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Programs of
* Signal Transduction and
Oncogenes and Tumor Suppressors, Burnham Institute, La Jolla, CA 92037
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Most cultured T cell lines, including the commonly used model for T cell Ag receptor signaling, the Jurkat T leukemia cell line, lack PTEN protein due to frame-shift mutations in both PTEN alleles (13). Mutations in PTEN are also frequently found in leukemias and lymphomas freshly isolated from patients (14, 15, 16) and likely contribute greatly to the initial malignant transformation and/or tumor progression and survival of the leukemic cells. In Jurkat cells the lack of PTEN manifests itself as a constitutively elevated level of D3-phosphoinositides and increased activity of enzymes that depend on these lipids, such as the Itk tyrosine kinase (17). Reconstitution of PTEN expression reduced cell survival by inducing apoptosis (6). This effect was prevented by a constitutively active form of protein kinase B (PKB),3 one of the best-characterized effectors for phosphatidylinositol 3-kinase (18, 19). Expression of PTEN also reduced the TCR-induced activation of the mitogen-activated kinase Erk 2 (6, 17), as did inhibition of phosphatidylinositol 3-kinase (20).
Because Ag-induced cell death is an important part of the down-regulation of an immune response, regulation of PTEN expression or function would be expected to be important for the immune system. Reduced PTEN function may lead to prolonged or exaggerated immune responses and autoimmunity, as suggested by the lymphoproliferative disorder induced by increased phosphatidylinositol 3-kinase activity (21). Therefore, we set out to study the regulation of PTEN in T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The anti-influenza hemagglutinin tag epitope mAb 12CA5 was from Boehringer Mannheim (Indianapolis, IN); 12CA5 conjugated to rhodamine isothiocyanate was from Roche Molecular Biochemicals (Indianapolis, IN); and anti-PKB was from New England Biolabs (Beverly, MA). Anti-phosphoSer380 was from Cell Signaling Technology (Beverly, MA), and anti-PKB-phosphoSer473 was from Upstate Biotechnology (Lake Placid, NY). The cDNAs for PTEN and the catalytically inactive PTEN-C124G were in the pEF vector with a C-terminal hemagglutinin epitope tag (6). The S380A, S385A, and double S380A/S385A (SA/SA) mutants were generated using the QuickChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) and were verified by sequencing. The plasmid encoding a fusion protein between green fluorescent protein (GFP) and the pleckstrin homology (PH) domain of the Bruton’s tyrosine kinase (Btk) was a kind gift from L. C. Cantley (Harvard Medical School, Boston, MA). myrPKB was a kind gift from T. Franke (Columbia University, New York, NY).
Cells
Peripheral blood lymphocytes were isolated from buffy coats from healthy volunteers by gradient centrifugation. The cells were cultured in RPMI medium with 10% heat-inactivated FCS, 10 µg/ml phytohemagglutinin, and 100 U/ml IL-2 for 72 h before use. Jurkat cells were kept at logarithmic growth in RPMI supplemented with 10% heat-inactivated FCS, L-glutamine, and antibiotics.
Transfections, labeling, and peptide mapping
Transient transfections were conducted by electroporation as before (6, 22, 23). A total of 20 x 106 transfected cells were labeled with 4 mCi/ml 32Pi in phosphate-free RPMI medium for 4 h as before (22, 23) or with 0.5–2 mCi/ml [35S]Met/Cys in methionine-free RPMI medium for 4 h. Cell lysis, immunoprecipitation, and immunoblotting were conducted as earlier (6, 22, 23).
Tryptic peptide mapping was performed as before (22, 23) with the protocol of Luo et al. (24). Phosphoamino acid analysis was performed by complete acid hydrolysis in 1 M HCl at 110°C and separation in two dimensions in the presence of unlabeled standards.
For cycloheximide treatments, cells were washed 24 h after transfection and resuspended in RPMI, and cycloheximide was added to 50 µg/ml. After incubation at 37°C for the indicated brief times, the cells were harvested, lysed, and analyzed by SDS-PAGE and immunoblotting.
Confocal microscopy
Double immunofluorescence staining was done as before (8). Briefly, cells were washed in PBS and fixed in freshly made 3.7% formaldehyde. Fixed cells were permeabilized with 0.05% saponin, 0.5% BSA in PBS for 10 min at room temperature, and then incubated with primary and secondary Ab diluted in the same buffer for 1 h each at room temperature. After three washes with PBS, the cells were mounted onto glass slides and viewed under a confocal laser scanning microscopy MRC-1024 (Bio-Rad, Hercules, CA). A Nomarski differential interference contrast image was also taken of the same cells.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether PTEN is a phosphoprotein, Jurkat cells were
transfected with catalytically inactive PTEN-C124G, metabolically
labeled with 32Pi
and PTEN immunoprecipitated with the anti-hemagglutinin mAb.
Autoradiography showed that PTEN was heavily labeled with
32P (Fig. 1
A). As a control, another
phosphatase (Lyp1) was expressed and immunoprecipitated with the
same mAb in parallel radiolabeled cell samples but did not yield the
55- to 60-kDa band seen in the PTEN-transfected cells (Fig. 1B). A brief treatment of the cells with phorbol ester had
no effect on PTEN or Lyp1 phosphorylation.
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, C and D). There
was also a trace of phosphothreonine (<1%) but no phosphotyrosine,
even on much longer exposures. Treatment of the cells with phorbol
ester had no significant effect on serine vs threonine labeling.
The bands shown in Fig. 1A were also digested with trypsin.
The resulting peptides were separated in two dimensions on thin layer
plates and exposed to film. Interestingly, there was only one peptide
with a tail toward the origin and with very little migration in the
second dimension (Fig. 1, E and F). Thus, PTEN
contains nearly all of its phosphate within what appears to be a single
peptide.
Tryptic peptide mapping does not per se allow a phosphorylated residue
to be identified, but the properties and behavior of a peptide can give
valuable hints. First, we scraped out the peptide and subjected it to
phosphoamino acid analysis, which showed that the spot contained mostly
phosphoserine, but also a trace of phosphothreonine (Fig. 1G). This phosphorylated peptide is also unusual in that it
remained very low on the plate in the second dimension. Because
migration in the organic solvent mix used in the ascending
chromatography is directly proportional to the hydrophobicity of a
peptide, the lack of migration suggests that the peptide is remarkably
hydrophilic. Of the tryptic peptides of PTEN, fragment 379–402 is
extraordinarily hydrophilic, with 19 hydrophilic amino acid residues of
24. This peptide contains two serine and several threonine residues
(Fig. 2A), a property shared
with only five other peptides, which are all 40–60% hydrophobic and
are expected to migrate quite high on the plate. Thus, it is very
likely that the major phosphorylated site is one or both serine
residues in this most C-terminal fragment of PTEN and that the minor
threonine site is in the same peptide. Because phosphorylation reduces
the migration of a peptide in the first dimension, the left-pointing
tail of the spot may represent the same peptide with two or three
phosphates.
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To identify the phosphorylated serine residue(s), we mutated
Ser380, Ser385, or both to
alanines and repeated the metabolic
32Pi labeling experiments
with the mutant proteins and the double mutant PTEN-SA/SA (Fig. 2B). Tryptic peptide mapping of the mutant proteins showed
that both PTEN-S380A (Fig. 2D) and PTEN-S385A (Fig. 2E) had a reduced labeling of the peptide, particularly the
former. In the S380A mutant, the peptide migrated less in the first
dimension but more in the second, as would be expected of a more
hydrophobic peptide (Ala is more hydrophobic than PSer). The spot in
the map of PTEN-S385A also had a reduced migration. The double mutant
did not give rise to any detectable peptides at all (Fig. 2F). These results indicate that both serines can be
phosphorylated in vivo, with Ser380 apparently
being phosphorylated to a higher stoichiometry than
Ser385.
PTEN is phosphorylated at Ser380 in normal human T lymphocytes
An Ab specific for phospho-Ser380 of PTEN
has recently become commercially available. We first tested its
specificity by expressing PTEN-C124G or its serine mutants in Jurkat T
cells, followed by immunoblotting with the
anti-phospho-Ser380 Ab. As shown in Fig. 3A, the Ab reacted very
strongly with PTEN-C124G and with the S385A mutant, but not with the
S380A or double SA/SA mutants. Thus, the phospho-specific Ab does have
the appropriate specificity and does not cross-react with
phosphorylated Ser385.
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B,
lane 1), precluding any analysis of in vivo phosphorylation.
However, upon polyclonal activation for 72 h PTEN expression
increased severalfold (Fig. 3B, lane 2) and also
became reactive with the anti-phospho-Ser380
Ab (Fig. 3B, lane 3). This reactivity was
increased somewhat upon anti-CD3 plus anti-CD28 stimulation of
the cells (Fig. 3B, lane 4) and was essentially
extinguished by treatment of the cells with 50 nM wortmannin for 15 min
(Fig. 3B, lane 5). Phosphorylation of PTEN in response to D3-phosphoinositide levels
To determine whether the phosphorylation of PTEN is regulated, we
first compared the phosphorylation of PTEN-C124G with that of wild-type
PTEN and repeatedly found that the latter was less phosphorylated even
at time points well before any signs of cell death could be detected.
This suggested to us that the activity of PTEN may be involved and that
the resulting levels of cellular D3-phosphorylated inositol
phospholipids may affect the phosphorylation of PTEN. To test this, we
transfected Jurkat cells with either active or C124G-mutated PTEN and
then either treated the cells with the phosphatidylinositol 3-kinase
inhibitor wortmannin or coexpressed the constitutively active myrPKB.
After metabolic 32Pi
labeling of the cells, the phosphorylation of PTEN was analyzed (Fig. 4A, upper panel).
These experiments showed that active PTEN was phosphorylated much less
than the catalytically inactive PTEN-C124G and that wortmannin reduced
the phosphorylation of active PTEN even a bit further. In contrast,
myrPKB increased the phosphorylation of active PTEN to the level of
inactive PTEN, which was only slightly reduced by wortmannin and
increased by myrPKB. Similar results were obtained with the
anti-phospho-Ser380 Ab (Fig. 4B),
except that the level of phosphorylation was more even in the samples
with high D3-phosphoinositides and PKB activity (Fig. 4B,
lanes 4–7). The relatively small differences between the
autoradiogram (Fig. 4A, upper panel) and the
anti-phospho-Ser380 immunoblot (Fig. 4B) may be due to phosphorylation at
Ser385, which would contribute to the signal only
in the autoradiogram. Together, these results demonstrate that
Ser380 and Ser385 are
involved in the observed changes in phosphorylation in response to
D3-phosphoinositides and PKB.
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A, lower
panel) also revealed that the PTEN protein ran as two molecular
mass forms, 
55 and 59 kDa. Of these two bands, only the upper
comigrated exactly with the 32P-labeled band in
the autoradiogram and its intensity correlated very well with the
amount of radioactivity. Thus, serine phosphorylation apparently causes
a shift in the apparent molecular mass of PTEN on SDS gels. As an
additional control, we immunoblotted the same lysates with Abs against
phosphorylated PKB, which reflects the levels of PKB activity in the
cells. The strength of the phospho-PKB band (Fig. 4C)
correlated with the phosphorylation or PTEN, particularly with the
phospho-Ser380 blot, and with the presence of the
upper PTEN band (Fig. 4A, lower panel).
To verify that D3-phosphoinositide in the intact cells levels changed
as predicted, we expressed a fusion protein consisting of GFP plus the
PH domain of the Btk kinase, GFP-Btk-PH, in the cells and visualized
the fluorescent protein by confocal microscopy. The PH domain of Btk is
highly specific for phosphatidylinositol-3,4,5-trisphosphate
(25). As shown in Fig. 4D, the fluorescence was
mostly at the plasma membrane in the absence of any treatments,
indicating that phosphatidylinositol-3,4,5-trisphosphate was present in
the plasma membrane. Upon treatment of the cells with 50 nM wortmannin
for 15 min, the membrane localization of the GFP-Btk-PH protein was
lost and it became diffusely distributed throughout the cells (Fig. 4D, lower panels). Similarly, in cells expressing
active PTEN, the Btk-PH domain was diffusely cytoplasmic (Fig. 4E), indicating that the level of D3-phosphoinositides in
the plasma membrane was low. In contrast, the inactive PTEN-C124G did
not cause loss of these lipids. Instead, much of PTEN-C124G colocalized
with the GFP-Btk-PH domain at the plasma membrane (Fig. 4F).
Fig. 4, E and F, also shows that active and
inactive PTEN both have the same intracellular, plasma
membrane-enriched localization.
We conclude that Jurkat cells possess a mechanism for phosphorylation of PTEN, which is very sensitive to the cellular levels of D3-phosphoinositides. The simplest model supported by our data would suggest that the kinase that phosphorylates PTEN is PKB or a kinase activated by PKB.
The half-life of PTEN is regulated by D3-phosphoinositides and phosphorylation
The identification of a major in vivo phosphorylation site to the last 50 amino acids of PTEN suggested to us that this phosphorylation may influence the turnover of the PTEN protein because this region is known to control the degradation of PTEN (26).
First, we tested whether treatments that alter the levels of
D3-phosphorylated inositol phospholipids also affect the half-life of
PTEN. Jurkat cells expressing PTEN-C124G were metabolically labeled
with [35S]methionine for 4 h, washed, and
kept at 37°C in medium with unlabeled methionine for various times
before PTEN was immunoprecipitated with the anti-hemagglutinin mAb.
The labeled protein was resolved by SDS PAGE, transferred to
nitrocellulose, and exposed to film (Fig. 5). The band corresponding to PTEN was
also excised and its radioactivity was quantitated in a beta counter.
The half-life of PTEN-C124G was determined to be 2 h but decreased
to 45 min upon addition of 50 nM wortmannin to the cells.
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1 h), while the
wild-type had a t1/2 of 2 h, the
S385A mutant had a t1/2 of 1.5 h,
and the double mutant had a t1/2 of
45 min (Fig. 5C). Thus, phosphorylation of
Ser380 and Ser385 appears
to be required for protection of PTEN from degradation. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Apparently, the constitutively elevated D3-phosphoinositide levels in
Jurkat (Fig. 4D) result in activation of the kinase that
normally phosphorylates PTEN on Ser380 and
Ser385. Thus, an introduced PTEN-C124G becomes
highly phosphorylated. In contrast, active PTEN lowers
D3-phosphoinositide levels and thereby inactivates the kinase, which
could be PKB or a kinase regulated by it. Jurkat cells also express a
much higher level of PTEN mRNA than normal T cells,
presumably also in an attempt to synthesize the missing PTEN protein.
In agreement with this notion, Jurkat cells have a high basal level of
PTEN transcription, as measured with a luciferase reporter
gene driven by 2 kb of 5' genomic PTEN sequence (D. Birle
and T. Mustelin, unpublished observation).
Our model supports the notion that PTEN plays a key role in maintaining the normal low level of D3-phosphoinositides in T cells. Jurkat T cells, which lack PTEN protein (17), have elevated levels of D3-phosphoinositides, which likely contribute to the long-term survival of this cell line. It is not known whether PTEN was missing in the initial passages of Jurkat, which was established in 1975 from a 14-year-old boy with an acute T lymphoblastoid leukemia (30), but this appears very likely because PTEN mutations are frequent in lymphomas and leukemias (14, 15, 16). If so, loss of PTEN may have been a key step in tumorigenesis in the patient.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Tomas Mustelin, Laboratory of Signal Transduction, Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037. E-mail address: tmustelin{at}burnham-inst.org
3 Abbreviations used in this paper: PKB, protein kinase B; GFP, green fluorescent protein; PH, pleckstrin homology; Btk, Bruton’s tyrosine kinase.
Received for publication March 22, 2002. Accepted for publication April 24, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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