From the Instituto de Investigaciones Citológicas, 46010 Valencia, Spain
Received for publication, August 25, 2000
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ABSTRACT |
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The tumor suppressor phosphatase PTEN regulates
cell migration, growth, and survival by dephosphorylating
phosphatidylinositol second messengers and signaling phosphoproteins.
PTEN possesses a C-terminal noncatalytic regulatory domain that
contains multiple putative phosphorylation sites, which could play an
important role in the control of its biological activity. The protein
kinase CK2 phosphorylated, in a constitutive manner, a cluster of
Ser/Thr residues located at the PTEN C terminus.
PTEN-phosphorylated defective mutants showed decreased stability
in comparison with wild type PTEN and were more rapidly degraded by the
proteasome. Inhibition of PTEN phosphorylation by the CK2 inhibitor
5,6-dichloro-1- The tumor suppressor gene PTEN (also named as
MMAC1 or TEP-1) (1-3) encodes a phosphatase with
enzymatic activity toward both protein substrates and the lipid second
messenger, phosphatidylinositol-3,4,5-triphosphate (4-6). PTEN
regulates distinct signal transduction pathways, including the
phosphatidylinositol 3-kinase/ protein kinase B cell survival-
and integrin-triggered signaling pathways (for recent reviews, see Ref.
7-10). Structurally, PTEN protein is composed of an N-terminal dual
specificity phosphatase-like enzyme domain and a C-terminal regulatory
domain, which binds to phospholipid membranes (11). Mutations in the
PTEN gene are present in a great number of tumors, as well
as in the germ line cells of patients with several inherited cancer
syndromes (reviewed in Refs. 12 and 13). The importance of PTEN
catalytic activity in its tumor suppressor function is underscored by
the fact that the majority of PTEN missense mutations
detected in tumor specimens target the phosphatase domain and cause a
loss in PTEN phosphatase activity. In addition, a large number of
PTEN nonsense or frame-shift mutations found in tumors are
targeted to the C-terminal domain of the protein, suggesting an
important role for this domain in the regulation of the PTEN tumor
suppressor activity. In this regard, the C-terminal region of PTEN has
been shown to be important in the regulation of the stability and
half-life of the molecule (14, 15). Also, the C-terminal PTEN amino
acid sequence possesses a putative PDZ binding motif, which has been proposed to modulate PTEN functions by
association to PDZ domain-containing proteins (16-19). Finally, the
C-terminal PTEN domain is rich in putative phosphorylation sites, and
phosphorylation of the PTEN C terminus has been recently reported to
affect PTEN protein stability and function (20); however, the kinase
responsible for such phosphorylation remains unidentified.
Protein kinase CK21 (formerly casein kinase II) is a highly
conserved, ubiquitously expressed, messenger-independent
serine/threonine-kinase that phosphorylates a wide variety of
substrates involved in essential cell processes, including cell cycle
and cell growth (21-23). In mammals, CK2 is a heterotetramer in
vivo, composed of two catalytic ( In this report, we demonstrate that the C terminus of the tumor
suppressor PTEN is constitutively phosphorylated by CK2 at several
residues in intact cells. An insight is provided on the putative role
of phosphorylation as a regulatory mechanism of PTEN biological
activity by modulating its stability to proteasome-mediated degradation.
Plasmids, Antibodies, and Reagents--
The cDNA encoding
full-length human PTEN was obtained by reverse transcriptase-polymerase
chain reaction amplification of poly(A)+ mRNA from MCF-7 cells
using primers flanking the human PTEN coding region (1-3), and the
PTEN nucleotide sequence was confirmed by DNA sequencing. pRK5
mammalian expression vectors were made by polymerase chain reaction
amplification of PTEN or GST cDNAs and subcloning. For the
construction of bacteria expression plasmids encoding GST-PTEN fusion
proteins, PTEN cDNAs were cloned into the pGEX-4T1 or pGEX-5X1
expression vectors. PTEN C terminus truncation mutants and amino acid
substitution mutants were made by polymerase chain reaction
oligonucleotide site-directed mutagenesis, and mutations were confirmed
by DNA sequencing. The 12CA5 anti-HA and the 4G10 anti-phosphotyrosine
mAb have been described previously (34). The rabbit polyclonal
anti-PTEN CS486 and BT166 antibodies were from R. Parsons and C. Eng,
respectively (35, 36). The anti-GST mAb (clone 22.A) was obtained in
our laboratory and is directed against Schistosoma
japonicum GST. Horseradish peroxidase-conjugated goat anti-mouse
secondary antibody was from Promega (Madison, WI). The following
activating agents were used: phorbol 12-myristate 13-acetate (Sigma
Chemical), 10 ng/ml, 30 min; EGF (Life Technologies, Inc.), 50 ng/ml, 10 min; insulin (Sigma), 1 µg/ml, 10 min; okadaic acid (Roche
Molecular Biochemicals), 1 µM, 30 min; sodium pervanadate (Sigma), 10 µM, 30 min; A23187 calcium ionophore (Roche
Molecular Biochemicals), 10 µM, 30 min. All incubations
were made at 37 °C. Human recombinant CK2 holoenzime was from Roche
Molecular Biochemicals. The following CK2 modulators were used:
spermine (Sigma), 1 mM; heparine (Sigma), 1 µg/µl;
5,6-dichloro-1- Cell Culture, Transfections, and Isotope Cell
Labeling--
Human breast carcinoma MCF-7 cells were grown in RPMI
medium supplemented with 10% heat-inactivated FCS (Life Technologies, Inc.). COS-7 cells were grown in Dulbecco's minimal essential medium
containing high glucose (4.5 g/l) supplemented with 5% heat-inactivated FCS. COS-7 cells were transfected with the
DEAE-dextran method and processed 48-72 h after transfection. For
[35S]methionine labeling, cells were incubated for 1 h in methionine/cysteine-free Dulbecco's minimal essential medium
containing 2% dialyzed FCS and then labeled with
[35S]methionine/cysteine (50 µCi/ml) for 2 h
(pulse). Chases were performed by substituting the
[35S]methionine/cysteine-containing medium for
Dulbecco's minimal essential medium, 2% FCS containing an excess of
cold methionine (1 mM). For 32P labeling, cells
were incubated in phosphate-free medium with 2% FCS for 1 h and
then labeled with [32P]orthophosphate (50 µCi/ml) for
4 h. Cell lysis, immunoprecipitation, and immunoblot were
performed as described (34). For protein turnover measurements, HA-PTEN
radiolabeled bands, resolved by SDS-PAGE after immunoprecipitation with
the anti-HA 12CA5 mAb, were quantitatively analyzed using a
phosphorImager. Results were plotted, and linear fits were performed
(r > 0.97).
GST Fusion Proteins and in Vitro Kinase Assays--
GST-PTEN
fusion proteins overexpressed in bacteria were purified with
glutathione-Sepharose using standard procedures. For in
vitro CK2 kinase assays, GST-PTEN fusion proteins (0.5-1 µg) were incubated with 0.2 milliunits/µl CK2 for 30 min at
30 °C in buffer A (20 mM Tris-HCl, pH 7.5, 0.3 µM ATP, 5 mM MgCl2, 0.5 mM dithiothreitol, 150 mM KCl; 30 µl final
volume) containing 2 µCi of PTEN Is a Phosphoprotein under Standard Cell Growth
Conditions--
To study the putative role of post-translational
modifications in the regulation of PTEN biological functions, the
possibility that PTEN could be phosphorylated in intact cells was
investigated. MCF-7 cells, known to carry a wild type PTEN
gene (1), were labeled with 32P, and cell lysates were
subjected to immunoprecipitation using two distinct anti-PTEN
antibodies (Fig. 1A). As
shown, both anti-PTEN antibodies immunoprecipitated a radiolabeled
protein that migrated at about 55 kDa (Fig. 1A, lanes
2 and 3), as expected for PTEN (36), whereas no signal
was detected using a control serum (Fig. 1A, lane
1). Next, the region of PTEN that is phoshorylated in intact cells
was analyzed using recombinant forms of this molecule. 32P
labeling was carried out in COS-7 cells transfected with HA-tagged PTEN
wild type (residues 1-403) or C-terminal truncated forms (residues
1-343, 1-369, or 1-386), followed by immunoprecipitation with the
anti-HA 12CA5 mAb. As observed for the endogenous PTEN in MCF-7 cells,
the recombinant HA-PTEN-(1-403) was also phosphorylated in
COS-7 cells (Fig. 1B, upper panel, lane 2). A
similar extent of phosphorylation was detected on the truncated
HA-PTEN-(1-386) molecule (Fig. 1B, upper panel, lane 5),
whereas the truncated HA-PTEN-(1-343) and -(1-369) forms were not
phosphorylated under these conditions (Fig. 1B, upper panel,
lanes 3 and 4). The lower panel in
Fig. 1B shows the expression of the distinct HA-PTEN molecules. The difference in the intensity of the endogenous and the
recombinant PTEN-phosphorylated bands is explained by the difference in
the amount of PTEN protein immunoprecipitated in our assays from MCF-7
or transfected COS-7 cells, as indicated by
[35S]methionine labeling experiments (data not shown).
These results indicate that PTEN is a phosphoprotein under normal
conditions of cell growth, suggesting that PTEN phosphorylation takes
place between residues 369 and 386.
To ascertain the identity of the kinase(s) involved in the
phosphorylation of PTEN, 32P-labeled COS-7 cells were
transfected with HA-PTEN and incubated in the presence of stimulators
of several signal transduction pathways, including okadaic acid, sodium
pervanadate, A23187 calcium ionophore, insulin, phorbol 12-myristate
13-acetate, and EGF. As shown, none of these stimuli significantly
modified the phosphorylation level of HA-PTEN (Fig.
2A, lanes 1-7). We
also tested the possibility that PTEN could be phosphorylated on
tyrosine residues. For these experiments, a GST-PTEN fusion protein was overexpressed in COS-7 cells, precipitated in one step using
glutathione-Sepharose, and subjected to immunoblot with the
anti-phosphotyrosine 4G10 mAb (Fig. 2B). No reactivity of
the 4G10 mAb toward GST-PTEN was detected (Fig. 2B, upper panel,
lanes 1 and 2), whereas a strong signal was observed
toward GST-ERK2 (included as a positive control of tyrosine
phosphorylation) upon EGF cell stimulation (Fig. 2B, upper
panel, lane 4). The lower panel in Fig.
2B shows the expression of the GST fusion proteins. The
phosphorylation of GST-PTEN, precipitated from transfected COS-7 cells
labeled with 32P, is also shown as a control (Fig.
2C, lane 2). Together, these results indicate
that the C-terminal region of PTEN is constitutively phosphorylated on
serine and/or threonine residues.
PTEN Is Phosphorylated by CK2 Both in Vitro and in Vivo--
The
results described above suggest that a messenger-independent
serine/threonine kinase may phosphorylate PTEN in intact cells.
Residues 369-386 of the PTEN amino acid sequence include several
consensus phosphorylation sites for protein kinase CK2 (S/TXXD/E/S(P)/T(P)) (residues Ser-370,
Ser-380, Thr-382, Thr-383, and Ser-385; see Fig.
3B), which is known to
phosphorylate in vivo, in a constitutive manner, a wide
array of substrates (23). Thus, the possibility was tested that PTEN
could be phosphorylated by CK2. First, in vitro CK2 kinase
assays were performed, using bacteria-purified GST-PTEN molecules (wild
type or truncated forms) as substrates and purified CK2 as the kinase.
Both, GST-PTEN-(1-403) and GST-PTEN-(202-403) (full-length and
C-terminal domain of PTEN, respectively) were strongly phosphorylated
by CK2 (Fig. 3A, lanes 2 and 4,
respectively), whereas this kinase did not phosphorylate GST alone or
GST-PTEN-(1-202) (N-terminal domain of PTEN) (Fig. 3A,
lanes 1 and 3, respectively). Furthermore, the
C-terminal truncated GST-PTEN-(1-386), but not GST-PTEN-(1-369), was
also phosphorylated by CK2 (Fig. 3A, lanes 6 and
7, respectively). Next, amino acid substitution mutants were
generated that replaced to Ala the CK2 putative phosphorylation sites
within the 369-386 PTEN region, and the mutant GST-PTEN-(1-403)
fusion proteins were also tested for in vitro
phosphorylation by CK2. As shown in Fig. 3B, CK2
phosphorylation of S370A and S385A mutants was greatly reduced,
suggesting that Ser-370 and Ser-385 are major determinants for in
vitro phosphorylation of PTEN by CK2. A partial contribution of
the Ser-380, Thr-382, and Thr-383 PTEN residues to the in
vitro phosphorylation by CK2 was also observed (Fig.
3B). In addition, stoichiometric analysis revealed that
GST-PTEN-(202-403) incorporated about 4 mol of phosphate/mol of
protein after extensive in vitro phosphorylation by CK2.
To test the involvement of CK2 in the phosphorylation of PTEN
observed in intact cells, crude extracts of COS-7 cells were incubated,
in the presence of inhibitors or activators of CK2, with the GST-PTEN
fusion proteins purified from bacteria, and in vitro kinase
assays were performed. In keeping with the results obtained after
in vitro phosphorylation by CK2, GST-PTEN-(1-403) and
GST-PTEN-(202-403) were specifically phosphorylated by the kinase
activity present in the cell extracts, whereas GST-PTEN-(1-202) or GST
alone were not (Fig. 3C, lane 1, and data not
shown). Remarkably, this phosphorylation was substantially increased in
the presence of spermine, an activator of CK2 (Fig. 3C). On
the other hand, in the presence of any of two CK2 inhibitors, DRB or
heparin, the phosphorylation of GST-PTEN was inhibited (Fig.
3C). No effect was observed in the presence of other protein
kinase effectors, such as the MEK1/2 inhibitor PD98059 or the protein
kinase A activator dibutyryl cAMP (data not shown). These
results sustain the notion that the kinase activity that phosphorylates
PTEN in vivo is attributable to CK2. Interestingly,
treatment of cells with DRB resulted in a decrease in both the in
vivo phospholabeling and the protein amount of PTEN, suggesting
that DRB cell treatment could diminish PTEN phosphorylation and/or PTEN
protein content (data not shown; and see Fig. 5C).
PTEN phosphorylation was also analyzed on 32P-labeled COS-7
cells overexpressing the distinct HA-PTEN CK2 phosphorylation mutants (Fig. 4). A significant reduction in the
in vivo PTEN phosphorylation was observed on the S370A and
S385A mutants (Fig. 4, upper panel, lanes 2 and
6, respectively). Also, a consistently reduced labeling was
observed on the S380A mutant (Fig. 4, upper panel,
lane 3), whereas the effect on the phosphorylation of the
individual T382A and T383A mutants was less manifest (Fig. 4,
upper panel, lanes 4 and 5,
respectively). Because these results suggested the involvement of
multiple residues on PTEN phosphorylation in intact cells, combined
mutations were generated and tested for in vivo
phosphorylation. The phosphorylation of the double mutant S370A/S385A
(DMA) was nearly absent (Fig. 4, upper panel,
lanes 8 and 12), and the phosphorylation of the
triple mutant S380A/T382A/T383A (TMA) was markedly decreased (Fig. 4, upper panel, lane 11), indicating an
additive contribution of all these residues to PTEN phosphorylation in
intact cells. The phosphorylation of a double phosphomimetic
S370E/S385E (DME) mutant was also analyzed. As shown, the
S370E/S385E mutant was hyperphosphorylated in comparison with the
S370A/S385A mutant (Fig. 4, upper panel, lane
13), suggesting that phosphorylation of these two PTEN residues
might prime for additional phosphorylation of nearby residues. Finally,
the putative in vivo phosphorylation of the Thr-401 residue,
located within the PDZ binding consensus motif of PTEN, was also
tested. As shown, the phosphorylation level of the T401A mutant was
almost indistinguishable from the wild type PTEN (Fig. 4, upper
panel, lane 7). Together, these results support the
notion that CK2 constitutively phosphorylates in COS-7 cells a cluster
of Ser/Thr residues at the C terminus of PTEN.
Phosphorylation of PTEN C Terminus Is Important for PTEN Protein
Stability to Proteasome-mediated Degradation--
To analyze the
effect of PTEN phosphorylation on the stability of the molecule,
pulse-chase experiments were performed on [35S]methionine-labeled COS-7 cells transfected with
HA-PTEN wild type or phosphorylation mutants. The kinetics of
degradation of HA-PTEN wild type was two to three times slower than
that showed by the distinct Ser/Thr to Ala phosphorylation mutants
(Fig. 5A). Comparative plots
of degradation of wild type PTEN with the S370A, T380A, T382A, T383A,
S385A, and S370A/S385A (DMA) mutants are shown (Fig.
5A). These results suggested that the presence of the
phosphorylated residues could be important for PTEN stability. To test
this possibility, the degradation of the phosphomimetic S370E/S385E
(DME) mutant was analyzed. Interestingly, the stability of
the S370E/S385E mutant was almost identical to that obtained with the
wild type PTEN (Fig. 5A, right panel), reinforcing the hypothesis of a role of phosphorylation of PTEN in protein
stabilization.
To ascertain the degradative pathway of PTEN under these conditions,
transfected COS-7 cells were pulsed with [35S]methionine,
and chases were performed in the presence of the proteasome inhibitor
MG132 or the lysosome inhibitors leupeptin + NH4Cl. As
shown, the proteasome inhibitor MG132 inhibited the degradation of both
PTEN wild type and S370A/S385A mutant (Fig. 5B, lanes
3 and 7), whereas no inhibition was observed in the presence of leupeptin + NH4Cl (Fig. 5B,
lanes 4 and 8). The effect of MG132 on the
degradation of the S370A/S385A mutant was consistently less pronounced
than that observed for the wild type PTEN. Finally, experiments were
carried out to elucidate whether the effect of DRB cell treatment in
the decrease of 32P-radiolabeled PTEN detected in our
studies was, in fact, because of the inhibition of CK2. Cells were left
untreated or were preincubated with the proteasome inhibitor MG132 to
prevent PTEN degradation, were then labeled with 32P or
[35S]methionine in the presence of DRB, and the amount of
radiolabeled PTEN was detected as above. Upon DRB cell treatment, both
the 32P phospholabeling and the amount of
[35S]methionine-labeled PTEN were decreased (Fig.
5C, lane 2). Remarkably, in the presence
of DRB plus MG132 the phosphorylation of PTEN was decreased without
affecting the amount of PTEN protein detected by
[35S]methionine labeling (Fig. 5C, lane
3). Together, these results indicate that defective PTEN
phosphorylation by CK2 accelerates the proteasome-mediated degradation
of PTEN.
On the basis that many CK2 substrates are oncogene products or
tumor suppressor proteins as well as key signal transduction proteins,
a role for CK2 in the regulation of cell growth- and cell cycle-related
processes has been proposed (22, 23). In this report, we provide
evidence that the tumor suppressor phosphatase PTEN is phosphorylated
by CK2 both in vitro and in intact cells. CK2
phosphorylation sites in PTEN were located within a C-terminal cluster
of Ser/Thr residues, which were found to be important for PTEN
stability to proteasome-mediated degradation. The CK2 phosphorylation
sites were identified as the PTEN residues Ser-370, Ser-380, Thr-382,
Thr-383, and Ser-385. Because some of these sites can be generated
after previous phosphorylation of nearby residues, and because the
phosphomimetic S370E/S385E mutant is phosphorylated more efficiently
than the S370A/S385A mutant, hierarchic CK2 multisite phosphorylation
of this region of PTEN is likely to exist (37). Several findings
support the notion that CK2 is the major kinase involved in the
phosphorylation of PTEN in intact cells. First, cell treatment with a
panel of different stimuli did not substantially affect the
phosphorylation of PTEN, suggesting that such phosphorylation is
constitutive and messenger-independent, as is known for most
CK2-mediated phosphorylations. Second, tyrosine phosphorylation was not
detected on PTEN, suggesting that tyrosine kinases do not directly
phosphorylate PTEN. Third, phosphorylation of PTEN by the kinase
activity present in COS-7 cell extracts was activated or inhibited by
CK2 activators or inhibitors, respectively. Fourth, phosphorylation of
PTEN in intact COS-7 cells was inhibited by cell treatment with the CK2
inhibitor DRB. Fifth, mutation to Ala of the PTEN CK2-phosphorylation
sites, as well as deletion of the C-terminal portion of PTEN including
these sites, resulted in an almost complete lack of PTEN
phosphorylation in intact cells. However, the possibility cannot be
ruled out that other kinase(s) not monitored in our assays, using
overexpressed HA-PTEN in COS-7 cells, may contribute to the
phosphorylation of endogenous PTEN in vivo. Remarkably, the
CK2 phosphorylation sites in PTEN are conserved in species from mammals
to Xenopus laevis, and clusters of putative CK2
phosphorylation sites are also present at the C terminus of PTEN from
Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae.
We found that PTEN suffers a rapid degradation in COS-7 cells, which
was inhibited by the proteasome inhibitor MG132 but not by lysosome
inhibitors, indicating that the turnover of PTEN in cultured COS-7
cells depends mainly on proteasome-mediated degradation. The
involvement of the proteasome in the degradation and regulation of the
functions of short-lived proteins, including oncoproteins, tumor
suppressors, and cell cycle proteins has been described extensively
(for reviews, see Refs. 38-40). Thus, regulated degradation of PTEN
via the proteasome could be envisaged as a major physiological mechanism that controls the amount of PTEN in specific cell types and
tissues. In this context, it has been reported recently that C-terminal
truncation mutants of PTEN have shorter half-lives than the wild type
molecule (14, 15). In addition, we show that Ser/Thr to Ala amino acid
mutations at the PTEN C terminus, leading to defective phosphorylation
by CK2, accelerate the proteasome-mediated degradation of PTEN.
However, a double Ser to Glu mutation (the S370E/S385E mutant), that
mimicked constitutive CK2 phosphorylation of PTEN at these sites showed
a degradation rate comparable with the wild type molecule. Furthermore,
cell treatment with the CK2 inhibitor DRB, which inhibited PTEN
constitutive phosphorylation, resulted in a diminution in PTEN protein
content but only in the absence of the proteasome inhibitor MG132.
While this manuscript was in preparation, Vazquez et al.
(20) also showed that phosphorylation of some of the PTEN C-terminal
residues analyzed in our study regulate protein stability as well as
PTEN-mediated G1 cell cycle arrest. Thus, constitutive phosphorylation
of PTEN by CK2 may play a key role in the regulation of PTEN biological
functions upon normal cell growth conditions.
Our findings suggest that phosphorylation of the PTEN C terminus itself
does not provide enough PTEN protein stability (see below) but rather
could be important to acquire the proper, stable conformation of the
PTEN C-terminal tail. For instance, it is well documented that
phosphorylation of the C terminus of I-D-ribofuranosyl-benzimidazole also diminished the PTEN protein content. Our results support the
notion that proper phosphorylation of PTEN by CK2 is important for PTEN
protein stability to proteasome-mediated degradation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and/or
') and two
regulatory (
) subunits. Alterations in CK2 expression have been
found in distinct types of tumors (24-26), and overexpression of the
catalytic or regulatory CK2 subunits differentially affects cell growth
and transformation (27-31). Also, antibody-mediated CK2 depletion
inhibited cell cycle progression and growth of fibroblasts (32), and
CK2 antisense oligonucleotide treatment blocked neurite outgrowth in
neuroblastoma cells (33).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-D-ribofuranosyl-benzimidazole (DRB)
(BIOMOL Research Laboratories Inc., Plymouth Meeting, PA), 100 µM. The MEK1/2 inhibitor PD98059 (New England Biolabs
Inc., Beverly, MA) was used at 10 µM, and dibutyryl cAMP
(Roche Molecular Biochemicals) was used at 50 µM. The
proteasome inhibitor MG132 (Peptide Institute Inc., Osaka, Japan) was
used at 50 µM, leupeptin (Peptide Institute Inc.) was
used at 0.1 mM, and NH4Cl was used at 20 mM. For the experiments of inhibition of degradation,
chases were performed in the continuous presence of the inhibitors.
-[32P]ATP/sample. For
kinase assays using COS-7 cell extracts, cells were lysed in lysis
buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 100 mM NaF, 2 mM
Na3VO4, and 20 mM
Na4P207). Lysates were centrifuged for 10 min at 14,000 r.p.m., and supernatant aliquots (5 µg) were left untreated or incubated with the distinct agents for 5 min at
30 °C in buffer B (20 mM HEPES, pH 7.5, 50 µM ATP, 10 mM MgCl2, 1 mM dithiothreitol, 2 mM
Na3VO4; 20 µl/sample). Then, 20 µl of buffer B containing the GST fusion proteins used as substrates (1 µg/sample) and [
-32P]ATP (0.25 µCi/µl) were
added, and samples were incubated further for 30 min at 30 °C. The
reactions were stopped by adding SDS-PAGE sample buffer and boiling
followed by SDS-PAGE and autoradiography, and radiolabeled bands were
quantified in a PhosphorImager. For quantitative analysis of phosphate
incorporation into GST-PTEN 202-403, 1 µg of the fusion protein was
phosphorylated by CK2 (0.2 milliunits/µl) for 16 h at 30 °C
in buffer B containing 0.5 mM [
-32P]ATP
(70 cpm/pmol) and 1 mM spermine (40 µl final volume). Gel segments containing the labeled bands were dissolved in 0.2 ml of 30%
H202 for 3 h at 95 °C, diluted in
scintillation liquid, and counted.
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Fig. 1.
PTEN is a phosphoprotein under normal cell
growth conditions. A, MCF-7 cells were labeled with
32P, and cell lysates were immunoprecipitated with a
control antibody (lane 1) or anti-PTEN BT166 or CS486 sera
(lanes 2 and 3, respectively). The
arrowhead indicates the migration of phosphorylated PTEN.
B, COS-7 cells were mock-transfected (pRK5 vector alone)
(lane 1) or transfected with pRK5 HA-PTEN-(1-403)
(lane 2), pRK5 HA-PTEN-(1-343) (lane 3), pRK5
HA-PTEN-(1-369) (lane 4), or pRK5 HA-PTEN-(1-386)
(lane 5). In the upper panel, cells were labeled
with 32P, and cell lysates were immunoprecipitated with the
anti-HA 12CA5 mAb. In the lower panel, 10 µg of total cell
lysates were loaded and subjected to immunoblot with the anti-HA 12CA5
mAb. All samples were resolved by 10% SDS-PAGE under reducing
conditions followed by autoradiography (A and B,
upper panel) or immunoblot (B, lower
panel).
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Fig. 2.
Constitutive phosphorylation of PTEN in
intact cells. A, COS-7 cells were transfected with pRK5
HA-PTEN-(1-403) and labeled with 32P. Cells were left
untreated (lane 1) or were treated for 30 min (okadaic acid,
sodium pervanadate, calcium ionophore A23187, and phorbol 12-myristate
13-acetate) or 10 min (insulin and EGF) with the distinct stimuli, as
indicated (lanes 2-7), and cell lysates were
immunoprecipitated with the anti-HA 12CA5 mAb. B, COS-7
cells were transfected with pRK5 GST-PTEN-(1-403) (lanes 1 and 2) or pRK5 GST-ERK2 (lanes 3 and
4). Cells were left untreated (lanes 1 and
3) or were treated with EGF (50 ng/ml, 10 min) (lanes
2 and 4), and cell lysates were precipitated with
glutathione-Sepharose followed by immunoblot analysis with the
anti-phosphotyrosine 4G10 (upper panel) or an anti-GST mAb
(lower panel). C.,COS-7 cells were transfected
with pRK5 GST (lanes 1 and 3) or pRK5
GST-PTEN-(1-403) (lanes 2 and 4). In the
left panel, cells were labeled with 32P, and
cell lysates were precipitated with glutathione-Sepharose. In the
right panel, 10 µg of total cell lysates were loaded and
subjected to immunoblot with an anti-GST mAb. All samples were resolved
by 10% SDS-PAGE under reducing conditions followed by autoradiography
(A and C, left panel) or immunoblot
(B and C, right panel).
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Fig. 3.
CK2 phosphorylates the C terminus of
PTEN. A, recombinant CK2 holoenzime was mixed with
purified GST (lane 1), GST-PTEN-(1-403) (lanes 2 and 5), or truncated GST-PTEN-(1-202) (lane 3), -(202-403)
(lane 4), -(1-386) (lane 6), or -(1-369)
(lane 7) fusion proteins (1 µg), and in vitro
kinase assays were carried out in the presence of
[ -32P]ATP. Samples were resolved by 10% SDS-PAGE
followed by autoradiography. B, recombinant CK2 holoenzyme
was mixed with purified GST-PTEN wild type or mutant fusion proteins
(0.5 µg), as indicated, and kinase assays were performed as described
in A. Data are presented as the incorporated radioactivity
with respect to GST-PTEN wild type. Values represent the mean ± S.D. of two separate experiments. The top of the figure
shows the PTEN amino acid sequence (residues 369-388) containing the
CK2 phosphorylation sites (shown in uppercase letters). The
distinct CK2 phosphorylation consensus motifs are
underlined. Amino acids are indicated using the
single-letter code. C, left panel,
crude extracts from COS-7 cell lysates (5 µg) were left untreated
(lane 1) or were preincubated separately with 1 mM spermine (lane 2), 100 µM DRB
(lane 3), or 1 µg/µl heparin (lane 4) in
buffer B and were then mixed with purified GST-PTEN-(202-403) (1 µg)
in the presence of [
-32P]ATP and subjected to in
vitro kinase assay (upper panel). The lower
panel shows the identical amount of GST-PTEN-(202-403) substrate
after all incubations, as detected by Coomassie blue staining. The
right panel shows the quantification of GST-PTEN-(202-403)
phospholabeling obtained after phosphorylation in the presence of the
distinct CK2 modulators. Data are presented as the incorporated
radioactivity with respect to control conditions. Values represent the
mean ± S.D. of two separate experiments. For quantification,
radiolabeled substrate bands were excised from the gel and counted in a
scintillation counter or were analyzed using a PhosphorImager.
View larger version (28K):
[in a new window]
Fig. 4.
Identification of the PTEN-phosphorylated
residues in intact cells. COS-7 cells were mock-transfected
(lane 9) or transfected with pRK5 HA-PTEN wild type
(lanes 1 and 10) or mutants (lanes
2-8 and 11-13), as indicated. DMA, double
mutant S370A/S385A; DME, double mutant S370E/S385E;
TMA, triple mutant S380A/T382A/T383A. In the upper
panel, cells were labeled with 32P, and cell lysates
were immunoprecipitated with the anti-HA 12CA5 mAb. In the lower
panel, 10 µg of total cell lysates were loaded and subjected to
immunoblot with the anti-HA 12CA5 mAb. Samples were resolved by 10%
SDS-PAGE under reducing conditions followed by autoradiography
(upper panel) or immunoblot with the anti-HA 12CA5 mAb
(lower panel).
View larger version (33K):
[in a new window]
Fig. 5.
Stability of PTEN wild type and
phosphorylation mutants. A, COS-7 cells were
transfected with pRK5 HA-PTEN wild type or mutants, as indicated. Cells
were pulsed for 2 h in the presence of
[35S]methionine and then chased at different times, as
indicated. Cell lysates were immunoprecipitated with the anti-HA 12CA5
mAb, and radiolabeled bands were resolved by 10% SDS-PAGE under
reducing conditions and analyzed using a PhosphorImager. Kinetics
degradation plots of PTEN wild type, single Ser/Thr to Ala mutants, and
double S370A/S385A (DMA) and S370E/S385E (DME)
mutants are shown. To facilitate comparison, results were grouped in
sets of mutants; representative experiments are shown. Experiments were
performed at least twice, and the difference between independent
experiments was always less than 10%. B, COS-7 cells were
transfected with pRK5 HA-PTEN wild type (lanes 1-4) or
S370A/S385A (DMA) mutant (lanes 5-8). Cells were
pulsed for 2 h in the presence of [35S]methionine;
then, cells were lysed (lanes 1 and 5) or were
chased for 4 h in the absence (lanes 2 and
6) or presence (lanes 3, 4, 7, 8) of
inhibitors, as indicated, followed by cell lysis. Cell lysates were
immunoprecipitated with the anti-HA 12CA5 mAb, and samples were
analyzed by 10% SDS-PAGE under reducing conditions. C,
COS-7 cells were transfected with pRK5 HA-PTEN wild type and labeled
with 32P (upper panel) or
[35S]methionine (lower panel) for 4 h in
the absence (lane 1) or in the continuous presence of DRB
(lane 2) or DRB plus MG132 (lane 3). Cells were
lysed and processed as described in B.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B
by CK2 affects the
stability and constitutive turnover of this NF-
B regulator (41-44).
In addition, the possibility exists that phosphorylation of the C
terminus of PTEN could modulate its subcellular location and function
by affecting its association with regulatory and/or stabilizing
molecules. In this regard, deletion of the C-terminal PDZ binding motif
of PTEN results in the impairment of some PTEN cellular functions (16).
Also, PTEN has been found to associate, through its PDZ binding motif,
with PDZ domain-containing proteins, including hMAST205 and MAGUK
scaffolding proteins (17-19). Interestingly, MAGUKs might regulate the
functions of PTEN through the assembly and stabilization of
multiprotein signaling complexes at specific subcellular compartments,
as known for other related PDZ-containing proteins (45). It has been
suggested that phosphorylation of the Thr-401 residue within the PDZ
binding motif of PTEN could regulate its binding to PDZ-containing
proteins (17). However, our results suggest that the PTEN Thr-401
residue is not phosphorylated in vivo. Mutations that did
not abrogate PTEN phosphorylation, but are predicted to disrupt the
association with PDZ-containing proteins (PTEN-(1-386) and
T401A mutant), also diminished the stability of PTEN (data not shown),
favoring the hypothesis of a putative role for phosphorylation by CK2
in PTEN stabilization through protein association. Frame-shift
mutations at the end of the PTEN coding region have been
found in tumors, indicating that such mutations could produce
C-terminal truncated PTEN molecules with altered tumor suppressor
function (13). Furthermore, by using anti-PTEN antibodies, variable
expression levels of PTEN protein are detected in tumor specimens as
compared with normal tissues (46-49), suggesting that mechanisms other
than loss of the PTEN gene may account for alterations on
PTEN protein expression in some tumors. The results presented here
provide evidence that the CK2 phosphorylation sites contained within
the C terminus of PTEN play an important role in its stabilization and
turnover, in a process mediated by the proteasome. Our findings support a model in which changes in the phosphorylation status of the C
terminus of PTEN might alter the basal expression levels of this tumor
suppressor. In this regard, we have not detected dephosphorylation of
PTEN after incubation with crude cell extracts, and PTEN
phosphorylation status did not change upon cell incubation with the
PP2A inhibitor, okadaic acid (Fig.
2).2 The possibility exists that dephosphorylation of PTEN
takes place only under particular conditions of activation of an
unidentified phosphatase and/or under specific subcellular location of
PTEN. Further work will be necessary to ascertain whether regulated dephosphorylation events could affect PTEN phosphorylation and function
in vivo.
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ACKNOWLEDGEMENTS |
---|
We thank C. Eng and R. Parsons for providing antibodies, E. Knecht for helpful discussions and critical reading of the manuscript, G. Fuertes for help with the degradation experiments, and I. Roglá for expert technical assistance.
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FOOTNOTES |
---|
* This work was supported by grants from the Ministerio de Educación y Cultura (PB96-0278) and the Generalitat Valenciana (GV-C-VS-20-047-96).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.
Supported by a fellowship from Generalitat Valenciana.
§ To whom correspondence should be addressed: Instituto de Investigaciones Citológicas, c/Amadeo de Saboya 4, 46010, Valencia, Spain. Tel.: 96-3391256; Fax: 96-3601453; E-mail: rpulido@ochoa.fib.es.
Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M009134200
2 J. Torres and R. Pulido, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
CK2, protein
kinase CK2 or casein kinase II;
DRB, 5,6-dichloro-1--D-ribofuranosyl-benzimidazole;
GST, glutathione S-transferase;
HA, hemagglutinin;
mAb, monoclonal antibody;
EGF, epidermal growth factor;
FCS, fetal calf
serum;
PAGE, polyacrylamide gel electrophoresis.
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