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The Tumor Suppressor PTEN Regulates T Cell Survival and Antigen Receptor Signaling by Acting as a Phosphatidylinositol 3-Phosphatase¹

The Burnham Institute, La Jolla Cancer Research Center, La Jolla, CA 92037

	Abstract

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The tumor suppressor gene PTEN encodes a 55-kDa enzyme that hydrolyzes both protein phosphotyrosyl and 3-phosphorylated inositol phospholipids in vitro. We have found that the latter activity is physiologically relevant in intact T cells. Expression of active PTEN lead to a 50% loss of transfected cells due to increased apoptosis, which was completely prevented by coexpression of a constitutively active, membrane-bound form of protein kinase B. A mutant of PTEN selectively lacking lipid phosphatase activity, but retaining protein phosphatase activity, had no effects on cell number. Active (but not mutant) PTEN also decreased TCR-induced activation of the mitogen-activated protein kinase ERK2 (extracellular signal-related kinase 2), as seen after inhibition of phosphatidylinositol 3-kinase. Our data indicate that PTEN is a phosphatidylinositol 3-phosphatase in T cells, and we suggest that PTEN may play a role in the regulation of T cell survival and TCR signaling by directly opposing phosphatidylinositol 3-kinase.

	Introduction

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The loss of genes with antitumor activity often accompanies malignant transformation and tumor progression. One such tumor suppressor gene, PTEN (1, 2, 3), is located on chromosome 10 at q22–23, a locus that is altered in almost half of all endometrial cancers, in a third of glioblastomas, and at lesser frequencies in a wide range of other human neoplasms, such as prostate, brain, breast, and kidney cancers (1, 3). Abnormalities at 10q are also found in many lymphoproliferative diseases (4, 5), and mutations of PTEN have been demonstrated in many leukemic cell lines (6, 7). Three human autosomal dominant diseases, Cowden disease, Lhermitte-Duclos disease, and Bannayan-Zonana syndrome, are caused by germline mutations in PTEN (8, 9). These genetic disorders have similar pathological features, including the formation of multiple benign tumors, and an increased incidence of malignant cancers (8, 9). Deletion of the PTEN gene in mice resulted in embryonic lethality probably due to hyperproliferation of embryonic cells (10). Together, all these findings suggest that disruption of PTEN sensitizes cells to malignant transformation and thus implicate the PTEN protein in a pathway that controls normal cell physiology.

PTEN encodes a 55-kDa protein, which contains the cysteine- and arginine-based signature motif of PTPases in an amino acid context that mostly resembles the dual-specificity phosphatases (11). In vitro, PTEN dephosphorylates phosphotyrosine (and to a lesser extent phosphothreonine), but the specific activity is quite low unless extremely acidic substrates are used (11). This unusual selectivity may reflect either a preference for substrates that are phosphorylated on multiple sites (10) or, more likely, the recent finding that PTEN readily dephosphorylates phosphatidylinositol (3, 4, 5)-trisphosphate specifically at position 3 of the inositol ring (12, 13). Overexpression of PTEN also reduced the insulin-induced increase in cellular phosphatidylinositol (3, 4, 5)-trisphosphate in human 293 cells without effecting insulin-induced activation of the enzyme that produces these lipids, phosphatidylinositol 3-kinase (PI3K).⁴ Thus, PTEN may act in vivo as a specific polyphosphoinositide 3-phosphatase and thereby directly counteract PI3K. Because PI3K is involved in a positive manner in a multitude of signaling pathways that promote cell growth, proliferation, differentiation, motility, and cytoskeletal organization (14), this would explain much of the pathology seen in cancer cells with a disrupted PTEN.

In support of the notion that phosphatase activity is important for the tumor-suppressing activity of PTEN, many of the point mutations in PTEN that have been found in tumor samples and cell lines, as well as the inherited diseases, result in impaired or reduced phosphatase activity (1, 3, 9, 10, 15, 16, 17). Thus, the enzymatic activity of PTEN is necessary for its ability to act as a tumor suppressor (18, 19).

We have addressed the function of PTEN in T cells. We find that the gene is expressed in Jurkat T cells and that the protein affects cell survival as well as signaling from the TCR. Our data support the notion that PTEN primarily acts by dephosphorylating 3-phosphorylated inositol phospholipids and thereby counteracts the versatile effects of PI3K.

	Materials and Methods

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Abs and reagents

The anti-CD3 mAb, OKT3, and the anti-myc tag epitope mAb 9E10 were purified from ascites fluid by protein A Sepharose chromatography. The anti-influenza hemagglutinin (HA) tag epitope mAb 12CA5 was from Boehringer Mannheim (Indianapolis, IN), polyclonal anti-extracellular signal-related kinase 2 (ERK2) was from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-phospho-ERK and anti-protein kinase B (PKB) Abs from New England Biolabs (Beverly, MA).

PCR and plasmids

Two primer pairs used for amplification of PTEN were 5'-CAT CTC TCT CCT CCT TTT TCT TCA-3' and 5'-ATA TCA TTA CAC CAG TTC GTC CCT-3' (pair no. 1) and 5'-GAA ACT ATT CCA ATG TTC AGT GGC-3' and 5'-CTG ATC TTC ATC AAA AGG TTC ATT CTC-3' (pair no. 2). Green fluorescent protein (GFP) was from Clontech (San Diego, CA) and was subcloned into the pEF/HA vector (20). The myc-tagged ERK2 was in the pEF vector as before (21). Myristylated PKB in pCMV6 was from T. F. Franke (Columbia University, New York, NY). The catalytic p110ß subunit of PI3K and the interSH2 region of its p85 subunit were in pEF/HA (20).

PTEN expression plasmids

The cDNA for PTEN (a kind gift from F. Furnari and W. Cavenee, University of California at San Diego, La Jolla, CA) was cloned into the eukaryotic expression vector pEF with a C-terminal HA epitope tag (see Fig. 1A). Two point mutants, the catalytically inactive C124G mutant and the mutant G129E, which is inactive toward 3-phosphorylated inositols, but retains 70% of the activity toward protein substrates (13, 22), were also from F. Furnari and W. Cavenee.

View larger version (36K): [in this window] [in a new window]   FIGURE 1. PTEN constructs. A, Schematic representation of the PTEN constructs used in this study. The location of the two primer pairs (no. 1 and no. 2) are indicated. B, PCR amplification of two fragments of PTEN from a Jurkat cDNA library using 5 µl (50 ng) or 15 µl (150 ng) of primer pairs no. 1 and no. 2, as indicated. C, Anti-HA tag immunoblot of lysates from Jurkat cells transfected with PTEN, PTEN-C124G, or PTEN-G129E.

Jurkat T leukemia cells were kept at logarithmic growth in RPMI supplemented with 10% heat-inactivated FCS, L-glutamine, and antibiotics. Transient transfections were conducted by electroporation as described previously (21, 23, 24). Electroporation conditions typically contained 20 x 10⁶ cells and a total of 15 µg of plasmid DNA (1–5 µg of each plasmid), and in each transfection the DNA amount was kept constant by the addition of empty vector. Immunoprecipitation, immunoblotting, and ERK2 kinase assays were performed as described previously (21, 23, 24). Briefly, cells were lysed in 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA containing 1% Nonidet P-40, 1 mM Na₃VO₄, 10 µg/ml aprotinin and leupeptin, 100 µg/ml soybean trypsin inhibitor, and 1 mM PMSF and clarified by centrifugation at 15,000 rpm for 20 min. The clarified lysates were preabsorbed on protein G Sepharose and then incubated with Ab for 2 h, followed by protein G Sepharose beads. Immune complexes were washed three times in lysis buffer, once in lysis buffer with 0.5 M NaCl, again in lysis buffer, and either suspended in SDS sample buffer or used for in vitro kinase assays. ERK2 kinase reactions were performed for 30 min at 30°C in 20 µl kinase buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 20 mM MgCl₂, 10 µg myelin basic protein (MBP), 1 µM ATP, and 10 µCi of [-³²P]ATP. The reactions were terminated by adding 20 µl 2x SDS sample buffer and heating to 95°C for 2 min. The samples were run on 12% SDS-polyacrylamide gels, transferred onto nitrocellulose filters, and the labeled proteins visualized by autoradiography. The presence of equal amounts of the immunoprecipitated ERK2 was verified by Western blotting using the anti-ERK2 Ab at 1:1000 dilution, anti-mouse-Ig-peroxidase, and the blots developed by the enhanced chemiluminescence technique (ECL kit, Amersham, Arlington Heights, IL) according to the manufacturer’s instructions.

FACS analysis and apoptosis assay

Cells transfected with GFP (plus PTEN and/or myristylated PKB (myrPKB)) were washed in PBS and analyzed (without fixation) in a FACSCalibur instrument (Becton Dickinson Immunocytometry Systems, San Jose, CA). For visualization of apoptosis, the transfected cells were washed in PBS and suspended in 3.7% formaldehyde in PBS and kept at room temperature for 10 min, washed once in PBS, and resuspended in 0.1 µg/ml 4',6'-diamidino-2-phenylindole (DAPI) in PBS for staining at room temperature for 15 min. Subsequently, cells were washed three times and mounted on glass slides. The number of DAPI-stained cells with apoptotic nuclei that were also GFP⁺ were quantitated using a Nikon (Melville, NY) fluorescence microscope.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
PTEN is expressed in Jurkat T cells

We began our studies of PTEN by determining whether PTEN is expressed in the Jurkat T leukemia cell line that we use as a model for studying TCR signaling. For these experiments, we designed two oligonucleotide primer pairs expected to yield a 447-bp fragment from the 5' half of the cDNA (pair no. 1) and a nonoverlapping 587-bp fragment from its 3' end (pair no. 2), respectively. The location of the primers are indicated in Fig. 1A. Using these primers and a Jurkat cDNA library as template in a PCR, we obtained both of the expected amplification products (Fig. 1B), which were sequenced to ensure that they were truly derived from the reverse-transcribed mRNA for PTEN. We conclude that PTEN mRNA is expressed in Jurkat cells.

PTEN reduces T cell viability, which is overcome by myrPKB

When Jurkat T cells were transiently transfected with the three PTEN expression plasmids, we consistently observed that the active PTEN was expressed at much lower levels than the two mutants PTEN-C124G and PTEN-G129E (Fig. 1C). To determine whether this was due to less PTEN protein per cell or to a reduced number of cells expressing PTEN, we cotransfected the cells with GFP in the same pEF/HA vector. Forty-eight hours after transfection, the cells were analyzed by FACS for GFP-derived fluorescence. As shown in Fig. 2 and in Table I, there were clearly fewer positive cells when active PTEN was used. In addition, the remaining 50% GFP⁺ cells were primarily in the lower end of the expression spectrum. Coexpression of PTEN-C124G or PTEN-G129E with GFP did not affect the number of GFP⁺ cells. The effect of active PTEN may be due to reduced proliferation, increased apoptosis, or both, of the cells expressing higher levels of active enzyme. When viewed under the microscope, the PTEN-expressing cells did not appear or behave different from control, nor did they aggregate or adhere abnormally.

View larger version (45K): [in this window] [in a new window]   FIGURE 2. Effects of PTEN constructs on the number of transfected cells. A, FACS of Jurkat T cells transfected with GFP alone or in combination with PTEN and myrPKB constructs. The experiment shown is the same as experiment 2 in Table I. B, Amount of expressed PTEN assessed by anti-HA immunoblotting of total lysates from the same transfectants as in A.

Active PTEN induces an increased rate of apoptosis

To directly test whether the loss of cells induced by active PTEN was due to increased rates of programmed cell death, we transfected the cells with GFP and the same plasmids as for FACS analysis. Subsequently, the cells were fixed and stained with DAPI to visualize cells with apoptotic nuclei. These experiments revealed that the population of GFP⁺ cells expressing active PTEN had a much higher percentage of apoptotic cells than the cells expressing mutant PTEN or the combination of active PTEN and myrPKB. In the presence of active PTEN, nearly half of the GFP⁺ cells had apoptotic nuclei, compared with less than 5% of the GFP⁺ cells in the vector controls (Fig. 3, Table I). Coexpression of myrPKB reduced the frequency of apoptosis nearly to the level seen in vector controls, as did the mutant PTEN constructs (Table I). These results suggest that the 50% loss of GFP⁺ cells seen in the presence of active PTEN in Fig. 2 is due to an increased rate of cell death that is still elevated at 48 h. Because cells with higher expression are preferentially lost (Fig. 2), it seems likely that individual cells initiate apoptosis at the point in time when the plasmid-derived PTEN expression reaches a critical threshold. Thus, cells that have taken up more DNA (reflected by higher GFP-derived fluorescence) die sooner, while dull cells remain alive to later time points. This critical threshold may reflect the rate at which PI3K can replenish the dephosphorylated inositol lipids. However, although cell death was prevented by myrPKB, we cannot entirely exclude the possibility that PTEN reduced the number of cells by additional mechanisms, e.g., secretion of soluble mediators.

View larger version (124K): [in this window] [in a new window]   FIGURE 3. Apoptotic nuclei in cells transfected with active PTEN. A, GFP-derived fluorescence ("green") in a subset of the cell population transfected with GFP plus empty pEF/HA vector. B, DAPI stain ("DAPI") of the same field of cells, showing the nuclei of both GFP+ and GFP- cells. C, GFP-derived fluorescence in cells transfected with GFP plus wild-type PTEN. D, DAPI stain of the same field of cells as in C. Note that the nuclei are intact in vector-transfected cells, but largely apoptotic in the cells that express PTEN (and GFP). See Table I for a statistical analysis of these assays.

The mitogen-activated kinases ERK1 and ERK2 are important integration points in signaling by growth factor and cytokine receptors, as well as lymphocyte Ag receptors. We have previously shown that PI3K activity is required for efficient activation of ERK2 activation in Jurkat T cells (20). Approximately 50% of the induced ERK2 activity is lost in cells expressing a dominant-negative PI3K p85iSH2 construct or in cells treated with wortmannin (20), a fungal PI3K inhibitor. In contrast, ERK2 activation was somewhat augmented in cells with increased PI3K activity (20). Based on these results, we reasoned that if PTEN acts as a phosphatidylinositol 3-phosphatase in intact T cells, it should have the same effect on ERK2 activation as the inhibition of PI3K. Indeed, when PTEN was coexpressed with a myc-tagged ERK2, the anti-CD3-induced activation of ERK2 was clearly reduced (Fig. 4). In contrast, PTEN-C124G and PTEN-G129E had no effect. In addition, we confirmed in several control experiments that the coexpressed myrPKB did not affect ERK2 activation, in agreement with PKB not being involved in the signaling cascade that leads to ERK activation in these cells.

View larger version (56K): [in this window] [in a new window]   FIGURE 4. Effects of PTEN on the CD3-induced activation of ERK2 in T cells. Top panel, In vitro kinase assay with MBP as a substrate and immunoprecipitates obtained with the anti-tag mAb 9E10 from Jurkat cells transiently cotransfected with vector alone or tagged ERK2 plus vector or the indicated PTEN constructs. The cells were stimulated for 5 min with anti-CD3 as indicated. Middle panel, Anti-ERK2 blot of the same filter. Bottom panel, Anti-HA immunoblot of lysates of the same transfectants. myrPKB (1 µg) was included in all transfections. Similar results were obtained in four independent experiments.

PTEN also blocks the TCR-induced phosphorylation of ERK

We also performed experiments with an Ab that only recognizes the activated phospho-form of ERK. This Ab cannot be used in transient transfection experiments with regular Jurkat cells (due to background from untransfected cells that respond to anti-CD3), but readily with the Lck-negative variant of Jurkat, JCaM1, which requires cotransfection of Lck to respond to TCR stimulation by ERK activation (Fig. 5A; Refs. 21, 24). Thus, a measurable phospho-ERK response comes only from the population of cells that express Lck and the cotransfected gene(s) (Fig. 5B). When JCaM1 cells were cotransfected with Lck and active PTEN, stimulated for 5 min with anti-CD3, and subsequently immunoblotted with anti-phospho-ERK Abs, it was clear that PTEN reduced the appearance of phospho-ERK, whereas PTEN-C124G and PTEN-G129E did not. Again, expression of myrPKB alone or with Lck had no effect (lanes 11 and 12) (except for rescuing the cells expressing active PTEN from apoptosis). The amount of phospho-ERK (Fig. 5B) correlated with the catalytic activity of ERK (Fig. 5A). As before (20), expression of more PI3K caused a small increase in ERK activation (Fig. 5A, lanes 9 and 10).

View larger version (53K): [in this window] [in a new window]   FIGURE 5. Effects of PTEN on ERK2 activation and phosphorylation in JCaM1 cells. A, top panel, In vitro kinase assay with MBP as a substrate and immunoprecipitates obtained with the anti-tag mAb 9E10 from JCaM1 cells transiently cotransfected with tagged ERK2 plus vector or plus Lck and the indicated PTEN or PI3K constructs. Second panel, Anti-ERK2 blot of the same immunoprecipitates. Third panel, Anti-HA blot of the lysates of the same transfectants. Bottom panel, Anti-Lck blot of the same filter. myrPKB (1 µg) was included in all samples. p110ß, the catalytic subunit of PI3K; iSH2, the interSH2 domain region of PI3K p85. B, top panel, Anti-phospho-ERK immunoblot of lysates from a similar experiment with JCaM1 cells transiently cotransfected with myrPKB, Lck, and PTEN, as indicated. Second panel, Anti-ERK2 blot of the same filter. Third panel, Anti-HA blot of the same filter. Bottom panel, Anti-Lck blot of the same filter.

Although our experiments do not exclude the possibility that PTEN may act on protein substrates, three independent lines of evidence indicate that it acts as a phosphatidylinositol 3-phosphatase in T cells. First, the reduction in the number of transfected cells and the increased rate of apoptosis in the presence of active PTEN were completely abolished by a constitutively active PKB, one of the best characterized effectors for PI3K (25, 26). Second, the effects of PTEN on the activation of ERK, but not JNK, mimics those seen after PI3K inhibition (20). Third, the G129E mutation, which abolishes lipid phosphatase activity, but not PTPase activity, resulted in a functionally inactive PTEN in all our experiments. Thus, we suggest that PTEN is primarily, perhaps exclusively, a polyphosphoinositide 3-phosphatase that directly counteracts the function of PI3K in intact T cells. The physiological role of PTEN is therefore closely related to that of PI3K, which participates in the regulation of T cell activation, costimulation and survival.

	Acknowledgments

 
We are grateful to Frank Furnari and Webster Cavenee for the kind gift of PTEN cDNAs, and to Thomas Franke for the myrPKB construct.

	Footnotes

 
¹ This study was supported by a fellowship from the Swedish Cancer Foundation (to A.G.-W.) and Grants AI35603, AI41481, and AI40552 (to T.M.) from the National Institutes of Health.

² Current address: Department of Medical Chemistry, University of Lund, Lund, Sweden.

³ Address correspondence and reprint requests to Dr. Tomas Mustelin, Laboratory of Signal Transduction, The Burnham Institute, La Jolla Cancer Research Center, 10901 North Torrey Pines Road, La Jolla, CA 92037. E-mail address:

⁴ Abbreviations used in this paper: PI3K, phosphatidylinositol 3-kinase; GFP, green fluorescent protein; HA, hemagglutinin; MBP, myelin basic protein; ERK, extracellular signal-related kinase; PKB, protein kinase B; myrPKB, myristylated PKB; DAPI, 4',6'-diamidino-2-phenylindole.

Received for publication October 13, 1999. Accepted for publication December 3, 1999.

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