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Phosphatidylinositol 3-Kinase Links the Interleukin-2 Receptor to Protein Kinase B and p70 S6 Kinase

Phosphatidylinositol 3-Kinase Links the Interleukin-2 Receptor to Protein Kinase B and p70 S6 Kinase THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 22, Issue of May 30, pp. 14426 –14433, 1997 © 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Phosphatidylinositol 3-Kinase Links the Interleukin-2 Receptor to Protein Kinase B and p70 S6 Kinase* (Received for publication, February 18, 1997, and in revised form, March 24, 1997) Karin Reif‡, Boudewijn M. T. Burgering§, and Doreen A. Cantrell From the Lymphocyte Activation Laboratory, Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London WC2A 3PX, United Kingdom and the §Laboratory for Physiological Chemistry, Utrecht University, Universiteitsweg 100, Utrecht 3584 CG , The Netherlands Phosphatidylinositol 3-kinase (PI 3-kinase) is acti- lyzes the phosphorylation of phosphoinositides at the D-3 hy- vated by the cytokine interleukin-2 (IL-2). We have used droxyl of the myo-inositol ring generating PI 3-phosphate, PI a constitutively active PI 3-kinase to identify IL-2-medi- 3,4-bisphosphate, and PI 3,4,5-trisphosphate (13, 14). The form ated signal transduction pathways directly regulated by of PI 3-kinase involved in protein-tyrosine kinase-dependent PI 3-kinase in lymphoid cells. The serine/threonine pro- receptor signal transduction comprises a regulatory 85-kDa tein kinase B (PKB)/Akt can act as a powerful oncogene subunit that contains two Src homology 2 domains and at its N in T cells, but its positioning in normal T cell responses terminus one Src homology 3 domain and a catalytic 110-kDa has not been explored. Herein, we demonstrate that subunit. Following IL-2R stimulation, several mechanisms PKB is activated by IL-2 in a PI 3-kinase-dependent have been proposed to recruit PI 3-kinase to the plasma mem- fashion. Importantly, PI 3-kinase signals are sufficient brane, where its cellular substrate PI 4,5-bisphosphate is lo- for PKB activation in IL-2-dependent T cells, and PKB is cated: engagement of the IL-2R leads to binding of the p85 a target for PI 3-kinase signals in IL-2 activation path- regulatory subunit of PI 3-kinase to tyrosine 392 in the IL-2R ways. The present study establishes also that PI 3-kinase b-chain (15); in addition, interleukin-2 (IL-2) stimulation re- signals or PKB signals are sufficient for activation of sults in the interaction of PI 3-kinase with the Src family p70 S6 kinase in T cells. PI 3-kinase can contribute to, kinases Fyn (16) and Lck (17). but is not sufficient for, activation of extracellular sig- The activation of PI 3-kinase is a response that IL-2 shares nal-regulated kinases (Erks) and Erk effector pathways. with other cytokines that control lymphoid cell growth and Therefore, PI 3-kinase is a selective regulator of serine/ development such as IL-4 and IL-7 (18, 19). It is also clear that threonine kinase signal transduction pathways in T PI 3-kinase activation is necessary for the growth- and differ- lymphocytes, and this enzyme provides a crucial link entiation-inducing properties of these cytokines (20 –23). How- between the interleukin-2 receptor, the protooncogene PKB, and p70 S6 kinase. ever, despite the pivotal role of PI 3-kinase in lymphoid cells, there is only a preliminary and incomplete understanding of the targets for this enzyme in the mitogenic signaling path- The high affinity interleukin-2 receptor (IL-2R), which com- ways regulated by the hematopoietin family of cytokines. To prises a-, b-, and g-subunits controls G to S progression, T cell date, the identification of biochemical targets for PI 3-kinase in clonal expansion, and functional differentiation (1–3). The T cells stems mainly from studies employing the PI 3-kinase IL-2R orchestrates downstream effector pathways by protein inhibitor wortmannin or the LY294002 compound (10, 20). tyrosine kinase-dependent activation mechanisms engaging Hence, IL-2 activation of the mitogen-activated protein (MAP) the Src family tyrosine kinases Lck and Fyn (4) and the Janus kinase Erk is sensitive to wortmannin (10). Similarly, IL-2 kinases 1 and 3 (5–7). Signaling cascades integrated by the activation of the serine/threonine kinase p70 S6 kinase action of these tyrosine kinases include activation of the Ras/ (p70S6k) is prevented by these PI 3-kinase inhibitors (20). In Raf/extracellular-signal regulated kinase (Erk) pathway (8 – addition, IL-2 activation of p70S6k is impeded by the immuno- 10), activation of the transcription factors STAT3 and STAT5 supressant rapamycin, which targets another member of the PI (11), and the regulation of phosphatidylinositol 3-kinase (PI 3-kinase family of enzymes, Frap (for FKBP12-rapamycin-as- 3-kinase) (12). sociated protein) also termed “mammalian target of rapamy- PI 3-kinase is a ubiquitously expressed enzyme that cata- cin” (mTor) (24, 25). Observations that wortmannin and rapa- mycin have identical inhibitory effects on IL-2 activation of p70S6k generated a model for the p70S6k signaling pathway in * This work was supported by the Imperial Cancer Research Fund and by Human Capital Mobility Program Grant ERB CHRX CT 94- which PI 3-kinase acts as an upstream regulator of Frap (24, 0537. The costs of publication of this article were defrayed in part by the 25). However, this model has been challenged by a recent study payment of page charges. This article must therefore be hereby marked showing that the action of Frap is directly inhibited by wort- “advertisement” in accordance with 18 U.S.C. Section 1734 solely to mannin and LY294002 (26). These results raise the issue of indicate this fact. whether PI 3-kinase itself has any upstream regulatory role in ‡ Supported by a Boehringer Ingelheim Fellowship. To whom corre- spondence should be addressed. Tel.: 0171-269-3307; Fax: 0171-269- p70S6k activation in T lymphocytes. Similar caution must be 3479; E-mail: [email protected]. applied to interpretations of data involving PI 3-kinase in Erk The abbreviations used are: IL-2R, interleukin-2 receptor; Erk, activation in T cells. In this context, expression of an active PI extracellular-signal regulated kinase; PI, phosphatidylinositol; IL, in- 3-kinase is sufficient for Erk activation in Xenopus oocytes (27), terleukin; MAP, mitogen-activated protein; p70S6k, p70 S6 kinase; Frap, FKBP12-rapamycin-associated protein; PKB, protein kinase B; but it would be fallacious to extrapolate data obtained in Xe- PdBu, phorbol 12,13-dibutyrate; Ab, antibody; mAb, monoclonal anti- nopus cells to T cells, since the role of PI 3-kinase as an body; CAT, chloramphenicol acetyltransferase; PKC, protein kinase C; upstream regulator of kinase pathways can vary depending on Mek, Erk kinase; HA, hemagglutinin; rIL-2, recombinant IL-2; Mops, the cell system; to this end, PI 3-kinase signals did not stimu- 4-morpholinepropanesulfonic acid; PAGE, polyacrylamide gel electro- phoresis; H2B, histone 2B; rCD2, rat CD2. late Erk activity in a variety of fibroblasts and in a monoblast 14426 This paper is available on line at http://www-jbc.stanford.edu/jbc/ This is an Open Access article under the CC BY license. Phosphatidylinositol 3-Kinase Targets in Lymphoid Cells 14427 cell line (28 –31). Whether PI 3-kinase signals are sufficient to stimulate p70S6k or Erk activation in T cells awaits analysis. We and others have recently reported that targeting the catalytic p110 subunit of PI 3-kinase to the plasma membrane generates a constitutively active enzyme that induces cellular accumulation of D-3 phosphoinositides (28 –31). A constitu- tively active PI 3-kinase finally allows assessment of the rela- tive contribution of PI 3-kinase-derived signals to a certain effector pathway, in particular whether PI 3-kinase activation is sufficient to promote a specific cellular response. In the present study, we have used a membrane-localized p110 con- struct, rCD2p110, that induces accumulation of cellular levels of PI 3,4-bisphosphate and PI 3,4,5-trisphosphate (29) as a tool to explore the regulation of serine/threonine kinase pathways by PI 3-kinase in T lymphocytes. We show that activation of PI 3-kinase is sufficient to stimulate p70S6k activity, although PI 3-kinase signals were not sufficient to induce activation of the MAP kinase Erk2 in T cells. The present study also character- izes a previously unrecognized IL-2-mediated signal transduc- tion pathway in T cells that involves the serine/threonine pro- tein kinase B (PKB) also known as c-Akt or Rac protein kinase (32–34). PKB was originally identified as the cellular homo- logue of the directly transforming oncogene of the murine ret- rovirus AKT8, which causes thymic lymphomas (35). Herein, we demonstrate that PKB is rapidly activated by IL-2 via a wortmannin- and LY294002-sensitive but rapamycin-insensi- tive pathway. PI 3-kinase signals alone were sufficient to acti- vate PKB in T cells, and expression of a constitutively active PKB could stimulate the activity of p70S6k. Therefore, PI 3-kinase is a selective regulator of serine/threonine kinase sig- nal transduction pathways in T lymphocytes, and this enzyme is an upstream regulator of the IL-2-activated kinases PKB and p70S6k. EXPERIMENTAL PROCEDURES Reagents—Phorbol 12,13-dibutyrate (PdBu) and wortmannin were FIG.1. Interleukin-2 activates p70S6k, which can be mimicked from Calbiochem. LY294002 was a gift from Zeneca. PD098059 was by co-expression of membrane-localized constitutively active PI from New England Biolabs. Rapamycin was a gift from G. Thomas 3-kinase, rCD2p110. A, Kit225 cells were deprived of rIL-2 for 68 h 14 32 (FMI, Basel). [ C]Acetyl coenzyme A (at 50 mCi/mmol), [g- P]ATP and treated with 20 ng/ml rIL-2 or 50 ng/ml PdBu for the indicated (5000 Ci/mmol), and I-conjugated protein A were from Amersham times, and p70S6k activation/phosphorylation was assessed by electro- Corp. mobility shift assays (top). p70S6k was precipitated from lysates with Antibodies—Ox34 monoclonal antibody (mAb) is raised against rat M5 Abs, and p70S6k activity was analyzed in immune complex kinase CD2 (rCD2) (29); 12CA5 mAb is reactive with hemagglutinin (HA), and assays using S6 as a substrate. [ P]phosphate incorporation into S6 9E10 mAb is reactive with the Myc epitope (36); anti-human S6 kinase (middle) was quantified (graph) using a PhosphorImager and is ex- M5 antiserum (37) was from Santa Cruz Biotechnology; M1 antiserum pressed in arbitrary units. Protein levels of p70S6k present in immune reactive with p70S6k (37) was a gift from G. Thomas; Rac-PK-CT Ab complexes were assessed in parallel by Western blotting with M1 Abs (Upstate Biotechnology, Inc.) is reactive with PKB. (bottom). B and C, HA-p70S6k activity was analyzed from extracts of Plasmids and Reporter Constructs—HA-p70S6k (pBJ5) (38); HA- untreated cells (2) or cells stimulated with either 20 ng/ml rIL-2 or 50 ng/ml PdBu for 15 min. B, before stimulation, Kit225 cells were co- PKB (pSG5) and gagPKB (pSG5) (32); HA-Erk2 (pCEP4) (39); Myc- transfected with HA-p70S6k plasmids and vector plasmid (empty) or V12Rac (pEF), Myc-V12Cdc42 (pEF), and Myc-V14Rho (pEF) (40); and with plasmids encoding for rCD2p110 as indicated. C, Kit225 cells were Ha-v-ras (pEF) (29) vector constructs have been described. The de- co-transfected with HA-p70S6k plasmids and 15 mg each of vector scribed rCD2p110, rCD2p110-R/P, and rCD2p85 chimeras (29) were plasmid (empty) or plasmids encoding for rCD2p110 or rCD2p110-R/P. subcloned into the pEF-BOS expression vector. The reporter plasmids B, S6 substrate phosphorylation from S6 kinase assays (top) was ana- Nlex.Elk-1 (pEF) and 2lexoptk.CAT (41) as well as Nlex.C2 (pMLV) (42) lyzed by autoradiography. Levels of p70S6k in immune complexes (bot- have been described. tom) were analyzed by immunoblotting using M1 antibodies followed by Cell Culture and Transient Transfections—The Kit225 T leukemic I-conjugated protein A and autoradiography. C, the data were ana- cell line (43) was maintained in RPMI 1640 medium containing 10% lyzed as in B and quantified using a PhosphorImager. Data are pre- heat-inactivated fetal calf serum supplemented with 20 ng/ml of recom- 32 125 sented as the ratio of [ P]phosphate incorporated into S6 to I- binant IL-2 (rIL-2) (Eurocetus) under normal growth conditions. For conjugated protein A bound to p70S6k (expressed in arbitrary units). IL-2 activation assays of endogenous proteins, Kit225 cells were washed The data are from a representative experiment. Similar results were three times with phosphate-buffered saline A to remove the IL-2 and obtained in two (A and B) or five (C) more experiments. cultured further in RPMI supplemented with 5% fetal calf serum in the absence of rIL-2 for 48 –72 h prior to IL-2 activation assays. When Kit225 cells were transfected, cells were treated as above but only V12Rac, V12Cdc42, gagPKB, or rCD2p85; 7.5 mg of 2lexoptk.CAT; and deprived of rIL-2 for 24 h prior to transfection. 15 mg of pEFNlex.Elk-1 or pMLVNlex.C2. For gene reporter assays, Kit225 cells were transfected by electroporation with 20 – 40 mgof cells were stimulated as indicated 2– 4 h after transfection. Cells were plasmid DNA. The amounts of plasmid DNA were kept constant per collected 14 –18 h after transfection. cuvette by adding vector plasmid. Kit225 cells (1.5 3 10 cells/0.625 ml) Immunoprecipitation, p70S6k Assays, and Western Blot Analysis— were pulsed at 320 V and 960 microfarads using a Gene Pulser (Bio- After stimulations as indicated, Kit225 cells were lysed in lysis buffer 1 Rad). The amounts of plasmid used were as follows (unless indicated (120 mM NaCl, 50 mM Tris pH 8.0, 20 mM NaF, 1 mM benzamidine, 1 mM otherwise): 7.5 mg of HA-p70S6k; 12.5 mg of HA-PKB; 10 mg of HA-Erk2; EDTA, 6 mM EGTA, 7.5 mM PP ,15mM p-nitrophenyl phosphate, 1% 20 mg of the plasmid pEF empty, rCD2p110, rCD2p110-R/P, Ha-v-ras, Nonidet P-40, 0.1 mM phenylmethylsulfonyl fluoride, and 0.1 mM 14428 Phosphatidylinositol 3-Kinase Targets in Lymphoid Cells Na VO ). Cell extracts corresponding to 3 3 10 cell equivalents (non- 3 4 transfected cells) or 1.5 3 10 live cell equivalents (transfected cells) were used for each immunoprecipitation. Postnuclear lysates were pre- cleared with protein A cell suspension (Sigma) prior to incubation with 2 mg of 12CA5 mAbs or, for endogenous proteins, 1 mg of M5 Abs. Immune complexes were precipitated with protein G-Sepharose beads (Sigma) or, when M5 Abs were used, with protein A-Sepharose beads (Pharmacia Biotech Inc.). The immunoprecipitates were washed three times in lysis buffer 1 and once in p70S6k assay buffer (50 mM Mops, pH 7.2, 5 mM MgCl , 0.1% Triton X-100) and assayed as described (37) using S6 as a substrate (a gift from G. Thomas). Proteins were resolved by SDS-PAGE. The lower part of the gel was dried, and P-labeled S6 proteins were detected by autoradiography. The levels of p70S6k pro- tein in each immunoprecipitate were assessed by transferring the pro- teins in the upper part of the gel onto polyvinylidene difluoride mem- branes and performing Western blot analysis with 12CA5 mAbs or M1 Abs using the ECL detection system (Amersham). If the p70S6k protein levels in the immunoprecipitate were not equal, activities were normal- ized for p70S6k expression levels by quantitation of Western blots probed with M1 Abs followed by I-conjugated protein A (Amersham). 32 125 Quantitation of incorporated P into S6 or of bound I-conjugated protein A was performed using a PhosphorImager (Molecular Dynam- ics). To test for effector protein expression in transfected cells, post- nuclear cell extracts corresponding to 3 3 10 cell equivalents of the same extract as above were analyzed by Western blotting as described (44) using 9E10, Ox34, or specific Abs. PKB Assays—Cells were treated as for p70S6k assays except that lysis buffer 2 (120 mM NaCl, 50 mM Hepes, pH 7.4, 10 mM NaF, 1 mM EDTA, 40 mM b-glycerophosphate, pH 7.5, 1% Nonidet P-40, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM Na VO ) was used to lyse cells. 3 4 To immunoprecipitate endogenous PKB, 2 mg of Rac-PK-CT Abs were used. The immunoprecipitates were washed twice in lysis buffer 2, twice in high salt wash buffer (500 mM LiCl, 100 mM Tris, pH 7.5, 1 mM EDTA, pH 7.5), and once in PKB assay buffer (50 mM Tris, pH 7.5, 10 mM MgCl ,1mM dithiothreitol). The reaction was initiated by the addition of 15 ml of PKB reaction buffer containing 3 mCi of [g- P]ATP, 50 mM ATP, 7.3 mM MgCl , 730 mM dithiothreitol, 500 nM protein kinase inhibitor (Sigma), 40 mM Tris, pH 7.5, and 2.5 mg of histone 2B (H2B) (Boehringer Mannheim). After 30 min at 25 °C, the reaction was ter- minated by adding reducing SDS-PAGE sample buffer and boiling. Proteins were resolved by SDS-PAGE, and the gel was treated as for p70S6k assays. To detect PKB proteins, Western blot analysis was performed with Rac-PK-CT Abs. Erk Assays—Cells and cell extracts were processed as for p70S6k assays. HA-tagged Erk2 was immunoprecipitated with 12CA5 mAbs. Precipitated immune complexes were washed three times with lysis buffer 1 and once with Erk wash buffer (30 mM Tris, pH 8.0, 20 mM MgCl ,2mM MnCl ). The reaction was initiated by the addition of 10 ml 2 2 of Erk reaction buffer containing 4 mCi of [g- P]ATP, 20 mM ATP, 20 mM MgCl ,2mM MnCl ,5mM p-nitrophenyl phosphate, 500 nM protein 2 2 kinase inhibitor, 30 mM Tris, pH 8.0, and 15 mg of myelin basic protein (Sigma). After 30 min at 37 °C, the reaction was terminated by adding reducing SDS-PAGE sample buffer and boiling. Proteins were resolved by SDS-PAGE, and the gel was treated as for p70S6k assays. To detect Erk proteins, Western blot analysis was performed with 12CA5 mAbs as primary Ab, rabbit anti-mouse IgG as secondary Ab, and I-conju- gated protein A. Gene Expression Analysis—Fourteen to 16 h after inductions, as indicated, Kit225 T cells were harvested and cells were lysed in 200 ml of lysis buffer (0.65% Nonidet P-40, 10 mM Tris, pH 8, 1 mM EDTA, 150 FIG.2. Interleukin-2 activates PKB: PI 3-kinase activity is nec- mM NaCl). Gene expression assays were carried out as described (45). essary for IL-2-mediated activation of PKB, and PI 3-kinase The data are presented as percentage of conversion. signals are sufficient to stimulate PKB activity in Kit225 T cells. A, interleukin-2 activates PKB. Kit 225 cells were deprived of rIL-2 for 68 h and treated with 20 ng/ml rIL-2 or 50 ng/ml PdBu for the indicated times, and PKB activation/phosphorylation was assessed by electromo- bility shift assays (top). PKB was precipitated from lysates with Rac- the times indicated before lysis. PKB activity was measured in immune PK-CT Abs and PKB activity was analyzed in immune complex kinase complex kinase assays using H2B as a substrate. C, PI 3-kinase signals assays using H2B as a substrate. [ P]Phosphate incorporation into trigger a potent stimulation of PKB activity. Kit225 cells were co- H2B (middle) was quantified (graph) using a PhosphorImager and is transfected with HA-PKB plasmids and vector plasmid (empty), or expressed in arbitrary units. Protein levels of PKB present in immune plasmids encoding for rCD2p110, rCD2p110-R/P, Ha-v-ras, or V12Rac complexes were assessed in parallel by Western blotting with Rac- as indicated. HA-PKB activity was analyzed in anti-HA tag immune PK-CT Abs (bottom). B, the PI 3-kinase inhibitors wortmannin and complex kinase assays using H2B as a substrate. B and C,[ P]phos- LY294002, but not rapamycin, inhibit IL-2-dependent activation of phate incorporation into H2B (top) was quantified (graph) using a PKB. Kit225 cells starved of rIL-2 for 72 h were pretreated for 30 min PhosphorImager and is expressed in arbitrary units. Protein levels of with the vehicle dimethyl sulfoxide (DMSO), 20 ng/ml rapamycin, 5 mM PKB (B) or HA-PKB (C) present in immune complexes were assessed in LY294002, or 100 nM wortmannin and then stimulated with rIL-2 for parallel by Western blotting with Rac-PK-CT Abs (bottom). Phosphatidylinositol 3-Kinase Targets in Lymphoid Cells 14429 expression of the rCD2p110 chimera was confirmed by flow cytometric immunofluorescence analysis with rCD2 mAbs (data not shown). The HA-tagged p70S6k was immunoprecipi- tated from transiently transfected cells and assayed for its ability to phosphorylate S6 ribosomal subunits (Fig. 1B). Ex- pression of the active PI 3-kinase, rCD2p110, resulted in con- stitutive IL-2-independent p70S6k activation (Fig. 1B). p70S6k was not constitutively activated in cells expressing “kinase- dead” rCD2p110-R/P, confirming that the p70S6k activation requires the kinase activity of the p110 subunit (Fig. 1C). The expression of rCD2p110-R/P was noted in some experiments to suppress rIL-2 inducibility of p70S6k, indicating that this chi- mera may be an inhibitory mutant of PI 3-kinase pathways. IL-2 and Membrane-localized PI 3-Kinase Activate PKB— PKB can be activated by receptor tyrosine kinases such as the platelet-derived growth factor receptor and has been identified as a target of PI 3-kinase in fibroblasts (28, 32, 33). However, whether this pathway is conserved in the hematopoietic system has not been explored. In particular, although PKB can become oncogenic and initiate thymic tumors, its regulation and sig- nificance for normal T cell growth processes is not known. Since cytokine receptors have essential functions in the development and maintenance of the hematopoietic system, we were inter- ested to assess whether members of the hematopoietin receptor family, such as the prototypical IL-2R, regulate PKB. To exam- ine whether IL-2 activates PKB, immunoprecipitates of this kinase were prepared from rIL-2-deprived and rIL-2-activated FIG.3. Co-expression of constitutively active forms of PKB Kit225 cells and subjected to in vitro kinase assays using H2B and PI 3-kinase but not of the GTPases Rac and Cdc42 stimu- as a substrate. The data in Fig. 2A show that IL-2 induced a lates p70S6k activity in Kit225 cells. A, HA-p70S6k activity was rapid activation of PKB. A 2–3-fold increase in enzyme activity analyzed from extracts of untreated cells (2) or cells stimulated with 20 ng/ml rIL-2 for 15 min. Before stimulation, Kit225 cells were co-trans- over basal levels was sustained for more than 60 min in re- fected with HA-p70S6k plasmids and vector plasmid (empty) or plas- sponse to rIL-2. PKB activity is regulated by phosphorylation mids encoding for rCD2p110, gagPKB, V12Rac, V12Cdc42, or rCD2p85 as indicated by the reduced electrophoretic mobility of PKB as indicated. p70S6k assays were performed as described under “Ex- isolated from rIL-2-activated cells (Fig. 2A). PKB activity was perimental Procedures.” The data were quantified using a PhosphorIm- ager and are presented as the ratio of [ P]phosphate incorporated into not induced by exposure of Kit225 cells to phorbol esters that S6 to I-conjugated protein A bound to p70S6k (expressed in arbitrary activate PKC (Fig. 2A). The data in Fig. 2B show the failure of units). B, Kit225 cells were transfected with the LexA operator-con- rIL-2 to stimulate PKB in cells pretreated with LY294002 or trolled CAT reporter plasmid (lexOP-CAT) and the expression plasmid wortmannin, two well characterized PI 3-kinase inhibitors that producing the LexA-C2 fusion protein Nlex.C2 together with vector plasmid (empty) or the expression plasmids for V14Rho, V12Rac, and bind to the ATP or lipid binding sites on the p110 catalytic V12Cdc42 as indicated. After 18 h, Kit225 T cells were harvested, and subunit, respectively. These inhibitors also prevent the autoki- CAT activity was analyzed as described under “Experimental Proce- nase activity of Frap/mTor (26), a member of the PI 3-kinase dures.” The CAT activity is presented as percentage of conversion. The family (47), which is the cellular target for the drug rapamycin data are from a representative experiment. Similar results were ob- tained in one further experiment. and which prevents IL-2-coordinated cell cycle progression and proliferation of T lymphocytes (24, 25). Frap activity is abso- RESULTS lutely required for p70S6k action in T cells (24, 25). We there- fore assessed whether Frap function was necessary for IL-2- IL-2 and PI 3-Kinase Signals Activate p70S6k in Kit225 induced stimulation of PKB. Rapamycin had no effect on IL-2- Cells—For our studies, we used the well characterized human triggered activation of PKB (Fig. 2B), although rapamycin IL-2-dependent T cell line Kit225. The data in Fig. 1A show completely abolished IL-2- or PI 3-kinase-controlled induction that p70S6k activity is low in quiescent rIL-2-deprived Kit225 of p70S6k (data not shown). Thus, the inhibition of PKB by cells but can be rapidly stimulated by rIL-2. The activity of wortmannin and LY294002 cannot be caused by prevention of p70S6k is increased in response to phorbol esters that stimu- Frap activity and indicate that IL-2 regulation of PKB employs late protein kinase C (PKC) (Fig. 1A). We asked whether PI PI 3-kinase. 3-kinase signals could substitute for IL-2 in inducing p70S6k To investigate directly whether PI 3-kinase signals are suf- activity. We have shown recently that plasma membrane tar- ficient to activate PKB, rIL-20-deprived Kit225 cells were co- geting of p110, the catalytic subunit of PI 3-kinase, generates transfected with either rCD2p110 or rCD2p110-R/P expression an enzyme that is constitutively active in vivo (29). Our mem- vectors together with an expression vector encoding HA brane-targeted active PI 3-kinase construct comprises a chi- epitope-tagged PKB. In addition, the ability of activated forms mera of the extracellular and transmembrane domains of the rCD2 antigen fused to the p110a catalytic domain of PI 3-ki- of the small GTPases Ha-v-ras and V12Rac to activate PKB was assessed. Immunoprecipitates of HA-tagged PKB were nase, rCD2p110. As a control, we used a rCD2p110 molecule that contained in the catalytic subunit of PI 3-kinase an inac- assayed for kinase activity using H2B as a substrate. The tivating point mutation (R1130P) in the ATP binding site (46) constitutively active PI 3-kinase rCD2p110 induced a robust that abolishes its in vivo and in vitro lipid kinase activity, activation of PKB (Fig. 2C). This stimulatory effect of rCD2p110-R/P. To assess the effects of PI 3-kinase signals on rCD2p110 was dependent on the kinase activity of the chimera, p70S6k activity, rIL-2-starved Kit225 cells were co-transfected since co-expression of kinase-inactive rCD2p110-R/P did not with HA epitope-tagged p70S6k and rCD2p110. Cell surface stimulate PKB activity. As observed previously in other cell 14430 Phosphatidylinositol 3-Kinase Targets in Lymphoid Cells FIG.4. Interleukin-2 but not rCD2p110 induces Elk-1-dependent gene expression. A–E, Kit225 cells were transfected with the LexA operator-con- trolled CAT reporter plasmid (lexOP- CAT) plus the expression plasmid pro- ducing the LexA-Elk-1 fusion protein Nlex.Elk. In E the Nlex.Elk/lexOP-CAT reporter plasmids were co-transfected to- gether with vector plasmid (empty) or the expression plasmids for rCD2p110, rCD2p110-R/P, Ha-v-ras, or gagPKB as indicated. Kit225 cells were treated over night with various concentrations of rIL-2 (A), 20 ng/ml rIL-2 plus various concen- trations of PD098059 (B), 20 ng/ml rIL-2 plus various concentrations of wortman- nin (C), 20 ng/ml rIL-2 plus 20 ng/ml of rapamycin (D), or 50 ng/ml PdBu (E), or they were left untreated as indicated. CAT activity was analyzed as described under “Experimental Procedures.” The data are from a representative experi- ment. Similar results were obtained in one (A–D) or three (E) further experi- ments. The CAT activity is presented as percentage of conversion. systems (28, 33, 48), co-expression of activated Ha-v-ras but not activity in Kit225 T cells (Fig. 3B). This response was specific, of active V12Rac led to a moderate rise in PKB activity in since the expression of V14Rho, which does not regulate stress- Kit225 T cells. activated protein kinases (50, 51), did not induce ATF-2/LexA- Co-expression of an Activated Form of PKB Stimulates C2-controlled gene expression. Therefore, the GTPases V12Rac p70S6k in Kit225 T Cells—p70S6k is activated by multiple and V12Cdc42 are active and can stimulate Rac-/Cdc42-regu- serine/threonine phosphorylation in response to mitogenic lated signaling pathways in Kit225 T cells. stimuli. The retroviral oncogene v-Akt is a chimeric molecule, IL-2 Regulates the Transcription Factor Elk-1 in Kit225 Cells consisting of the retroviral Gag protein fused to the N terminus in a PI 3-Kinase-dependent Fashion—In T cells, a PI 3-kinase- of c-Akt, which is myristoylated, and hence v-Akt is predomi- sensitive pathway for regulating the activity of Erk kinase nantly found at the plasma membrane, which may give raise to (Mek) and the Erks has been reported to co-exist alongside the its oncogenicity (49). The expression of constitutively active PI 3-kinase/p70S6k pathway (10). While p70S6k is thought to PKB, gagPKB, has been described as activating p70S6k in exert its mitogenic function by controlling translation initiation Rat-1 cells (32) and COS1 cells (33). Nevertheless, the ability of and protein synthesis, the MAP kinase Erk is implicated in phorbol esters to stimulate p70S6k without any discernible regulating the phosphorylation and activity of certain tran- activation of PKB indicated that PKB-independent pathways scription factors. One well characterized cellular substrate for for activation of p70S6k must exist in T cells. To determine the Erks in fibroblasts and T cells is the transcription factor Elk-1 role of PKB in p70S6k activation in T cells, rIL-2-deprived (40, 52, 53). We therefore tested the ability of IL-2 to regulate Kit225 cells were co-transfected with a gagPKB expression Elk-1 transcriptional activity and hence Erk in Kit225 cells. To vector together with an expression vector encoding HA epitope- monitor Elk-1 transcriptional activity, a fusion protein com- tagged p70S6k. p70S6k activity was analyzed in anti-HA tag prising the C terminus of Elk-1 linked to the LexA repressor immune complexes with S6 ribosomal subunits as a substrate. (41) was co-transfected into Kit225 cells with a LexA operator- Co-expression of constitutively active PKB induced a strong controlled CAT reporter gene. The data in Fig. 4A demonstrate activation of p70S6k that was comparable with increases in that IL-2 can regulate Elk-1 transcriptional activity in Kit225 p70S6k activity seen by co-expression of rCD2p110 (Fig. 3A). A cells. To confirm that Elk-1 transactivation is induced by a rCD2p85 construct that does not regulate cellular levels of D-3 Mek/Erk-sensitive pathway, we investigated the ability of the phosphoinositides (29) did not stimulate p70S6k. In contrast to well characterized inhibitor of Mek activation, PD098059 (54), data described in fibroblasts (48), co-expression of V12Rac and to prevent IL-2-mediated activation of Elk-1. Treatment of V12Cdc42 had no effect on p70S6k activity in Kit225 cells (Fig. Kit225 cells with the PD098059 component inhibited stimula- 3A). To confirm that V12Rac and V12Cdc42 are active in tion of Elk-1 transcriptional activity triggered by rIL-2 (Fig. Kit225 cells, we tested their ability to activate the stress- 4B). Moreover, rIL-2-induction of Elk-1 activity was prevented activated protein kinases, also known as c-Jun N-terminal by the PI 3-kinase inhibitor wortmannin in a dose-dependent kinases (50, 51). Stress-activated protein kinase and hence manner (Fig. 4C), which corroborates earlier studies indicating Rac/Cdc42 activity can be measured by the ability of stress- that Erk activation by IL-2 requires PI 3-kinase function (10). activated protein kinases to phosphorylate the transcription Treatment of Kit225 cells with rapamycin did not affect Elk-1 factor ATF-2 (42). To monitor ATF-2 transcriptional activity, a transactivation in Kit225 cells (Fig. 4D). To assess whether fusion protein comprising the N terminus of ATF-2 (termed C2) constitutively active PI 3-kinase and the in vivo production of linked to the LexA repressor (42) was co-transfected into D-3 phosphoinositides could induce MAP kinase signaling Kit225 cells together with a LexA operator-controlled chloram- pathways in T cells, the ability of rCD2p110 to induce tran- phenicol acetyltransferase (CAT) reporter gene. V12Rac and scriptional activation of Elk-1 was analyzed. LexA-Elk-1 tran- V12Cdc42 potently stimulated ATF-2/LexA-C2 transcriptional scriptional activity was low in quiescent Kit225 cells but could Phosphatidylinositol 3-Kinase Targets in Lymphoid Cells 14431 FIG.5. PI 3-kinase signals are not sufficient to stimulate Erk2 activity but can synergize with phorbol esters to give an in- crease in Erk2 activity in Kit225 cells. HA-Erk2 activity was ana- lyzed from extracts of untreated cells (2) or cells stimulated with 50 ng/ml PdBu for 5 min. Before stimulation, Kit225 cells were co-trans- fected with HA-Erk2 plasmids and vector plasmid (empty) or plasmids encoding for rCD2p110, rCD2p110-R/P, or Ha-v-ras as indicated. Erk assays were performed as described under “Experimental Procedures.” FIG.6. A schematic representation of the IL-2-regulated sig- The data were quantified using a PhosphorImager and are presented as 32 125 naling pathways that involve PI 3-kinase. Binding of IL-2 to its the ratio of [ P]phosphate incorporated into S6 to I-conjugated pro- receptor activates PI 3-kinase, PKB, p70S6k, and the Ras/Raf/Erk tein A bound to Erk (expressed in arbitrary units). The data are from a effector pathway. PI 3-kinase signals are sufficient to stimulate PKB representative experiment. Similar results were obtained in two more and p70S6k. Activated PKB is sufficient to propagate p70S6k activa- experiments. tion. Hence, the available evidence suggests that IL-2 activates PI 3-kinase, which subsequently leads to PKB activation, which in turn be instigated by co-expression of active Ha-v-ras and by stim- stimulates p70S6k. Activation of p70S6k by IL-2, PI 3-kinase, and PKB ulation with phorbol esters, whereas expression of rCD2p110 is sensitive to rapamycin, which indicates that the target of rapamycin, the Frap, is required for p70S6k activation either as a downstream did not stimulate Elk-1 transactivation (Fig. 4E). However, target of PKB (1) or in a parallel pathway (2). p70S6k can also be rCD2p110 signals could potentiate phorbol ester induction of stimulated by phorbol esters via classical or novel PKC isoforms the transcriptional activity of Elk-1. This potentiating effect (cPKC), whereas PKB cannot. p70S6k appears to exert its mitogenic was not observed in cells expressing the kinase-dead function by regulating translation initiation and protein biosynthesis. PI 3-kinase signals are not sufficient to stimulate the MAP kinase Erk rCD2p110-R/P and was thus dependent on the integrity of the and its cellular target, the transcription factor Elk-1. However, PI lipid kinase and the cellular production of D-3 phosphoinosi- 3-kinase signals can synergize with phorbol esters to induce Erk or tides. Moreover, gagPKB cannot mimic the effects of PI 3-ki- Elk-1 activation. Erk and Elk-1 activity is not inhibited by rapamycin, nase on the Erk/Elk-1 pathway (Fig. 4E). and activated PKB does not potentiate phorbol ester induction of Elk-1 PI 3-Kinase Signals Synergize with Phorbol Esters to Induce transcriptional activity. Hence, PI 3-kinase signals bifurcate to activate the PKB/rapamycin-sensitive/p70S6k pathway and independently con- Erk Activity in Kit225 Cells—To assess the effect of membrane- tribute to the Mek/Erk/Elk-1 pathway. PI 3-kinase signals are impli- localized PI 3-kinase on Erk activity directly, rIL-2-deprived cated to contribute to the Ras/Raf/Mek/Erk pathway at the level of Mek, Kit225 cells were co-transfected with expression vectors encod- since IL-2 induction of Mek and Erk but not Ras and Raf are sensitive ing rCD2p110 and HA epitope-tagged p42 Erk2, and cells were to wortmannin. Possible mediators of PI 3-kinase action on Mek/Erk are the novel/atypical members of the PKC family (n/aPKC)(1)orthe stimulated with phorbol esters or left untreated. Co-expression GTPase Rac (2). of rCD2p110 did not stimulate Erk2 activity, although Erk2 could be activated by co-expressing the activated Ras, Ha-v-ras nal pleckstrin homology domain that can directly bind D-3 (Fig. 5). These results thus confirm the data in Fig. 4E indicat- phosphoinositides (33, 55, 56), which may contribute to the ing that PI 3-kinase signals are not sufficient to activate the regulation of the enzyme. Since PI 3-kinase signals are suffi- Erk/Elk-1 pathway. The data in Fig. 5 demonstrate that active cient to substitute for IL-2 in PKB activation, PKB could be a PI 3-kinase markedly potentiated the level of Erk2 activation direct target for PI 3-kinase signals during IL-2 signal trans- triggered by phorbol esters, an effect that was not observed in duction. PKB/c-Akt is highly expressed in the thymus (57), and cells expressing the kinase-dead rCD2p110-R/P. PI 3-kinase the oncogenic form of this kinase causes thymic malignancies. signals did not enhance IL-2 activation responses on Erk (data Therefore, PKB has a pivotal role in controlling T cell prolifer- not shown). Taken together, the results in Figs. 4E and 5 ation/differentiation. The present data identify one function for clearly demonstrate that although PI 3-kinase signals are not PKB in T cells; PKB action is sufficient to stimulate p70S6k. sufficient for Erk/Elk-1 activation, they can synergize with Moreover, PI 3-kinase signals are sufficient for activation of phorbol esters to induce a maximal response. These results are p70S6k, which stresses the close link between PI 3-kinase and concordant with a model where PI 3-kinase signals bifurcate to PKB in regulating p70S6k activity in T cells. Questions regard- activate the PKB/rapamycin-sensitive/p70S6k pathway and in- ing the selectivity of the inhibitors that were first used to define dependently contribute to the Mek/Erk/Elk-1 pathway via an a role for PI 3-kinase in T cell biology have challenged the as yet undefined mechanism (see Fig. 6). involvement of this enzyme in the regulation of p70S6k in T DISCUSSION cells (26). The present data resolve this controversy and pro- The present study has used a membrane-targeted, constitu- vide unequivocal evidence that PI 3-kinase can function as an tively active, catalytic subunit of PI 3-kinase as a tool to iden- upstream regulator of p70S6k in T cells. tify direct targets of PI 3-kinase action in IL-2 signal transduc- Results obtained recently with p110 constructs that were tion pathways. We demonstrate that the serine/threonine membrane-targeted by myristoylation or farnesylation signals kinase PKB/Akt can be activated by the cytokine IL-2 via a PI showed that PI 3-kinase signals are sufficient to activate PKB 3-kinase-dependent pathway. Importantly, PI 3-kinase signals and p70S6k in COS cells (28). It has also been shown in fibro- alone are sufficient to activate PKB in T cells, demonstrating blasts that the GTPases Rac and Cdc42 induce p70S6k activa- that PI 3-kinase acts as an upstream regulator of this serine/ tion. We find no evidence for Rac/Cdc42 activation of p70S6k in threonine kinase in lymphoid cells. PKB contains an N-termi- T cells, indicating that cells of different lineages can differ 14432 Phosphatidylinositol 3-Kinase Targets in Lymphoid Cells markedly in their cellular mechanisms for kinase activation. (data not shown) or by active PKB (32, 33), thus indicating that Nevertheless, the present data show a striking conservation of PI 3-kinase or PKB activation signals cannot bypass the role of the PI 3-kinase/PKB/p70S6k link in human T cells and simian Frap in p70S6k activation pathways. A simple interpretation of fibroblasts. The conservation of the PI 3-kinase/PKB/p70S6k these data is that PI 3-kinase and PKB activation of p70S6k is mediated by Frap, although the possibility cannot be excluded signaling cascade in T cells implies a physiological importance of this pathway, which has guaranteed its evolutionary that Frap regulates p70S6k by an essential signaling pathway operating in parallel with PI 3-kinase/PKB signals (Fig. 6). conservation. Frap controls p70S6k activation by regulating the phosphoryl- The role of PI 3-kinase as an upstream regulator of the Erk ation of key residues in the enzyme (63, 64). Nevertheless, kinase pathways can also vary depending on the cell system; p70S6k is not a direct substrate for Frap, and some interme- expression of an active PI 3-kinase is sufficient for Erk activa- diate p70S6k kinase(s), as yet uncharacterized, must be in- tion in Xenopus oocytes (27) but not in fibroblasts or mono- voked to explain the role of Frap in p70S6k activation. Al- blasts (28 –31). The present data show directly that PI 3-kinase though the evidence that PKB mediates PI 3-kinase effects on can have a positive regulatory role in Erk activation in T cells p70S6k are compelling, these data do not exclude the possibil- (see Fig. 6). However, PI 3-kinase signals alone fail to stimulate ity that there are PKB-independent mechanisms for p70S6k Erk signaling pathways but markedly potentiate Erk re- activation of T cells. In this context, the present data show that sponses in combination with phorbol esters. Erk regulation of activation of PKC by phorbol esters stimulates p70S6k without downstream nuclear targets is hereby analyzed using the any discernible stimulatory effect on PKB. transactivation capacity of the ternary complex factor Elk-1, a Recent studies showing that cytokine activation of serine well characterized substrate for Erks in fibroblasts and Jurkat kinases is important for the regulation of apoptosis (65, 66) T cells (40, 53). We establish that Elk-1 is regulated by IL-2 via have focused attention on cytokine-induced serine kinase cas- Mek- and PI 3-kinase-sensitive pathways. Furthermore, as cades. PI 3-kinase and PKB have been implicated in the pre- observed in direct Erk activation assays, PI 3-kinase signals vention of apoptosis in other cell systems (67, 68). The present potently enhance phorbol ester induction of Elk-1 transcrip- study demonstrates that PI 3-kinase can couple the IL-2R to a tional activity. PI 3-kinase signals may thus be required for selective subset of serine/threonine kinase pathways in T cells, activation of MAP kinase pathways in IL-2-dependent T cells, and in this respect, the PI 3-kinase/PKB link is intriguing, but they are not sufficient and hence are one component of a since PKB mediates activation of the Frap/p70S6k pathway but more complex signaling network. We have not yet explored the may also regulate other kinase cascades that bifurcate from the PI 3-kinase effector pathways involved in Erk activation, al- PKB/p70S6k pathway including glycogen synthase kinase-3 though previous data have excluded the involvement of the (GSK3) signaling pathways (69). Therefore, PI 3-kinase and/or Frap/p70S6k pathway, since activation of Erk is not sensitive PKB have the potential for pleiotropic functions in T cells, and to rapamycin inhibition. Moreover, PKB, which is a potent their downstream effectors may include additional serine/thre- activator of p70S6k, cannot mimic the effects of activated PI onine kinases evoked by IL-2R engagement. 3-kinase on the Erk/Elk-1 pathway. These data best fit a model Finally, PI 3-kinase is activated by members of the cytokine in which PI 3-kinase regulation of MAP kinases and PKB/ receptor family such as the IL-2R, the IL-4 receptor, the IL-7 p70S6k bifurcate prior to activation of PKB (Fig. 6). Members receptor, and the IL-13 receptor. Signaling pathways regulated of the Rho family of GTPases can potentiate Erk activation by PI 3-kinase can hence have an impact on lymphocyte biology pathways in fibroblasts (39). Activation of PI 3-kinase is suffi- at multiple points. Accordingly, it is important to establish the cient to induce cytoskeletal rearrangements mediated by the function of this enzyme in lymphoid cells. The IL-2R is a GTPase Rac and Rho in Swiss 3T3 cells (29) and thus has the prototypical member of this hematopoietin receptor family. The potential to regulate Rac/Rho signaling pathways in T cells. present results directly define PI 3-kinase function in T cells Accordingly, it is possible that Rac/Rho family GTPases could and position PKB for the first time in a physiologically relevant mediate PI 3-kinase regulation of Erk. However, several other cytokine-induced signal transduction pathway in lymphoid candidate in vivo targets for D-3 phosphoinositides have been cells. The model described herein may also be applicable to proposed including members of the novel PKC family (58) and serine/threonine kinase pathways regulated by other receptors l (59) and PKC-z (60), which has the atypical PKC family, PKC- that activate PI 3-kinase in T cells. recently been implicated as a regulator of Mek and Erk activity in COS cells (61). Acknowledgments—We thank George Thomas and Richard Treis- man for reagents. p70S6k plays a key role in cellular growth control mecha- nisms by coordinating protein biosynthesis via phosphorylation Note Added in Proof—Recently, Alessi and colleagues described the of the S6 subunit of 40 S ribosomes or via regulation of the purification of two upstream kinases that are likely to mediate PKB activity of the eukaryotic initiation factor 4E binding protein, activation. The activity of at least one of these upstream kinases, PKD1, is regulated by binding D-3 polyphosphate phosphoinositides (Alessi, D. 4E-BP1 (24, 25, 62). Expression of an activated PKB can stim- R., James, S. R., Downes, C. P., Holmes, A. B., Gaffney, P. R. J., Reese, ulate p70S6k activity in T cells, indicating that PKB substrates C. B., and Cohen, P. (1997) Curr. Biol. 7, 261–269). are part of the p70S6k activation pathways. 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Phosphatidylinositol 3-Kinase Links the Interleukin-2 Receptor to Protein Kinase B and p70 S6 Kinase

Reif, Karin; Burgering, Boudewijn M.T.; Cantrell, Doreen A.
Journal of Biological Chemistry – May 1, 1997
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 22, Issue of May 30, pp. 14426 –14433, 1997 © 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Phosphatidylinositol 3-Kinase Links the Interleukin-2 Receptor to Protein Kinase B and p70 S6 Kinase* (Received for publication, February 18, 1997, and in revised form, March 24, 1997) Karin Reif‡, Boudewijn M. T. Burgering§, and Doreen A. Cantrell From the Lymphocyte Activation Laboratory, Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London WC2A 3PX, United Kingdom and the §Laboratory for Physiological Chemistry, Utrecht University, Universiteitsweg 100, Utrecht 3584 CG , The Netherlands Phosphatidylinositol 3-kinase (PI 3-kinase) is acti- lyzes the phosphorylation of phosphoinositides at the D-3 hy- vated by the cytokine interleukin-2 (IL-2). We have used droxyl of the myo-inositol ring generating PI 3-phosphate, PI a constitutively active PI 3-kinase to identify IL-2-medi- 3,4-bisphosphate, and PI 3,4,5-trisphosphate (13, 14). The form ated signal transduction pathways directly regulated by of PI 3-kinase involved in protein-tyrosine kinase-dependent PI 3-kinase in lymphoid cells. The serine/threonine pro- receptor signal transduction comprises a regulatory 85-kDa tein kinase B (PKB)/Akt can act as a powerful oncogene subunit that contains two Src homology 2 domains and at its N in T cells, but its positioning in normal T cell responses terminus one Src homology 3 domain and a catalytic 110-kDa has not been explored. Herein, we demonstrate that subunit. Following IL-2R stimulation, several mechanisms PKB is activated by IL-2 in a PI 3-kinase-dependent have been proposed to recruit PI 3-kinase to the plasma mem- fashion. Importantly, PI 3-kinase signals are sufficient brane, where its cellular substrate PI 4,5-bisphosphate is lo- for PKB activation in IL-2-dependent T cells, and PKB is cated: engagement of the IL-2R leads to binding of the p85 a target for PI 3-kinase signals in IL-2 activation path- regulatory subunit of PI 3-kinase to tyrosine 392 in the IL-2R ways. The present study establishes also that PI 3-kinase b-chain (15); in addition, interleukin-2 (IL-2) stimulation re- signals or PKB signals are sufficient for activation of sults in the interaction of PI 3-kinase with the Src family p70 S6 kinase in T cells. PI 3-kinase can contribute to, kinases Fyn (16) and Lck (17). but is not sufficient for, activation of extracellular sig- The activation of PI 3-kinase is a response that IL-2 shares nal-regulated kinases (Erks) and Erk effector pathways. with other cytokines that control lymphoid cell growth and Therefore, PI 3-kinase is a selective regulator of serine/ development such as IL-4 and IL-7 (18, 19). It is also clear that threonine kinase signal transduction pathways in T PI 3-kinase activation is necessary for the growth- and differ- lymphocytes, and this enzyme provides a crucial link entiation-inducing properties of these cytokines (20 –23). How- between the interleukin-2 receptor, the protooncogene PKB, and p70 S6 kinase. ever, despite the pivotal role of PI 3-kinase in lymphoid cells, there is only a preliminary and incomplete understanding of the targets for this enzyme in the mitogenic signaling path- The high affinity interleukin-2 receptor (IL-2R), which com- ways regulated by the hematopoietin family of cytokines. To prises a-, b-, and g-subunits controls G to S progression, T cell date, the identification of biochemical targets for PI 3-kinase in clonal expansion, and functional differentiation (1–3). The T cells stems mainly from studies employing the PI 3-kinase IL-2R orchestrates downstream effector pathways by protein inhibitor wortmannin or the LY294002 compound (10, 20). tyrosine kinase-dependent activation mechanisms engaging Hence, IL-2 activation of the mitogen-activated protein (MAP) the Src family tyrosine kinases Lck and Fyn (4) and the Janus kinase Erk is sensitive to wortmannin (10). Similarly, IL-2 kinases 1 and 3 (5–7). Signaling cascades integrated by the activation of the serine/threonine kinase p70 S6 kinase action of these tyrosine kinases include activation of the Ras/ (p70S6k) is prevented by these PI 3-kinase inhibitors (20). In Raf/extracellular-signal regulated kinase (Erk) pathway (8 – addition, IL-2 activation of p70S6k is impeded by the immuno- 10), activation of the transcription factors STAT3 and STAT5 supressant rapamycin, which targets another member of the PI (11), and the regulation of phosphatidylinositol 3-kinase (PI 3-kinase family of enzymes, Frap (for FKBP12-rapamycin-as- 3-kinase) (12). sociated protein) also termed “mammalian target of rapamy- PI 3-kinase is a ubiquitously expressed enzyme that cata- cin” (mTor) (24, 25). Observations that wortmannin and rapa- mycin have identical inhibitory effects on IL-2 activation of p70S6k generated a model for the p70S6k signaling pathway in * This work was supported by the Imperial Cancer Research Fund and by Human Capital Mobility Program Grant ERB CHRX CT 94- which PI 3-kinase acts as an upstream regulator of Frap (24, 0537. The costs of publication of this article were defrayed in part by the 25). However, this model has been challenged by a recent study payment of page charges. This article must therefore be hereby marked showing that the action of Frap is directly inhibited by wort- “advertisement” in accordance with 18 U.S.C. Section 1734 solely to mannin and LY294002 (26). These results raise the issue of indicate this fact. whether PI 3-kinase itself has any upstream regulatory role in ‡ Supported by a Boehringer Ingelheim Fellowship. To whom corre- spondence should be addressed. Tel.: 0171-269-3307; Fax: 0171-269- p70S6k activation in T lymphocytes. Similar caution must be 3479; E-mail: [email protected]. applied to interpretations of data involving PI 3-kinase in Erk The abbreviations used are: IL-2R, interleukin-2 receptor; Erk, activation in T cells. In this context, expression of an active PI extracellular-signal regulated kinase; PI, phosphatidylinositol; IL, in- 3-kinase is sufficient for Erk activation in Xenopus oocytes (27), terleukin; MAP, mitogen-activated protein; p70S6k, p70 S6 kinase; Frap, FKBP12-rapamycin-associated protein; PKB, protein kinase B; but it would be fallacious to extrapolate data obtained in Xe- PdBu, phorbol 12,13-dibutyrate; Ab, antibody; mAb, monoclonal anti- nopus cells to T cells, since the role of PI 3-kinase as an body; CAT, chloramphenicol acetyltransferase; PKC, protein kinase C; upstream regulator of kinase pathways can vary depending on Mek, Erk kinase; HA, hemagglutinin; rIL-2, recombinant IL-2; Mops, the cell system; to this end, PI 3-kinase signals did not stimu- 4-morpholinepropanesulfonic acid; PAGE, polyacrylamide gel electro- phoresis; H2B, histone 2B; rCD2, rat CD2. late Erk activity in a variety of fibroblasts and in a monoblast 14426 This paper is available on line at http://www-jbc.stanford.edu/jbc/ This is an Open Access article under the CC BY license. Phosphatidylinositol 3-Kinase Targets in Lymphoid Cells 14427 cell line (28 –31). Whether PI 3-kinase signals are sufficient to stimulate p70S6k or Erk activation in T cells awaits analysis. We and others have recently reported that targeting the catalytic p110 subunit of PI 3-kinase to the plasma membrane generates a constitutively active enzyme that induces cellular accumulation of D-3 phosphoinositides (28 –31). A constitu- tively active PI 3-kinase finally allows assessment of the rela- tive contribution of PI 3-kinase-derived signals to a certain effector pathway, in particular whether PI 3-kinase activation is sufficient to promote a specific cellular response. In the present study, we have used a membrane-localized p110 con- struct, rCD2p110, that induces accumulation of cellular levels of PI 3,4-bisphosphate and PI 3,4,5-trisphosphate (29) as a tool to explore the regulation of serine/threonine kinase pathways by PI 3-kinase in T lymphocytes. We show that activation of PI 3-kinase is sufficient to stimulate p70S6k activity, although PI 3-kinase signals were not sufficient to induce activation of the MAP kinase Erk2 in T cells. The present study also character- izes a previously unrecognized IL-2-mediated signal transduc- tion pathway in T cells that involves the serine/threonine pro- tein kinase B (PKB) also known as c-Akt or Rac protein kinase (32–34). PKB was originally identified as the cellular homo- logue of the directly transforming oncogene of the murine ret- rovirus AKT8, which causes thymic lymphomas (35). Herein, we demonstrate that PKB is rapidly activated by IL-2 via a wortmannin- and LY294002-sensitive but rapamycin-insensi- tive pathway. PI 3-kinase signals alone were sufficient to acti- vate PKB in T cells, and expression of a constitutively active PKB could stimulate the activity of p70S6k. Therefore, PI 3-kinase is a selective regulator of serine/threonine kinase sig- nal transduction pathways in T lymphocytes, and this enzyme is an upstream regulator of the IL-2-activated kinases PKB and p70S6k. EXPERIMENTAL PROCEDURES Reagents—Phorbol 12,13-dibutyrate (PdBu) and wortmannin were FIG.1. Interleukin-2 activates p70S6k, which can be mimicked from Calbiochem. LY294002 was a gift from Zeneca. PD098059 was by co-expression of membrane-localized constitutively active PI from New England Biolabs. Rapamycin was a gift from G. Thomas 3-kinase, rCD2p110. A, Kit225 cells were deprived of rIL-2 for 68 h 14 32 (FMI, Basel). [ C]Acetyl coenzyme A (at 50 mCi/mmol), [g- P]ATP and treated with 20 ng/ml rIL-2 or 50 ng/ml PdBu for the indicated (5000 Ci/mmol), and I-conjugated protein A were from Amersham times, and p70S6k activation/phosphorylation was assessed by electro- Corp. mobility shift assays (top). p70S6k was precipitated from lysates with Antibodies—Ox34 monoclonal antibody (mAb) is raised against rat M5 Abs, and p70S6k activity was analyzed in immune complex kinase CD2 (rCD2) (29); 12CA5 mAb is reactive with hemagglutinin (HA), and assays using S6 as a substrate. [ P]phosphate incorporation into S6 9E10 mAb is reactive with the Myc epitope (36); anti-human S6 kinase (middle) was quantified (graph) using a PhosphorImager and is ex- M5 antiserum (37) was from Santa Cruz Biotechnology; M1 antiserum pressed in arbitrary units. Protein levels of p70S6k present in immune reactive with p70S6k (37) was a gift from G. Thomas; Rac-PK-CT Ab complexes were assessed in parallel by Western blotting with M1 Abs (Upstate Biotechnology, Inc.) is reactive with PKB. (bottom). B and C, HA-p70S6k activity was analyzed from extracts of Plasmids and Reporter Constructs—HA-p70S6k (pBJ5) (38); HA- untreated cells (2) or cells stimulated with either 20 ng/ml rIL-2 or 50 ng/ml PdBu for 15 min. B, before stimulation, Kit225 cells were co- PKB (pSG5) and gagPKB (pSG5) (32); HA-Erk2 (pCEP4) (39); Myc- transfected with HA-p70S6k plasmids and vector plasmid (empty) or V12Rac (pEF), Myc-V12Cdc42 (pEF), and Myc-V14Rho (pEF) (40); and with plasmids encoding for rCD2p110 as indicated. C, Kit225 cells were Ha-v-ras (pEF) (29) vector constructs have been described. The de- co-transfected with HA-p70S6k plasmids and 15 mg each of vector scribed rCD2p110, rCD2p110-R/P, and rCD2p85 chimeras (29) were plasmid (empty) or plasmids encoding for rCD2p110 or rCD2p110-R/P. subcloned into the pEF-BOS expression vector. The reporter plasmids B, S6 substrate phosphorylation from S6 kinase assays (top) was ana- Nlex.Elk-1 (pEF) and 2lexoptk.CAT (41) as well as Nlex.C2 (pMLV) (42) lyzed by autoradiography. Levels of p70S6k in immune complexes (bot- have been described. tom) were analyzed by immunoblotting using M1 antibodies followed by Cell Culture and Transient Transfections—The Kit225 T leukemic I-conjugated protein A and autoradiography. C, the data were ana- cell line (43) was maintained in RPMI 1640 medium containing 10% lyzed as in B and quantified using a PhosphorImager. Data are pre- heat-inactivated fetal calf serum supplemented with 20 ng/ml of recom- 32 125 sented as the ratio of [ P]phosphate incorporated into S6 to I- binant IL-2 (rIL-2) (Eurocetus) under normal growth conditions. For conjugated protein A bound to p70S6k (expressed in arbitrary units). IL-2 activation assays of endogenous proteins, Kit225 cells were washed The data are from a representative experiment. Similar results were three times with phosphate-buffered saline A to remove the IL-2 and obtained in two (A and B) or five (C) more experiments. cultured further in RPMI supplemented with 5% fetal calf serum in the absence of rIL-2 for 48 –72 h prior to IL-2 activation assays. When Kit225 cells were transfected, cells were treated as above but only V12Rac, V12Cdc42, gagPKB, or rCD2p85; 7.5 mg of 2lexoptk.CAT; and deprived of rIL-2 for 24 h prior to transfection. 15 mg of pEFNlex.Elk-1 or pMLVNlex.C2. For gene reporter assays, Kit225 cells were transfected by electroporation with 20 – 40 mgof cells were stimulated as indicated 2– 4 h after transfection. Cells were plasmid DNA. The amounts of plasmid DNA were kept constant per collected 14 –18 h after transfection. cuvette by adding vector plasmid. Kit225 cells (1.5 3 10 cells/0.625 ml) Immunoprecipitation, p70S6k Assays, and Western Blot Analysis— were pulsed at 320 V and 960 microfarads using a Gene Pulser (Bio- After stimulations as indicated, Kit225 cells were lysed in lysis buffer 1 Rad). The amounts of plasmid used were as follows (unless indicated (120 mM NaCl, 50 mM Tris pH 8.0, 20 mM NaF, 1 mM benzamidine, 1 mM otherwise): 7.5 mg of HA-p70S6k; 12.5 mg of HA-PKB; 10 mg of HA-Erk2; EDTA, 6 mM EGTA, 7.5 mM PP ,15mM p-nitrophenyl phosphate, 1% 20 mg of the plasmid pEF empty, rCD2p110, rCD2p110-R/P, Ha-v-ras, Nonidet P-40, 0.1 mM phenylmethylsulfonyl fluoride, and 0.1 mM 14428 Phosphatidylinositol 3-Kinase Targets in Lymphoid Cells Na VO ). Cell extracts corresponding to 3 3 10 cell equivalents (non- 3 4 transfected cells) or 1.5 3 10 live cell equivalents (transfected cells) were used for each immunoprecipitation. Postnuclear lysates were pre- cleared with protein A cell suspension (Sigma) prior to incubation with 2 mg of 12CA5 mAbs or, for endogenous proteins, 1 mg of M5 Abs. Immune complexes were precipitated with protein G-Sepharose beads (Sigma) or, when M5 Abs were used, with protein A-Sepharose beads (Pharmacia Biotech Inc.). The immunoprecipitates were washed three times in lysis buffer 1 and once in p70S6k assay buffer (50 mM Mops, pH 7.2, 5 mM MgCl , 0.1% Triton X-100) and assayed as described (37) using S6 as a substrate (a gift from G. Thomas). Proteins were resolved by SDS-PAGE. The lower part of the gel was dried, and P-labeled S6 proteins were detected by autoradiography. The levels of p70S6k pro- tein in each immunoprecipitate were assessed by transferring the pro- teins in the upper part of the gel onto polyvinylidene difluoride mem- branes and performing Western blot analysis with 12CA5 mAbs or M1 Abs using the ECL detection system (Amersham). If the p70S6k protein levels in the immunoprecipitate were not equal, activities were normal- ized for p70S6k expression levels by quantitation of Western blots probed with M1 Abs followed by I-conjugated protein A (Amersham). 32 125 Quantitation of incorporated P into S6 or of bound I-conjugated protein A was performed using a PhosphorImager (Molecular Dynam- ics). To test for effector protein expression in transfected cells, post- nuclear cell extracts corresponding to 3 3 10 cell equivalents of the same extract as above were analyzed by Western blotting as described (44) using 9E10, Ox34, or specific Abs. PKB Assays—Cells were treated as for p70S6k assays except that lysis buffer 2 (120 mM NaCl, 50 mM Hepes, pH 7.4, 10 mM NaF, 1 mM EDTA, 40 mM b-glycerophosphate, pH 7.5, 1% Nonidet P-40, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM Na VO ) was used to lyse cells. 3 4 To immunoprecipitate endogenous PKB, 2 mg of Rac-PK-CT Abs were used. The immunoprecipitates were washed twice in lysis buffer 2, twice in high salt wash buffer (500 mM LiCl, 100 mM Tris, pH 7.5, 1 mM EDTA, pH 7.5), and once in PKB assay buffer (50 mM Tris, pH 7.5, 10 mM MgCl ,1mM dithiothreitol). The reaction was initiated by the addition of 15 ml of PKB reaction buffer containing 3 mCi of [g- P]ATP, 50 mM ATP, 7.3 mM MgCl , 730 mM dithiothreitol, 500 nM protein kinase inhibitor (Sigma), 40 mM Tris, pH 7.5, and 2.5 mg of histone 2B (H2B) (Boehringer Mannheim). After 30 min at 25 °C, the reaction was ter- minated by adding reducing SDS-PAGE sample buffer and boiling. Proteins were resolved by SDS-PAGE, and the gel was treated as for p70S6k assays. To detect PKB proteins, Western blot analysis was performed with Rac-PK-CT Abs. Erk Assays—Cells and cell extracts were processed as for p70S6k assays. HA-tagged Erk2 was immunoprecipitated with 12CA5 mAbs. Precipitated immune complexes were washed three times with lysis buffer 1 and once with Erk wash buffer (30 mM Tris, pH 8.0, 20 mM MgCl ,2mM MnCl ). The reaction was initiated by the addition of 10 ml 2 2 of Erk reaction buffer containing 4 mCi of [g- P]ATP, 20 mM ATP, 20 mM MgCl ,2mM MnCl ,5mM p-nitrophenyl phosphate, 500 nM protein 2 2 kinase inhibitor, 30 mM Tris, pH 8.0, and 15 mg of myelin basic protein (Sigma). After 30 min at 37 °C, the reaction was terminated by adding reducing SDS-PAGE sample buffer and boiling. Proteins were resolved by SDS-PAGE, and the gel was treated as for p70S6k assays. To detect Erk proteins, Western blot analysis was performed with 12CA5 mAbs as primary Ab, rabbit anti-mouse IgG as secondary Ab, and I-conju- gated protein A. Gene Expression Analysis—Fourteen to 16 h after inductions, as indicated, Kit225 T cells were harvested and cells were lysed in 200 ml of lysis buffer (0.65% Nonidet P-40, 10 mM Tris, pH 8, 1 mM EDTA, 150 FIG.2. Interleukin-2 activates PKB: PI 3-kinase activity is nec- mM NaCl). Gene expression assays were carried out as described (45). essary for IL-2-mediated activation of PKB, and PI 3-kinase The data are presented as percentage of conversion. signals are sufficient to stimulate PKB activity in Kit225 T cells. A, interleukin-2 activates PKB. Kit 225 cells were deprived of rIL-2 for 68 h and treated with 20 ng/ml rIL-2 or 50 ng/ml PdBu for the indicated times, and PKB activation/phosphorylation was assessed by electromo- bility shift assays (top). PKB was precipitated from lysates with Rac- the times indicated before lysis. PKB activity was measured in immune PK-CT Abs and PKB activity was analyzed in immune complex kinase complex kinase assays using H2B as a substrate. C, PI 3-kinase signals assays using H2B as a substrate. [ P]Phosphate incorporation into trigger a potent stimulation of PKB activity. Kit225 cells were co- H2B (middle) was quantified (graph) using a PhosphorImager and is transfected with HA-PKB plasmids and vector plasmid (empty), or expressed in arbitrary units. Protein levels of PKB present in immune plasmids encoding for rCD2p110, rCD2p110-R/P, Ha-v-ras, or V12Rac complexes were assessed in parallel by Western blotting with Rac- as indicated. HA-PKB activity was analyzed in anti-HA tag immune PK-CT Abs (bottom). B, the PI 3-kinase inhibitors wortmannin and complex kinase assays using H2B as a substrate. B and C,[ P]phos- LY294002, but not rapamycin, inhibit IL-2-dependent activation of phate incorporation into H2B (top) was quantified (graph) using a PKB. Kit225 cells starved of rIL-2 for 72 h were pretreated for 30 min PhosphorImager and is expressed in arbitrary units. Protein levels of with the vehicle dimethyl sulfoxide (DMSO), 20 ng/ml rapamycin, 5 mM PKB (B) or HA-PKB (C) present in immune complexes were assessed in LY294002, or 100 nM wortmannin and then stimulated with rIL-2 for parallel by Western blotting with Rac-PK-CT Abs (bottom). Phosphatidylinositol 3-Kinase Targets in Lymphoid Cells 14429 expression of the rCD2p110 chimera was confirmed by flow cytometric immunofluorescence analysis with rCD2 mAbs (data not shown). The HA-tagged p70S6k was immunoprecipi- tated from transiently transfected cells and assayed for its ability to phosphorylate S6 ribosomal subunits (Fig. 1B). Ex- pression of the active PI 3-kinase, rCD2p110, resulted in con- stitutive IL-2-independent p70S6k activation (Fig. 1B). p70S6k was not constitutively activated in cells expressing “kinase- dead” rCD2p110-R/P, confirming that the p70S6k activation requires the kinase activity of the p110 subunit (Fig. 1C). The expression of rCD2p110-R/P was noted in some experiments to suppress rIL-2 inducibility of p70S6k, indicating that this chi- mera may be an inhibitory mutant of PI 3-kinase pathways. IL-2 and Membrane-localized PI 3-Kinase Activate PKB— PKB can be activated by receptor tyrosine kinases such as the platelet-derived growth factor receptor and has been identified as a target of PI 3-kinase in fibroblasts (28, 32, 33). However, whether this pathway is conserved in the hematopoietic system has not been explored. In particular, although PKB can become oncogenic and initiate thymic tumors, its regulation and sig- nificance for normal T cell growth processes is not known. Since cytokine receptors have essential functions in the development and maintenance of the hematopoietic system, we were inter- ested to assess whether members of the hematopoietin receptor family, such as the prototypical IL-2R, regulate PKB. To exam- ine whether IL-2 activates PKB, immunoprecipitates of this kinase were prepared from rIL-2-deprived and rIL-2-activated FIG.3. Co-expression of constitutively active forms of PKB Kit225 cells and subjected to in vitro kinase assays using H2B and PI 3-kinase but not of the GTPases Rac and Cdc42 stimu- as a substrate. The data in Fig. 2A show that IL-2 induced a lates p70S6k activity in Kit225 cells. A, HA-p70S6k activity was rapid activation of PKB. A 2–3-fold increase in enzyme activity analyzed from extracts of untreated cells (2) or cells stimulated with 20 ng/ml rIL-2 for 15 min. Before stimulation, Kit225 cells were co-trans- over basal levels was sustained for more than 60 min in re- fected with HA-p70S6k plasmids and vector plasmid (empty) or plas- sponse to rIL-2. PKB activity is regulated by phosphorylation mids encoding for rCD2p110, gagPKB, V12Rac, V12Cdc42, or rCD2p85 as indicated by the reduced electrophoretic mobility of PKB as indicated. p70S6k assays were performed as described under “Ex- isolated from rIL-2-activated cells (Fig. 2A). PKB activity was perimental Procedures.” The data were quantified using a PhosphorIm- ager and are presented as the ratio of [ P]phosphate incorporated into not induced by exposure of Kit225 cells to phorbol esters that S6 to I-conjugated protein A bound to p70S6k (expressed in arbitrary activate PKC (Fig. 2A). The data in Fig. 2B show the failure of units). B, Kit225 cells were transfected with the LexA operator-con- rIL-2 to stimulate PKB in cells pretreated with LY294002 or trolled CAT reporter plasmid (lexOP-CAT) and the expression plasmid wortmannin, two well characterized PI 3-kinase inhibitors that producing the LexA-C2 fusion protein Nlex.C2 together with vector plasmid (empty) or the expression plasmids for V14Rho, V12Rac, and bind to the ATP or lipid binding sites on the p110 catalytic V12Cdc42 as indicated. After 18 h, Kit225 T cells were harvested, and subunit, respectively. These inhibitors also prevent the autoki- CAT activity was analyzed as described under “Experimental Proce- nase activity of Frap/mTor (26), a member of the PI 3-kinase dures.” The CAT activity is presented as percentage of conversion. The family (47), which is the cellular target for the drug rapamycin data are from a representative experiment. Similar results were ob- tained in one further experiment. and which prevents IL-2-coordinated cell cycle progression and proliferation of T lymphocytes (24, 25). Frap activity is abso- RESULTS lutely required for p70S6k action in T cells (24, 25). We there- fore assessed whether Frap function was necessary for IL-2- IL-2 and PI 3-Kinase Signals Activate p70S6k in Kit225 induced stimulation of PKB. Rapamycin had no effect on IL-2- Cells—For our studies, we used the well characterized human triggered activation of PKB (Fig. 2B), although rapamycin IL-2-dependent T cell line Kit225. The data in Fig. 1A show completely abolished IL-2- or PI 3-kinase-controlled induction that p70S6k activity is low in quiescent rIL-2-deprived Kit225 of p70S6k (data not shown). Thus, the inhibition of PKB by cells but can be rapidly stimulated by rIL-2. The activity of wortmannin and LY294002 cannot be caused by prevention of p70S6k is increased in response to phorbol esters that stimu- Frap activity and indicate that IL-2 regulation of PKB employs late protein kinase C (PKC) (Fig. 1A). We asked whether PI PI 3-kinase. 3-kinase signals could substitute for IL-2 in inducing p70S6k To investigate directly whether PI 3-kinase signals are suf- activity. We have shown recently that plasma membrane tar- ficient to activate PKB, rIL-20-deprived Kit225 cells were co- geting of p110, the catalytic subunit of PI 3-kinase, generates transfected with either rCD2p110 or rCD2p110-R/P expression an enzyme that is constitutively active in vivo (29). Our mem- vectors together with an expression vector encoding HA brane-targeted active PI 3-kinase construct comprises a chi- epitope-tagged PKB. In addition, the ability of activated forms mera of the extracellular and transmembrane domains of the rCD2 antigen fused to the p110a catalytic domain of PI 3-ki- of the small GTPases Ha-v-ras and V12Rac to activate PKB was assessed. Immunoprecipitates of HA-tagged PKB were nase, rCD2p110. As a control, we used a rCD2p110 molecule that contained in the catalytic subunit of PI 3-kinase an inac- assayed for kinase activity using H2B as a substrate. The tivating point mutation (R1130P) in the ATP binding site (46) constitutively active PI 3-kinase rCD2p110 induced a robust that abolishes its in vivo and in vitro lipid kinase activity, activation of PKB (Fig. 2C). This stimulatory effect of rCD2p110-R/P. To assess the effects of PI 3-kinase signals on rCD2p110 was dependent on the kinase activity of the chimera, p70S6k activity, rIL-2-starved Kit225 cells were co-transfected since co-expression of kinase-inactive rCD2p110-R/P did not with HA epitope-tagged p70S6k and rCD2p110. Cell surface stimulate PKB activity. As observed previously in other cell 14430 Phosphatidylinositol 3-Kinase Targets in Lymphoid Cells FIG.4. Interleukin-2 but not rCD2p110 induces Elk-1-dependent gene expression. A–E, Kit225 cells were transfected with the LexA operator-con- trolled CAT reporter plasmid (lexOP- CAT) plus the expression plasmid pro- ducing the LexA-Elk-1 fusion protein Nlex.Elk. In E the Nlex.Elk/lexOP-CAT reporter plasmids were co-transfected to- gether with vector plasmid (empty) or the expression plasmids for rCD2p110, rCD2p110-R/P, Ha-v-ras, or gagPKB as indicated. Kit225 cells were treated over night with various concentrations of rIL-2 (A), 20 ng/ml rIL-2 plus various concen- trations of PD098059 (B), 20 ng/ml rIL-2 plus various concentrations of wortman- nin (C), 20 ng/ml rIL-2 plus 20 ng/ml of rapamycin (D), or 50 ng/ml PdBu (E), or they were left untreated as indicated. CAT activity was analyzed as described under “Experimental Procedures.” The data are from a representative experi- ment. Similar results were obtained in one (A–D) or three (E) further experi- ments. The CAT activity is presented as percentage of conversion. systems (28, 33, 48), co-expression of activated Ha-v-ras but not activity in Kit225 T cells (Fig. 3B). This response was specific, of active V12Rac led to a moderate rise in PKB activity in since the expression of V14Rho, which does not regulate stress- Kit225 T cells. activated protein kinases (50, 51), did not induce ATF-2/LexA- Co-expression of an Activated Form of PKB Stimulates C2-controlled gene expression. Therefore, the GTPases V12Rac p70S6k in Kit225 T Cells—p70S6k is activated by multiple and V12Cdc42 are active and can stimulate Rac-/Cdc42-regu- serine/threonine phosphorylation in response to mitogenic lated signaling pathways in Kit225 T cells. stimuli. The retroviral oncogene v-Akt is a chimeric molecule, IL-2 Regulates the Transcription Factor Elk-1 in Kit225 Cells consisting of the retroviral Gag protein fused to the N terminus in a PI 3-Kinase-dependent Fashion—In T cells, a PI 3-kinase- of c-Akt, which is myristoylated, and hence v-Akt is predomi- sensitive pathway for regulating the activity of Erk kinase nantly found at the plasma membrane, which may give raise to (Mek) and the Erks has been reported to co-exist alongside the its oncogenicity (49). The expression of constitutively active PI 3-kinase/p70S6k pathway (10). While p70S6k is thought to PKB, gagPKB, has been described as activating p70S6k in exert its mitogenic function by controlling translation initiation Rat-1 cells (32) and COS1 cells (33). Nevertheless, the ability of and protein synthesis, the MAP kinase Erk is implicated in phorbol esters to stimulate p70S6k without any discernible regulating the phosphorylation and activity of certain tran- activation of PKB indicated that PKB-independent pathways scription factors. One well characterized cellular substrate for for activation of p70S6k must exist in T cells. To determine the Erks in fibroblasts and T cells is the transcription factor Elk-1 role of PKB in p70S6k activation in T cells, rIL-2-deprived (40, 52, 53). We therefore tested the ability of IL-2 to regulate Kit225 cells were co-transfected with a gagPKB expression Elk-1 transcriptional activity and hence Erk in Kit225 cells. To vector together with an expression vector encoding HA epitope- monitor Elk-1 transcriptional activity, a fusion protein com- tagged p70S6k. p70S6k activity was analyzed in anti-HA tag prising the C terminus of Elk-1 linked to the LexA repressor immune complexes with S6 ribosomal subunits as a substrate. (41) was co-transfected into Kit225 cells with a LexA operator- Co-expression of constitutively active PKB induced a strong controlled CAT reporter gene. The data in Fig. 4A demonstrate activation of p70S6k that was comparable with increases in that IL-2 can regulate Elk-1 transcriptional activity in Kit225 p70S6k activity seen by co-expression of rCD2p110 (Fig. 3A). A cells. To confirm that Elk-1 transactivation is induced by a rCD2p85 construct that does not regulate cellular levels of D-3 Mek/Erk-sensitive pathway, we investigated the ability of the phosphoinositides (29) did not stimulate p70S6k. In contrast to well characterized inhibitor of Mek activation, PD098059 (54), data described in fibroblasts (48), co-expression of V12Rac and to prevent IL-2-mediated activation of Elk-1. Treatment of V12Cdc42 had no effect on p70S6k activity in Kit225 cells (Fig. Kit225 cells with the PD098059 component inhibited stimula- 3A). To confirm that V12Rac and V12Cdc42 are active in tion of Elk-1 transcriptional activity triggered by rIL-2 (Fig. Kit225 cells, we tested their ability to activate the stress- 4B). Moreover, rIL-2-induction of Elk-1 activity was prevented activated protein kinases, also known as c-Jun N-terminal by the PI 3-kinase inhibitor wortmannin in a dose-dependent kinases (50, 51). Stress-activated protein kinase and hence manner (Fig. 4C), which corroborates earlier studies indicating Rac/Cdc42 activity can be measured by the ability of stress- that Erk activation by IL-2 requires PI 3-kinase function (10). activated protein kinases to phosphorylate the transcription Treatment of Kit225 cells with rapamycin did not affect Elk-1 factor ATF-2 (42). To monitor ATF-2 transcriptional activity, a transactivation in Kit225 cells (Fig. 4D). To assess whether fusion protein comprising the N terminus of ATF-2 (termed C2) constitutively active PI 3-kinase and the in vivo production of linked to the LexA repressor (42) was co-transfected into D-3 phosphoinositides could induce MAP kinase signaling Kit225 cells together with a LexA operator-controlled chloram- pathways in T cells, the ability of rCD2p110 to induce tran- phenicol acetyltransferase (CAT) reporter gene. V12Rac and scriptional activation of Elk-1 was analyzed. LexA-Elk-1 tran- V12Cdc42 potently stimulated ATF-2/LexA-C2 transcriptional scriptional activity was low in quiescent Kit225 cells but could Phosphatidylinositol 3-Kinase Targets in Lymphoid Cells 14431 FIG.5. PI 3-kinase signals are not sufficient to stimulate Erk2 activity but can synergize with phorbol esters to give an in- crease in Erk2 activity in Kit225 cells. HA-Erk2 activity was ana- lyzed from extracts of untreated cells (2) or cells stimulated with 50 ng/ml PdBu for 5 min. Before stimulation, Kit225 cells were co-trans- fected with HA-Erk2 plasmids and vector plasmid (empty) or plasmids encoding for rCD2p110, rCD2p110-R/P, or Ha-v-ras as indicated. Erk assays were performed as described under “Experimental Procedures.” FIG.6. A schematic representation of the IL-2-regulated sig- The data were quantified using a PhosphorImager and are presented as 32 125 naling pathways that involve PI 3-kinase. Binding of IL-2 to its the ratio of [ P]phosphate incorporated into S6 to I-conjugated pro- receptor activates PI 3-kinase, PKB, p70S6k, and the Ras/Raf/Erk tein A bound to Erk (expressed in arbitrary units). The data are from a effector pathway. PI 3-kinase signals are sufficient to stimulate PKB representative experiment. Similar results were obtained in two more and p70S6k. Activated PKB is sufficient to propagate p70S6k activa- experiments. tion. Hence, the available evidence suggests that IL-2 activates PI 3-kinase, which subsequently leads to PKB activation, which in turn be instigated by co-expression of active Ha-v-ras and by stim- stimulates p70S6k. Activation of p70S6k by IL-2, PI 3-kinase, and PKB ulation with phorbol esters, whereas expression of rCD2p110 is sensitive to rapamycin, which indicates that the target of rapamycin, the Frap, is required for p70S6k activation either as a downstream did not stimulate Elk-1 transactivation (Fig. 4E). However, target of PKB (1) or in a parallel pathway (2). p70S6k can also be rCD2p110 signals could potentiate phorbol ester induction of stimulated by phorbol esters via classical or novel PKC isoforms the transcriptional activity of Elk-1. This potentiating effect (cPKC), whereas PKB cannot. p70S6k appears to exert its mitogenic was not observed in cells expressing the kinase-dead function by regulating translation initiation and protein biosynthesis. PI 3-kinase signals are not sufficient to stimulate the MAP kinase Erk rCD2p110-R/P and was thus dependent on the integrity of the and its cellular target, the transcription factor Elk-1. However, PI lipid kinase and the cellular production of D-3 phosphoinosi- 3-kinase signals can synergize with phorbol esters to induce Erk or tides. Moreover, gagPKB cannot mimic the effects of PI 3-ki- Elk-1 activation. Erk and Elk-1 activity is not inhibited by rapamycin, nase on the Erk/Elk-1 pathway (Fig. 4E). and activated PKB does not potentiate phorbol ester induction of Elk-1 PI 3-Kinase Signals Synergize with Phorbol Esters to Induce transcriptional activity. Hence, PI 3-kinase signals bifurcate to activate the PKB/rapamycin-sensitive/p70S6k pathway and independently con- Erk Activity in Kit225 Cells—To assess the effect of membrane- tribute to the Mek/Erk/Elk-1 pathway. PI 3-kinase signals are impli- localized PI 3-kinase on Erk activity directly, rIL-2-deprived cated to contribute to the Ras/Raf/Mek/Erk pathway at the level of Mek, Kit225 cells were co-transfected with expression vectors encod- since IL-2 induction of Mek and Erk but not Ras and Raf are sensitive ing rCD2p110 and HA epitope-tagged p42 Erk2, and cells were to wortmannin. Possible mediators of PI 3-kinase action on Mek/Erk are the novel/atypical members of the PKC family (n/aPKC)(1)orthe stimulated with phorbol esters or left untreated. Co-expression GTPase Rac (2). of rCD2p110 did not stimulate Erk2 activity, although Erk2 could be activated by co-expressing the activated Ras, Ha-v-ras nal pleckstrin homology domain that can directly bind D-3 (Fig. 5). These results thus confirm the data in Fig. 4E indicat- phosphoinositides (33, 55, 56), which may contribute to the ing that PI 3-kinase signals are not sufficient to activate the regulation of the enzyme. Since PI 3-kinase signals are suffi- Erk/Elk-1 pathway. The data in Fig. 5 demonstrate that active cient to substitute for IL-2 in PKB activation, PKB could be a PI 3-kinase markedly potentiated the level of Erk2 activation direct target for PI 3-kinase signals during IL-2 signal trans- triggered by phorbol esters, an effect that was not observed in duction. PKB/c-Akt is highly expressed in the thymus (57), and cells expressing the kinase-dead rCD2p110-R/P. PI 3-kinase the oncogenic form of this kinase causes thymic malignancies. signals did not enhance IL-2 activation responses on Erk (data Therefore, PKB has a pivotal role in controlling T cell prolifer- not shown). Taken together, the results in Figs. 4E and 5 ation/differentiation. The present data identify one function for clearly demonstrate that although PI 3-kinase signals are not PKB in T cells; PKB action is sufficient to stimulate p70S6k. sufficient for Erk/Elk-1 activation, they can synergize with Moreover, PI 3-kinase signals are sufficient for activation of phorbol esters to induce a maximal response. These results are p70S6k, which stresses the close link between PI 3-kinase and concordant with a model where PI 3-kinase signals bifurcate to PKB in regulating p70S6k activity in T cells. Questions regard- activate the PKB/rapamycin-sensitive/p70S6k pathway and in- ing the selectivity of the inhibitors that were first used to define dependently contribute to the Mek/Erk/Elk-1 pathway via an a role for PI 3-kinase in T cell biology have challenged the as yet undefined mechanism (see Fig. 6). involvement of this enzyme in the regulation of p70S6k in T DISCUSSION cells (26). The present data resolve this controversy and pro- The present study has used a membrane-targeted, constitu- vide unequivocal evidence that PI 3-kinase can function as an tively active, catalytic subunit of PI 3-kinase as a tool to iden- upstream regulator of p70S6k in T cells. tify direct targets of PI 3-kinase action in IL-2 signal transduc- Results obtained recently with p110 constructs that were tion pathways. We demonstrate that the serine/threonine membrane-targeted by myristoylation or farnesylation signals kinase PKB/Akt can be activated by the cytokine IL-2 via a PI showed that PI 3-kinase signals are sufficient to activate PKB 3-kinase-dependent pathway. Importantly, PI 3-kinase signals and p70S6k in COS cells (28). It has also been shown in fibro- alone are sufficient to activate PKB in T cells, demonstrating blasts that the GTPases Rac and Cdc42 induce p70S6k activa- that PI 3-kinase acts as an upstream regulator of this serine/ tion. We find no evidence for Rac/Cdc42 activation of p70S6k in threonine kinase in lymphoid cells. PKB contains an N-termi- T cells, indicating that cells of different lineages can differ 14432 Phosphatidylinositol 3-Kinase Targets in Lymphoid Cells markedly in their cellular mechanisms for kinase activation. (data not shown) or by active PKB (32, 33), thus indicating that Nevertheless, the present data show a striking conservation of PI 3-kinase or PKB activation signals cannot bypass the role of the PI 3-kinase/PKB/p70S6k link in human T cells and simian Frap in p70S6k activation pathways. A simple interpretation of fibroblasts. The conservation of the PI 3-kinase/PKB/p70S6k these data is that PI 3-kinase and PKB activation of p70S6k is mediated by Frap, although the possibility cannot be excluded signaling cascade in T cells implies a physiological importance of this pathway, which has guaranteed its evolutionary that Frap regulates p70S6k by an essential signaling pathway operating in parallel with PI 3-kinase/PKB signals (Fig. 6). conservation. Frap controls p70S6k activation by regulating the phosphoryl- The role of PI 3-kinase as an upstream regulator of the Erk ation of key residues in the enzyme (63, 64). Nevertheless, kinase pathways can also vary depending on the cell system; p70S6k is not a direct substrate for Frap, and some interme- expression of an active PI 3-kinase is sufficient for Erk activa- diate p70S6k kinase(s), as yet uncharacterized, must be in- tion in Xenopus oocytes (27) but not in fibroblasts or mono- voked to explain the role of Frap in p70S6k activation. Al- blasts (28 –31). The present data show directly that PI 3-kinase though the evidence that PKB mediates PI 3-kinase effects on can have a positive regulatory role in Erk activation in T cells p70S6k are compelling, these data do not exclude the possibil- (see Fig. 6). However, PI 3-kinase signals alone fail to stimulate ity that there are PKB-independent mechanisms for p70S6k Erk signaling pathways but markedly potentiate Erk re- activation of T cells. In this context, the present data show that sponses in combination with phorbol esters. Erk regulation of activation of PKC by phorbol esters stimulates p70S6k without downstream nuclear targets is hereby analyzed using the any discernible stimulatory effect on PKB. transactivation capacity of the ternary complex factor Elk-1, a Recent studies showing that cytokine activation of serine well characterized substrate for Erks in fibroblasts and Jurkat kinases is important for the regulation of apoptosis (65, 66) T cells (40, 53). We establish that Elk-1 is regulated by IL-2 via have focused attention on cytokine-induced serine kinase cas- Mek- and PI 3-kinase-sensitive pathways. Furthermore, as cades. PI 3-kinase and PKB have been implicated in the pre- observed in direct Erk activation assays, PI 3-kinase signals vention of apoptosis in other cell systems (67, 68). The present potently enhance phorbol ester induction of Elk-1 transcrip- study demonstrates that PI 3-kinase can couple the IL-2R to a tional activity. PI 3-kinase signals may thus be required for selective subset of serine/threonine kinase pathways in T cells, activation of MAP kinase pathways in IL-2-dependent T cells, and in this respect, the PI 3-kinase/PKB link is intriguing, but they are not sufficient and hence are one component of a since PKB mediates activation of the Frap/p70S6k pathway but more complex signaling network. We have not yet explored the may also regulate other kinase cascades that bifurcate from the PI 3-kinase effector pathways involved in Erk activation, al- PKB/p70S6k pathway including glycogen synthase kinase-3 though previous data have excluded the involvement of the (GSK3) signaling pathways (69). Therefore, PI 3-kinase and/or Frap/p70S6k pathway, since activation of Erk is not sensitive PKB have the potential for pleiotropic functions in T cells, and to rapamycin inhibition. Moreover, PKB, which is a potent their downstream effectors may include additional serine/thre- activator of p70S6k, cannot mimic the effects of activated PI onine kinases evoked by IL-2R engagement. 3-kinase on the Erk/Elk-1 pathway. These data best fit a model Finally, PI 3-kinase is activated by members of the cytokine in which PI 3-kinase regulation of MAP kinases and PKB/ receptor family such as the IL-2R, the IL-4 receptor, the IL-7 p70S6k bifurcate prior to activation of PKB (Fig. 6). Members receptor, and the IL-13 receptor. Signaling pathways regulated of the Rho family of GTPases can potentiate Erk activation by PI 3-kinase can hence have an impact on lymphocyte biology pathways in fibroblasts (39). Activation of PI 3-kinase is suffi- at multiple points. Accordingly, it is important to establish the cient to induce cytoskeletal rearrangements mediated by the function of this enzyme in lymphoid cells. The IL-2R is a GTPase Rac and Rho in Swiss 3T3 cells (29) and thus has the prototypical member of this hematopoietin receptor family. The potential to regulate Rac/Rho signaling pathways in T cells. present results directly define PI 3-kinase function in T cells Accordingly, it is possible that Rac/Rho family GTPases could and position PKB for the first time in a physiologically relevant mediate PI 3-kinase regulation of Erk. However, several other cytokine-induced signal transduction pathway in lymphoid candidate in vivo targets for D-3 phosphoinositides have been cells. The model described herein may also be applicable to proposed including members of the novel PKC family (58) and serine/threonine kinase pathways regulated by other receptors l (59) and PKC-z (60), which has the atypical PKC family, PKC- that activate PI 3-kinase in T cells. recently been implicated as a regulator of Mek and Erk activity in COS cells (61). Acknowledgments—We thank George Thomas and Richard Treis- man for reagents. p70S6k plays a key role in cellular growth control mecha- nisms by coordinating protein biosynthesis via phosphorylation Note Added in Proof—Recently, Alessi and colleagues described the of the S6 subunit of 40 S ribosomes or via regulation of the purification of two upstream kinases that are likely to mediate PKB activity of the eukaryotic initiation factor 4E binding protein, activation. The activity of at least one of these upstream kinases, PKD1, is regulated by binding D-3 polyphosphate phosphoinositides (Alessi, D. 4E-BP1 (24, 25, 62). Expression of an activated PKB can stim- R., James, S. R., Downes, C. P., Holmes, A. B., Gaffney, P. R. J., Reese, ulate p70S6k activity in T cells, indicating that PKB substrates C. B., and Cohen, P. (1997) Curr. Biol. 7, 261–269). are part of the p70S6k activation pathways. 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Published: May 1, 1997

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