate, and a mixture of protease inhibitors
(Complete, Roche)) and disrupted by brief sonication. The resulting
homogenates were centrifuged at 400,000 x g for 15 min at 4
°C. Supernatants (
10 mg of protein/ml) were supplemented with 150
mM NaCl and 10 mM MgCl2 and used as a source
of kinases. HsPI3K-C2
immunoprecipitates were mixed with supernatants
in a final volume of 100 µl, and phosphorylation assays were initiated by
adding 1 mM ATP. Assays were carried out at 30 °C for 1 h and
terminated by the addition of ice-cold Tris-buffered saline containing 0.1% of
Triton X-100. HsPI3K-C2
immunoprecipitates were collected by
centrifugation, washed twice with Tris-buffered saline, and analyzed by
Western blotting as described above.
For in vitro phosphorylation GST-HsPI3K-C2
fusion
proteins were expressed in Escherichia coli strain (BL21) and
purified by absorption to glutathione-Sepharose beads (Amersham Biosciences).
Fusion proteins were left attached to the beads, and phosphorylation reactions
with cellular extracts were carried out as described above in the presence of
50 µCi of [
-32P]ATP and 1 mM ATP. In
vitro phosphorylation of GST-
HsPI3K-C2
and
GFP-HsPI3K-C2
fusion proteins by 10 units of purified recombinant human
Cdc2-cyclin B (Calbiochem) was performed in Cdc2-kinase buffer (50
mM Tris-HCl (pH 7.5), 2 mM dithiothreitol, 10
mM MgCl2, 1 mM EGTA) in the presence of 10
µCi of [
-32P]ATP and 100 µM ATP. Soluble
GST-
HsPI3K-C2
was used as substrate in phosphorylation assays
with immunoprecipitated Cdc2. Cdc2 was immunoprecipitated from cytosols of
HeLa cells as described above, immunocomplexes bound to GammaBind
Plus-Sepharose beads were additionally washed twice in the kinase buffer, and
the reaction was initiated by addition of purified GST-
HsPI3K-C2
and ATP (100 µM ATP, 10 µCi of [
-32P]
ATP).
ImmunofluorescenceImmunofluorescence experiments were performed as described previously (1) using the methanol fixation protocol.
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RESULTS |
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To investigate whether down-regulation of transcription caused by different
types of genotoxic stress results in phosphorylation of HsPI3K-C2, we
exposed cells to DNA-damaging treatment such as UV light or ionizing
radiation. Exposure of HeLa and MCF7 cells to UV irradiation induced a
collapse of nuclear HsPI3K-C2
-positive speckles
(Fig. 1A) similar to
that observed in actinomycin D-treated cells: speckles lose their irregular
shape, become round, and fuse into larger clusters
(1). This effect was associated
with the increased phosphorylation of HsPI3K-C2
, as measured by its
mobility shift on SDS-polyacrylamide gels
(Fig. 1B). When cell
extracts were treated with
-phosphatase the appearance of the slower
migrating band was abolished (not shown). In contrast to UV-treated cells,
exposure of cells to
-irradiation neither changed subnuclear
localization of HsPI3K-C2
(not shown) nor induced its phosphorylation
(Fig. 1B). These
results suggest that HsPI3K-C2
specifically participates in UV-induced
damage response.
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The observation that phosphorylation of HsPI3K-C2 correlates with
changes in its subnuclear localization let us to speculate that the
phosphorylation status of the kinase may also be cell cycle-dependent, because
in mitotic cells HsPI3K-C2
-positive speckles dissolve, and the kinase
becomes equally distributed over the cytoplasm
(Fig. 1A). We used
HeLa cells to examine whether HsPI3K-C2
demonstrates different
phosphorylation patterns during the cell cycle. Cells were synchronized in
different stages of the cell cycle as follows: at late G1 with
mimosine, at M with nocodazole, in early S phase by serum deprivation followed
by an aphidicolin block, and cells enriched in G2 phase were
obtained 8 h after release from aphidicolin block
(26). Proteins from
corresponding cell lysates were fractioned by SDS-PAGE, and HsPI3K-C2
was analyzed by immunoblotting (Fig.
2A). In mimosine-treated cells HsPI3K-C2
was
detected as a single band, in cells blocked in S phase a second slower
migrating band became visible. Two bands, a faster and a slower migrating, of
equal intensity were apparent in cells enriched in G2 phase. A
single slower migrating band was found in prometaphase-blocked mitotic cells.
To confirm that altered gel mobility of HsPI3K-C2
was the result of
phosphorylation, protein extracts from synchronized HeLa cells were treated
with
-phosphatase (Fig.
2). Phosphatase treatment resulted in the collapse of the slower
migrating band of the kinase, indicating that indeed retarded mobility of
HsPI3K-C2
during SDS-PAGE is a consequence of phosphorylation. These
results demonstrate that HsPI3K-C2
undergoes a cell cycle-regulated
phosphorylation that reaches its maximum in mitosis.
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HsPI3K-C2 Can Be Phosphorylated in Vitro by Kinases
Present in HeLa Cell ExtractsThe initial strategy to identify
residues on which HsPI3K-C2
becomes phosphorylated upon UV irradiation
and during cell cycle was to label HeLa cells metabolically in the presence of
32Pi. In several attempts we did not succeed to obtain
sufficient amounts of in vivo 32P-labeled HsPI3K-C2
by immunoprecipitation to perform phosphopeptide mapping analysis. To overcome
this problem, we developed an in vitro phosphorylation assay that
allowed the identification of potential phosphorylation sites. As a substrate
for phosphorylation we used HsPI3K-C2
immunoprecipitated from
mimosine-treated HeLa cells. Concentrated high speed supernatants (S100)
prepared from HeLa cells (asynchronously growing or mitotic) were used as
sources of kinases. The supernatants are devoid of HsPI3K-C2
as shown
previously (1), which excluded
any additional input of the already phosphorylated substrate into the assay.
Phosphorylation of HsPI3K-C2
was measured by mobility shift of the
protein during SDS-PAGE. Fig.
2B illustrates that HsPI3K-C2
can be
phosphorylated successfully in vitro by kinases present in cell
extracts, and it is not caused by autophosphorylation. Almost complete
phosphorylation of HsPI3K-C2
was achieved in vitro by kinases
from mitotic extracts, whereas the protein was less efficiently phosphorylated
by kinases present in extracts from asynchronously cycling cells. The
phosphorylation profile and the efficiency of phosphorylation of
HsPI3K-C2
in vitro appeared to be remarkably similar to that
seen in vivo (compare Fig. 2,
A and B, upper panel), suggesting that
in vitro phosphorylation assay accurately mimics the in vivo
situation.
Ser259 Is the Site of Mitotic as Well as UV-induced
PhosphorylationWe expressed different domains covering the entire
sequence of HsPI3K-C2
(1) as GFP fusion proteins in
HeLa cells and found that only fusion proteins containing the
NH2-terminal domain (amino acids 1482) exhibited an
electrophoretic mobility shift (not shown). This observation suggested that
phosphorylation site(s) is (are) localized within this domain and that shorter
fragments of the kinase can be used as reporters of phosphorylation. Therefore
we expressed different segments of the NH2-terminal domain of the
kinase as GST fusion proteins and used these proteins as substrates for in
vitro phosphorylation. The shortest domain (GST-
HsPI3K-C2
)
that showed retarded electrophoretic mobility upon phosphorylation in
vitro comprised amino acids 240275
(Fig. 2B).
Analysis of the amino acid sequence of HsPI3K-C2 between residues
240 and 275 revealed seven potential phosphorylation sites
(Fig. 3A). Among them,
Ser254 conforms to the consensus motif for phosphorylation by
casein kinase I (S251PKVS254), Thr243 is
within a recognition motif for phosphorylation by casein kinase II
(T243DLE), Ser259 followed by Pro is a potential target
for proline-directed protein kinases. To map the phosphorylation site(s)
within GST-
HsPI3K-C2
, we mutated Ser254,
Ser259, Ser262, and Ser266 by a single
substitution to alanine and subjected the resulting GST fusion proteins to
in vitro phosphorylation assays
(Fig. 3B).
Incorporation of 32P as well as the electrophoretic mobility shift
of the corresponding fusion protein was completely abolished when
Ser259 was mutated to alanine (S259A), indicating that this residue
is a prime target of phosphorylation. The mutation of Ser254
(S254A) did not affect phosphorylation but resulted in the loss of the
electrophoretic mobility shift of phosphorylated protein, whereas mutations of
either Ser262 (S262A) or Ser266 (S266A) resulted in a
complete mobility shift of phosphorylated proteins.
GST-
HsPI3K-C2
incorporated 32P into several slower
and faster migrating forms. To rule out the possibility that phosphorylation
on Ser259 might be a primary event necessary for subsequent
phosphorylation of other residues, we mutated this serine to either aspartic
acid (S259D) or glutamic acid (S259E) to mimic its phosphorylation status.
However, despite the fact that the corresponding mutant proteins did change
their electrophoretic mobility they failed to incorporate 32P. To
confirm further Ser259 as phosphorylation site,
32P-labeled forms of GST-
HsPI3K-C2
were analyzed by
mass spectrometry. MALDI-TOF measurements of tryptic fragments of
GST-
HsPI3K-C2
showed only for the peptide
Val253-Lys261 a mass shift of 80 Da consistent with the
hypothesis that Ser259 is phosphorylated. The peptide, however,
encompasses two serine residues, Ser254 and Ser259.
Electron spray ionization (ESI-TOF) mass spectrometry and MS/MS fragmentation
pattern analysis of the peptide revealed Ser259 as unique
phosphorylation site. The results excluded phosphorylation of other
serine/threonine residues within this sequence (240275). The
observation that phosphorylated GST-
HsPI3K-C2
migrates as
doublet (Figs. 3B and
6B) suggests that the
fusion protein can acquire different SDS-resistant conformations.
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Based on the results of the in vitro kinase assay, we transiently
expressed the wild-type and the mutated versions of HsPI3K-C2
(amino acids 240275) as GFP fusion proteins in HeLa cells and measured
their phosphorylation status in mitosis and after UV irradiation. As shown in
Fig. 4A, only
mutations of Ser259 (S259A and S259D) completely blocked the M
phase-dependent as well as UV-induced electrophoretic mobility shift of the
corresponding GFP fusion proteins. Similar results were obtained when
full-length wild-type and mutated (S259A and S259D) forms of HsPI3K-C2
were transiently expressed as GFP fusion proteins in COS-7 cells. In
asynchronously growing cells GFP-HsPI3K-C2
showed a marginal level of
phosphorylation, and UV irradiation further induced the phosphorylation of
this fusion protein, as judged by the increase in the level of the slower
migrating form (Fig.
4B). In contrast, the mutants GFP-HsPI3K-C2
/S259A
and GFP-HsPI3K-C2
/S259D were detected as single bands on
SDS-polyacrylamide gels and did not show any change in their electrophoretic
mobility upon UV irradiation. Analysis of nocodazole-arrested HEK-293 cells
stably expressing HsPI3K-C2
-GFP and HsPI3K-C2
/S259A-GFP
(Fig. 4C) confirmed
that Ser259 is also the site of mitotic phosphorylation.
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Taken together, these data demonstrate that Ser259 is a common
site of the mitosis phase-dependent and the UV-induced phosphorylation of
HsPI3K-C2. Moreover, the phosphorylation of this site induces a
conformational change in HsPI3K-C2
which alters its mobility during
SDS-PAGE. The latter conclusion is strengthened by observations that mutation
of Ser259 to aspartic acid or glutamic acid, which should mimic
phosphorylation on this residue, results in mutants with electrophoretic
mobility comparable to that of the phosphorylated protein. However, it can not
be totally excluded that HsPIK3-C2
may also become phosphorylated at
other sites.
In Vivo Phosphorylation of HsPI3K-C2 by
Cdc2The G2/M transition of the cell cycle is triggered
by activation of a protein kinase cascade. The major kinase,
p34cdc2, required for promotion of mitosis, belongs to a
family of proline-directed kinases. Because the identified site of mitotic
phosphorylation in HsPI3K-C2
resembles the consensus motif
(S-P-X-R/K) for phosphorylation by Cdc2, we tested whether in
vivo phosphorylation of HsPI3K-C2
in mitosis is mediated by Cdc2.
We used roscovitine, a highly selective inhibitor of cyclin-dependent kinases
(27,
28), to block Cdc2 activity in
mitotically synchronized cells. HeLa cells were synchronized in S phase,
released, and then allowed to proceed to mitosis under nocodazole block in the
presence or absence of roscovitine. After exposure to nocodazole for 15 h
approximately 98% of control cells were arrested in mitosis. In contrast, only
approximately 10% of roscovitine-treated cells were nonadherent mitotic cells,
whereas the majority remained adherent because of the arrest in G2
phase. Immunoblot analysis revealed that roscovitine treatment led to a
significant inhibition of phosphorylation of HsPI3K-C2
in mitotic and
in G2 phase-arrested cells (Fig.
5A).
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To test whether Cdc2 activity is indispensable for maintaining
phosphorylation of HsPI3K-C2 in mitosis, we treated the
nocodazole-blocked mitotic HeLa cells with roscovitine for short periods of
time and analyzed the phosphorylation state of HsPI3K-C2
by
immunoblotting. As shown in Fig.
5B, the amount of phosphorylated HsPI3K-C2
present
in the slower migrating form decreased with the duration of roscovitine
treatment, whereas simultaneously an increase in the amount of the faster
migrating form was observed. We speculated that the dephosphorylation of
HsPI3K-C2
caused by the inhibition of Cdc2 might involve an okadaic
acid-sensitive protein phosphatase. Indeed pretreatment of mitotic cells with
okadaic acid markedly attenuated the dephosphorylation of HsPI3K-C2
observed after roscovitine treatment (Fig.
5C). Taken together, these findings suggest that in
vivo Cdc2 is involved in the phosphorylation of HsPI3K-C2
during
the cell cycle.
In Vitro Phosphorylation of HsPI3K-C2 by
Cdc2To test whether Cdc2 can directly phosphorylate
HsPI3K-C2
, we performed in vitro kinase assays using Cdc2
immunoprecipitated from HeLa cells (asynchronously growing, enriched in
G2 phase or mitotic) and the GST-
HsPI3K-C2
as
substrate. Fig. 6A
shows that GST-
HsPI3K-C2
is phosphorylated by immunoprecipitated
Cdc2 in a roscovitine-sensitive manner (top panel). The efficiency of
phosphorylation correlates with cell cycle-dependent activation of Cdc2 as
detected by the loss of the inactive slower migrating hyperphosphorylated form
of p34cdc2 (bottom panel).
To confirm that Ser259 is a target of Cdc2, we determined
whether purified activated Cdc2/cyclinB could phosphorylate wild-type and
mutated GST-HsPI3K-C2
fusion proteins.
Fig. 6B shows that
mutation of Ser259 to either alanine or aspartic acid abolished
Cdc2-mediated phosphorylation. The same result was obtained when full-length
native and mutated GFP-HsPI3K-C2
-fusion proteins expressed in COS-7
cells were used as substrates for in vitro phosphorylation by
purified activated Cdc2/cyclinB (Fig.
6C).
Phosphorylation Controls the Turnover of HsPI3K-C2
during the Cell CycleA dramatic change in the phosphorylation
state of HsPI3K-C2
occurs at the M to G1 phase transition of
the cell cycle (Fig.
2A, compare G1 and M). We reasoned that
phosphorylation could regulate the stability of HsPI3K-C2
and
potentially provide a signal for degradation of the kinase during mitosis.
Therefore we performed pulse-chase experiments to determine whether
HsPI3K-C2
, which was phosphorylated in mitosis, becomes
dephosphorylated or degraded upon reentry of cells into subsequent
G1 phase. Fig.
7A (left panel) shows that HsPI3K-C2
metabolically labeled with [35S]methionine during interphase
becomes fully phosphorylated and remains stable in prometaphase-arrested
cells. However, upon release of mitotic cells from the nocodazole block, the
level of the 35S-labeled phosphorylated HsPI3K-C2
decreases.
Concomitantly unphosphorylated kinase appears, which is not labeled with
[35S]methionine, and therefore represents de novo
synthesized protein (Fig.
7A, right panel). To confirm that
HsPI3K-C2
is indeed newly synthesized at the M/G1 transition
of the cell cycle, unlabeled mitotic cells were released from nocodazole block
in the presence of [35S]methionine
(Fig. 7B). Progression
into G1 phase resulted in the appearance of the unphosphorylated
[35S]methionine-labeled form of HsPI3K-C2
.
|
To determine whether mitotic destruction HsPI3K-C2 is caused by the
proteasome activity, the proteasome inhibitor MG132
(29) was added to mitotic
cells during the release from nocodazole block.
Fig. 7C shows that
addition of MG132 caused significant stabilization of the phosphorylated form
of the kinase as well as stabilization of cyclin B1, a known target of
proteasome degradation in late mitosis. These results suggest that
M/G1 transition of the cell cycle triggers a proteasome-dependent
degradation of phosphorylated HsPI3K-C2
.
UV-induced Phosphorylation of HsPI3K-C2 at
Ser259 Is Mediated by JNKsCell cycle responses to DNA
damage induced by UV irradiation lead to G1 and G2 phase
delays as a result of the inhibition of cyclin-dependent kinases
(30,
31). In contrast, UV
irradiation causes the activation of MAP kinases, including ERKs, JNKs, and
p38 kinase
(3234).
To identify the possible role of MAP kinases in mediating UV-induced
phosphorylation of HsPI3K-C2
and to facilitate the identification of
the relevant pathway, we examined the influence of specific chemical
inhibitors on the phosphorylation status of HsPI3K-C2
. Pretreatment of
HeLa cells with either SB202190, a specific inhibitor of p38
(35,
36), or PD98059 a specific
inhibitor of MEK1 (37) failed
to abolish UV-induced phosphorylation of HsPI3K-C2
(Fig. 8A). In
contrast, UV-induced phosphorylation of the kinase was markedly attenuated by
pretreatment of cells with SP600125
(38), an inhibitor of JNK
catalytic activity (Fig.
8B). Analysis of UV-irradiated HEK-293 cells stably
expressing HsPI3K-C2
-GFP and HsPI3K-C2
/S259A-GFP
(Fig. 8C) gave similar
results, showing that UV-induced phosphorylation of HsPI3K-C2
-GFP at
Ser259 is sensitive only to SP600125.
|
To examine further whether JNKs are implicated in the phosphorylation of
HsPI3K-C2, we tested whether the transient expression of JNKK2/MKK7, a
specific JNK-activating MAPKK, influences the steady-state level of the
phosphorylation of HsPI3K-C2
. Transfection of JNKK2 into HEK-293 cells
stably expressing HsPI3K-C2
-GFP or HsPI3K-C2
/S259A-GFP resulted
in a significant increase of the steady-state level of phosphorylated
HsPI3K-C2
-GFP, which was close to that seen in UV-irradiated cells. As
expected, the mutant HsPI3K-C2
/S259A-GFP failed to show any change in
the electrophoretic mobility upon UV irradiation
(Fig. 9, upper panel).
The efficiency of JNK activation caused by transfection of JNKK2 was similar
in both cell lines as judged by the increase in c-Jun phosphorylation at
Ser73 (Fig. 9,
lower panel).
|
UV Irradiation Induces Phosphorylation-dependent Degradation of
HsPI3K-C2Our observation that mitotic
phosphorylation of HsPI3K-C2
was followed by its degradation at the M
to G1 transition of the cell cycle led us to speculate that
phosphorylation at Ser259 might be a common signal required to
activate HsPI3K-C2
proteolysis. Therefore, we examined the rate of
disappearance of phosphorylated HsPI3K-C2
after UV irradiation. HEK-293
cells expressing HsPI3K-C2
-GFP or HsPI3K-C2
/S259A-GFP were
exposed to UV light and allowed to recover for 224 h. Whole cell
extracts from equivalent numbers of cells were prepared and analyzed on
Western blots. Fig.
10A illustrates that the slower migrating phosphorylated
forms of HsPI3K-C2
and HsPI3K-C2
-GFP are induced within 2 h
after UV irradiation and diminish thereafter until they become barely
detectable after 24 h (compare recovery time between 2 and 24 h). In contrast,
the levels of faster migrating unphosphorylated forms of these proteins remain
unaffected during the recovery period. Over the time course after UV
irradiation the levels of both HsPI3K-C2
and HsPI3K-C2
-GFP
diminish by about 50% compared with those detected in unirradiated cells.
Phosphorylation-deficient HsPI3K-C2
/S259A-GFP remains stable after UV
irradiation (Fig.
10A), suggesting that mutation of Ser259 to
alanine protects the protein from proteolysis. We used the reversible
inhibitor MG132 to demonstrate that the reduction of the levels of
phosphorylated HsPI3K-C2
and HsPI3K-C2
-GFP is caused by
subsequent degradation by the proteasome. MG132 is a potent inhibitor of the
proteasome but also inhibits calpains. We therefore examined the effects of
additional protease inhibitors on the HsPI3K-C2
turnover. As shown in
Fig. 10B, treatment
of cells with MG132 (29) or
with lactacystin, an irreversible inhibitor specific for proteasome
(39,
40), for 16 h after UV
irradiation resulted in a significant stabilization of the phosphorylated
forms of HsPI3K-C2
and HsPI3K-C2
-GFP. Conversely, ALLM, a
peptide aldehyde that is more selective for calpain than to the proteasome
(41), caused only marginal
effects. Neither of the inhibitors affected stability of
HsPI3K-C2
/S259A-GFP.
|
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DISCUSSION |
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The phosphorylation status of HsPI3K-C2 during the cell cycle is
under the direct control of Cdc2. Several lines of evidence support this
conclusion. First, marginal phosphorylation of HsPI3K-C2
is detected
during interphase, whereas the phosphorylated form of the kinase appears at
the G2/M transition phase and reaches a maximum in mitosis, which
coincides with the timing of Cdc2 activation. Second, mitotic phosphorylation
of HsPI3K-C2
in both in vivo and in vitro assays was
sensitive to roscovitine, a highly selective inhibitor of cyclin-dependent
kinases (27,
28). Several cyclin-dependent
kinases, including Cdc2-cyclin B and Cdk2-cyclin A or E, are sensitive to
roscovitine. However, the main target of roscovitine in mitosis is Cdc2-cyclin
B because Cdk2-cyclin E and Cdk2-cyclin A kinases are active at the
G1/S transition and during S phase, respectively. Furthermore,
purified activated Cdc2/cyclin B was able to phosphorylate HsPI3K-C2
fusion proteins on Ser259 in vitro. Mutations of
Ser259 to either alanine or aspartate abolished Cdc2-mediated
phosphorylation of HsPI3K-C2
in vitro. In addition, Cdc2
activity appears to be indispensable for maintaining phosphorylation of
HsPI3K-C2
during mitosis, probably by antagonizing an okadaic
acid-sensitive phosphatase.
In interphase cells HsPI3K-C2 is concentrated in nuclear speckles
(1), which represent a
subnuclear compartment enriched in small nuclear ribonucleoprotein particles
and other splicing factors. In mitosis, at the end of prophase the break-down
of the nuclear envelope is accompanied by the disassembly and dispersal of all
major nuclear structures and down-regulation of transcription and splicing.
Following the changes in the nuclear structure, at the metaphase-anaphase
transition HsPI3K-C2
is found to disperse throughout the cytoplasm
concomitantly with its complete phosphorylation. A mechanism for mitotic
repression of transcription is phosphorylation, which leads to inactivation of
transcription factors and causes their release from mitotic chromatin
(42,
43). Whether phosphorylation
of HsPI3K-C2
in early mitosis plays a similar role and controls the
enzyme activity in vivo is not clear. The finding that in
vitro lipid kinase activity of mitotic, fully phosphorylated
HsPI3K-C2
is similar to that isolated from interphase cells suggests
that phosphorylation per se does not alter the activity of the
enzyme. In addition, the activity of phosphorylation-deficient mutants (S259A
or S259D) of HsPI3K-C2
is similar to that of the wild-type
enzyme.2 Therefore, it is conceivable that phosphorylation of
HsPI3K-C2
induces modifications in protein-protein or protein-nucleic
acid interactions which affect localization of HsPI3K-C2
and perhaps
prevent the kinase from reaching potential substrates.
Analysis of the protein turnover suggests that mitotic phosphorylation on
Ser259 indirectly controls the activity of HsPI3K-C2 by
facilitating its degradation at the M/G1 transition of cell cycle.
Several examples of regulated proteolysis have been characterized. In many
cases, modification of the substrate by phosphorylation provides a recognition
signal for specific E3 ubiquitin-protein ligases, followed by subsequent
digestion by the proteasome
(44). The ability to inhibit
degradation of the phosphorylated form of HsPI3K-C2
with MG132 supports
the conclusion that degradation takes place in the proteasome. Cell
cycle-regulated proteolysis in anaphase depends on anaphase-promoting complex,
a multisubunit ubiquitin ligase (E3)
(45). Targets of the
anaphase-promoting complex contain destruction boxes necessary for
ubiquitination-mediated proteolysis
(46). Sequences which could
represent putative destruction boxes (RXXL) are found in
HsPI3K-C2
, one of them in close vicinity to the phosphorylation site
Ser259. In addition, a computer-assisted sequence analysis revealed
a putative PEST motif (amino acids 519531). PEST sequences, which are
found in numerous short lived proteins, are assumed to target proteins for
degradation via the proteasome, although the exact mechanism is unclear
(47). We are currently
investigating whether HsPI3K-C2
is a substrate for ubiquitination and
which domains of the protein are important for degradation.
Further studies are necessary to resolve the concise function of
HsPI3K-C2 in cell cycle progression. Our findings suggest that
elimination of the kinase at the exit from mitosis could be necessary to
ensure a proper entry into subsequent G1 phase. This view is
consistent with our observation that overexpression of wild-type
HsPI3K-C2
and its phosphorylation-deficient mutants (S259A and S259D)
in COS-7 and Chinese hamster ovary cells leads to mitotic defects such as
multipolar spindle assembly, which results in aberrant cytokinesis and
formation of multinucleated
cells.2 A similar
effect of defective cytokinesis was observed in cells expressing
constitutively active class I PI 3-kinase (p110CAAX)
(48). Thus, it is tempting to
speculate that down-regulation of PI 3-kinase activity could be an essential
step for execution of the mitotic program.
Cellular responses to UV irradiation include the activation of MAP kinase
signaling pathways. By using selective inhibitors of different MAP kinases, we
demonstrate that UV-induced phosphorylation of HsPI3K-C2 on
Ser259 appears to be dependent on the activation of JNK signaling
pathway and does not involve ERKs and p38. Accordingly, ectopic expression of
the upstream JNKK2/MKK7 increased the steady-state level of HsPIK3-C2
phosphorylated on Ser259, corroborating the involvement of JNKs in
UV irradiation-induced phosphorylation. UV responses meditated by JNK include
transcriptional output in the nucleus
(49,
50) and antiapoptotic
signaling events in the cytoplasm
(51,
52). Prominent changes in the
subnuclear localization of HsPI3K-C2
after UV irradiation and the
observation that only a fraction of HsPI3K-C2
is accessible to
phosphorylation suggest that JNK might target only nuclear
HsPI3K-C2
.
Similar to mitosis, UV-induced phosphorylation serves as a signal for
activation of proteasome-dependent HsPI3K-C2 proteolysis. This
conclusion is supported by the following observations: (i) there is an
apparent preferential disappearance of phosphorylated form of HsPI3K-C2
over time after UV irradiation; (ii) treatment of UV-irradiated cells with
proteasome inhibitors resulted in a significant stabilization of the
phosphorylated form; (iii) the level of phosphorylation-deficient mutant
GFP-HsPI3K-C2
/S259A remained unaffected after UV irradiation.
It is well known that RNA synthesis is down-regulated in response to DNA
damage to allow transcription-coupled repair. Inhibition of transcription
caused by DNA-damaging agents, such as -amanitin, actinomycin D,
cisplatin, and UV irradiation, leads to the degradation of the polymerase II
LS (53,
54). The irreversible
disassembly of transcription complexes as consequence of the degradation of
polymerase II LS has been proposed as a mechanism for down-regulation of
transcription (55). UV
irradiation, similar to
-amanitin and actinomycin D treatment, leads to
morphological changes of HsPI3K-C2
-positive speckles and induces its
phosphorylation, suggesting that these events are linked to transcriptional
repression. The fact that HsPI3K-C2
follows the fate of the polymerase
II LS and is degraded upon cell exposure to UV irradiation could therefore
reflect the disassembly of transcriptional complexes.
In summary, our findings suggest that the phosphorylation of
HsPI3K-C2 on Ser259 is critical for its subnuclear
localization and turnover and that two distinct signaling pathways
phosphorylate Ser259 depending on the physiological state of the
cell. Identification of proteins that associate with of HsPI3K-C2
and
analysis of the regulation of these interactions by phosphorylation are
necessary to determine the physiological role of serine phosphorylation in
HsPI3K-C2
function.
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FOOTNOTES |
---|
To whom correspondence should be addressed. E-mail:
marcus.thelen{at}irb.unisi.ch.
1 The abbreviations used are: PI 3-kinases, phosphoinositide 3-kinases; Cdk,
cyclin-dependent protein kinase; CMV, cytomegalovirus; DMEM, Dulbecco's
modified Eagle's medium; EGFP, enhanced green fluorescent protein; ERK,
extracellular signal regulated kinase; GFP, green fluorescent protein; GST,
glutathione S-transferase; HA, hemagglutinin; HEK, human embryonic
kidney; JNK, c-Jun NH2-terminal kinase; JNKK, JNK kinase;
MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MAP,
mitogen-activated protein; MAPKK, MAP kinase kinase; MEK, mitogen-activated
protein kinase/extracellular signal-regulated kinase kinase; MS/MS, tandem
mass spectrometry; PI3K, PI 3-kinases; PtdIns, phosphatidylinositol; SAPK,
stress-activated protein kinase.
2 S. A. Didichenko and M. Thelen, unpublished data.
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ACKNOWLEDGMENTS |
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REFERENCES |
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