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

Phosphatidylinositol 3-kinase (PI 3-kinase) is activated by the cytokine interleukin-2 (IL-2). We have used a constitutively active PI 3-kinase to identify IL-2-mediated signal transduction pathways directly regulated by PI 3-kinase in lymphoid cells. The serine/threonine protein kinase B (PKB)/Akt can act as a powerful oncogene in T cells, but its positioning in normal T cell responses has not been explored. Herein, we demonstrate that PKB is activated by IL-2 in a PI 3-kinase-dependent fashion. Importantly, PI 3-kinase signals are sufficient for PKB activation in IL-2-dependent T cells, and PKB is a target for PI 3-kinase signals in IL-2 activation pathways. The present study establishes also that PI 3-kinase signals or PKB signals are sufficient for activation of p70 S6 kinase in T cells. PI 3-kinase can contribute to, but is not sufficient for, activation of extracellular signal-regulated kinases (Erks) and Erk effector pathways. Therefore, PI 3-kinase is a selective regulator of serine/threonine kinase signal transduction pathways in T lymphocytes, and this enzyme provides a crucial link between the interleukin-2 receptor, the protooncogene PKB, and p70 S6 kinase.

The high affinity interleukin-2 receptor (IL-2R), 1 which comprises ␣-, ␤-, and ␥-subunits controls G 1 to S progression, T cell clonal expansion, and functional differentiation (1)(2)(3). The IL-2R orchestrates downstream effector pathways by protein tyrosine kinase-dependent activation mechanisms engaging the Src family tyrosine kinases Lck and Fyn (4) and the Janus kinases 1 and 3 (5)(6)(7). Signaling cascades integrated by the action of these tyrosine kinases include activation of the Ras/ Raf/extracellular-signal regulated kinase (Erk) pathway (8 -10), activation of the transcription factors STAT3 and STAT5 (11), and the regulation of phosphatidylinositol 3-kinase (PI 3-kinase) (12). PI 3-kinase is a ubiquitously expressed enzyme that cata-lyzes the phosphorylation of phosphoinositides at the D-3 hydroxyl of the myo-inositol ring generating PI 3-phosphate, PI 3,4-bisphosphate, and PI 3,4,5-trisphosphate (13,14). The form of PI 3-kinase involved in protein-tyrosine kinase-dependent receptor signal transduction comprises a regulatory 85-kDa subunit that contains two Src homology 2 domains and at its N terminus one Src homology 3 domain and a catalytic 110-kDa subunit. Following IL-2R stimulation, several mechanisms have been proposed to recruit PI 3-kinase to the plasma membrane, where its cellular substrate PI 4,5-bisphosphate is located: engagement of the IL-2R leads to binding of the p85 regulatory subunit of PI 3-kinase to tyrosine 392 in the IL-2R ␤-chain (15); in addition, interleukin-2 (IL-2) stimulation results in the interaction of PI 3-kinase with the Src family kinases Fyn (16) and Lck (17). The activation of PI 3-kinase is a response that IL-2 shares with other cytokines that control lymphoid cell growth and development such as IL-4 and IL-7 (18,19). It is also clear that PI 3-kinase activation is necessary for the growth-and differentiation-inducing properties of these cytokines (20 -23). However, 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 pathways regulated by the hematopoietin family of cytokines. To date, the identification of biochemical targets for PI 3-kinase in T cells stems mainly from studies employing the PI 3-kinase inhibitor wortmannin or the LY294002 compound (10,20). Hence, IL-2 activation of the mitogen-activated protein (MAP) kinase Erk is sensitive to wortmannin (10). Similarly, IL-2 activation of the serine/threonine kinase p70 S6 kinase (p70S6k) is prevented by these PI 3-kinase inhibitors (20). In addition, IL-2 activation of p70S6k is impeded by the immunosupressant rapamycin, which targets another member of the PI 3-kinase family of enzymes, Frap (for FKBP12-rapamycin-associated protein) also termed "mammalian target of rapamycin" (mTor) (24,25). Observations that wortmannin and rapamycin have identical inhibitory effects on IL-2 activation of p70S6k generated a model for the p70S6k signaling pathway in which PI 3-kinase acts as an upstream regulator of Frap (24,25). However, this model has been challenged by a recent study showing that the action of Frap is directly inhibited by wortmannin and LY294002 (26). These results raise the issue of whether PI 3-kinase itself has any upstream regulatory role in p70S6k activation in T lymphocytes. Similar caution must be applied to interpretations of data involving PI 3-kinase in Erk activation in T cells. In this context, expression of an active PI 3-kinase is sufficient for Erk activation in Xenopus oocytes (27), but it would be fallacious to extrapolate data obtained in Xenopus cells to T cells, since the role of PI 3-kinase as an upstream regulator of kinase pathways can vary depending on the cell system; to this end, PI 3-kinase signals did not stimulate Erk activity in a variety of fibroblasts and in a monoblast * This work was supported by the Imperial Cancer Research Fund and by Human Capital Mobility Program Grant ERB CHRX CT 94-0537. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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 constitutively active PI 3-kinase finally allows assessment of the relative 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 construct, 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 characterizes a previously unrecognized IL-2-mediated signal transduction pathway in T cells that involves the serine/threonine protein kinase B (PKB) also known as c-Akt or Rac protein kinase (32)(33)(34). PKB was originally identified as the cellular homologue of the directly transforming oncogene of the murine retrovirus 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-insensitive pathway. PI 3-kinase signals alone were sufficient to activate 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 signal transduction pathways in T lymphocytes, and this enzyme is an upstream regulator of the IL-2-activated kinases PKB and p70S6k.
Cell Culture and Transient Transfections-The Kit225 T leukemic cell line (43) was maintained in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum supplemented with 20 ng/ml of recombinant IL-2 (rIL-2) (Eurocetus) under normal growth conditions. For IL-2 activation assays of endogenous proteins, Kit225 cells were washed three times with phosphate-buffered saline A to remove the IL-2 and 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 deprived of rIL-2 for 24 h prior to transfection.
Immunoprecipitation, p70S6k Assays, and Western Blot Analysis-After stimulations as indicated, Kit225 cells were lysed in lysis buffer 1 (120 mM NaCl, 50 mM Tris pH 8.0, 20 mM NaF, 1 mM benzamidine, 1 mM EDTA, 6 mM EGTA, 7.5 mM PP i , 15 mM p-nitrophenyl phosphate, 1% Nonidet P-40, 0.1 mM phenylmethylsulfonyl fluoride, and 0.1 mM FIG. 1. Interleukin-2 activates p70S6k, which can be mimicked by co-expression of membrane-localized constitutively active PI 3-kinase, rCD2p110. A, Kit225 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 p70S6k activation/phosphorylation was assessed by electromobility shift assays (top). p70S6k was precipitated from lysates with M5 Abs, and p70S6k activity was analyzed in immune complex kinase assays using S6 as a substrate. [ 32 P]phosphate incorporation into S6 (middle) was quantified (graph) using a PhosphorImager and is expressed in arbitrary units. Protein levels of p70S6k present in immune complexes were assessed in parallel by Western blotting with M1 Abs (bottom). B and C, HA-p70S6k activity was analyzed from extracts of untreated cells (Ϫ) or cells stimulated with either 20 ng/ml rIL-2 or 50 ng/ml PdBu for 15 min. B, before stimulation, Kit225 cells were cotransfected with HA-p70S6k plasmids and vector plasmid (empty) or with plasmids encoding for rCD2p110 as indicated. C, Kit225 cells were co-transfected with HA-p70S6k plasmids and 15 g each of vector plasmid (empty) or plasmids encoding for rCD2p110 or rCD2p110-R/P. B, S6 substrate phosphorylation from S6 kinase assays (top) was analyzed by autoradiography. Levels of p70S6k in immune complexes (bottom) were analyzed by immunoblotting using M1 antibodies followed by 125 I-conjugated protein A and autoradiography. C, the data were analyzed as in B and quantified using a PhosphorImager. Data are presented as the ratio of [ 32 P]phosphate incorporated into S6 to 125 Iconjugated protein A bound to p70S6k (expressed in arbitrary units). The data are from a representative experiment. Similar results were obtained in two (A and B) or five (C) more experiments. Na 3 VO 4 ). Cell extracts corresponding to 3 ϫ 10 6 cell equivalents (nontransfected cells) or 1.5 ϫ 10 6 live cell equivalents (transfected cells) were used for each immunoprecipitation. Postnuclear lysates were precleared with protein A cell suspension (Sigma) prior to incubation with 2 g of 12CA5 mAbs or, for endogenous proteins, 1 g 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 2 , 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 32 P-labeled S6 proteins were detected by autoradiography. The levels of p70S6k protein in each immunoprecipitate were assessed by transferring the proteins in the upper part of the gel onto polyvinylidene difluoride membranes 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 normalized for p70S6k expression levels by quantitation of Western blots probed with M1 Abs followed by 125 I-conjugated protein A (Amersham). Quantitation of incorporated 32 P into S6 or of bound 125 I-conjugated protein A was performed using a PhosphorImager (Molecular Dynamics). To test for effector protein expression in transfected cells, postnuclear cell extracts corresponding to 3 ϫ 10 6 cell equivalents of the same extract as above were analyzed by Western blotting as described (44) using 9E10, Ox34, or specific 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 2 , 2 mM MnCl 2 ). The reaction was initiated by the addition of 10 l of Erk reaction buffer containing 4 Ci of [␥-32 P]ATP, 20 M ATP, 20 mM MgCl 2 , 2 mM MnCl 2 , 5 mM p-nitrophenyl phosphate, 500 nM protein kinase inhibitor, 30 mM Tris, pH 8.0, and 15 g 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 125 I-conjugated protein A.
Gene Expression Analysis-Fourteen to 16 h after inductions, as indicated, Kit225 T cells were harvested and cells were lysed in 200 l of lysis buffer (0.65% Nonidet P-40, 10 mM Tris, pH 8, 1 mM EDTA, 150 mM NaCl). Gene expression assays were carried out as described (45). The data are presented as percentage of conversion.

FIG. 2. Interleukin-2 activates PKB: PI 3-kinase activity is necessary for IL-2-mediated activation of PKB, and PI 3-kinase 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 electromobility shift assays (top). PKB was precipitated from lysates with Rac-PK-CT Abs and PKB activity was analyzed in immune complex kinase assays using H2B as a substrate. [ 32 P]Phosphate incorporation into H2B (middle) was quantified (graph) using a PhosphorImager and is expressed in arbitrary units. Protein levels of PKB present in immune complexes were assessed in parallel by Western blotting with Rac-PK-CT Abs (bottom). B, the PI 3-kinase inhibitors wortmannin and LY294002, but not rapamycin, inhibit IL-2-dependent activation of PKB. Kit225 cells starved of rIL-2 for 72 h were pretreated for 30 min with the vehicle dimethyl sulfoxide (DMSO), 20 ng/ml rapamycin, 5 M LY294002, or 100 nM wortmannin and then stimulated with rIL-2 for the times indicated before lysis. PKB activity was measured in immune complex kinase assays using H2B as a substrate. C, PI 3-kinase signals trigger a potent stimulation of PKB activity. Kit225 cells were cotransfected with HA-PKB plasmids and vector plasmid (empty), or plasmids encoding for rCD2p110, rCD2p110-R/P, Ha-v-ras, or V12Rac as indicated. HA-PKB activity was analyzed in anti-HA tag immune complex kinase assays using H2B as a substrate. B and C, [ 32 P]phosphate incorporation into H2B (top) was quantified (graph) using a PhosphorImager and is expressed in arbitrary units. Protein levels of PKB (B) or HA-PKB (C) present in immune complexes were assessed in parallel by Western blotting with Rac-PK-CT Abs (bottom).

IL-2 and PI 3-Kinase Signals Activate p70S6k in Kit225
Cells-For our studies, we used the well characterized human IL-2-dependent T cell line Kit225. The data in Fig. 1A show that p70S6k activity is low in quiescent rIL-2-deprived Kit225 cells but can be rapidly stimulated by rIL-2. The activity of p70S6k is increased in response to phorbol esters that stimulate protein kinase C (PKC) (Fig. 1A). We asked whether PI 3-kinase signals could substitute for IL-2 in inducing p70S6k activity. We have shown recently that plasma membrane targeting of p110, the catalytic subunit of PI 3-kinase, generates an enzyme that is constitutively active in vivo (29). Our membrane-targeted active PI 3-kinase construct comprises a chimera of the extracellular and transmembrane domains of the rCD2 antigen fused to the p110␣ catalytic domain of PI 3-kinase, rCD2p110. As a control, we used a rCD2p110 molecule that contained in the catalytic subunit of PI 3-kinase an inactivating point mutation (R1130P) in the ATP binding site (46) that abolishes its in vivo and in vitro lipid kinase activity, rCD2p110-R/P. To assess the effects of PI 3-kinase signals on p70S6k activity, rIL-2-starved Kit225 cells were co-transfected with HA epitope-tagged p70S6k and rCD2p110. Cell surface expression of the rCD2p110 chimera was confirmed by flow cytometric immunofluorescence analysis with rCD2 mAbs (data not shown). The HA-tagged p70S6k was immunoprecipitated from transiently transfected cells and assayed for its ability to phosphorylate S6 ribosomal subunits (Fig. 1B). Expression of the active PI 3-kinase, rCD2p110, resulted in constitutive IL-2-independent p70S6k activation (Fig. 1B). p70S6k was not constitutively activated in cells expressing "kinasedead" 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 chimera 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 significance 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 interested to assess whether members of the hematopoietin receptor family, such as the prototypical IL-2R, regulate PKB. To examine whether IL-2 activates PKB, immunoprecipitates of this kinase were prepared from rIL-2-deprived and rIL-2-activated Kit225 cells and subjected to in vitro kinase assays using H2B as a substrate. The data in Fig. 2A show that IL-2 induced a rapid activation of PKB. A 2-3-fold increase in enzyme activity over basal levels was sustained for more than 60 min in response to rIL-2. PKB activity is regulated by phosphorylation as indicated by the reduced electrophoretic mobility of PKB isolated from rIL-2-activated cells ( Fig. 2A). PKB activity was not induced by exposure of Kit225 cells to phorbol esters that activate PKC (Fig. 2A). The data in Fig. 2B show the failure of rIL-2 to stimulate PKB in cells pretreated with LY294002 or wortmannin, two well characterized PI 3-kinase inhibitors that bind to the ATP or lipid binding sites on the p110 catalytic subunit, respectively. These inhibitors also prevent the autokinase activity of Frap/mTor (26), a member of the PI 3-kinase family (47), which is the cellular target for the drug rapamycin and which prevents IL-2-coordinated cell cycle progression and proliferation of T lymphocytes (24,25). Frap activity is absolutely required for p70S6k action in T cells (24,25). We therefore assessed whether Frap function was necessary for IL-2induced stimulation of PKB. Rapamycin had no effect on IL-2triggered activation of PKB (Fig. 2B), although rapamycin completely abolished IL-2-or PI 3-kinase-controlled induction of p70S6k (data not shown). Thus, the inhibition of PKB by wortmannin and LY294002 cannot be caused by prevention of Frap activity and indicate that IL-2 regulation of PKB employs PI 3-kinase.
To investigate directly whether PI 3-kinase signals are sufficient to activate PKB, rIL-20-deprived Kit225 cells were cotransfected with either rCD2p110 or rCD2p110-R/P expression vectors together with an expression vector encoding HA epitope-tagged PKB. In addition, the ability of activated forms of the small GTPases Ha-v-ras and V12Rac to activate PKB was assessed. Immunoprecipitates of HA-tagged PKB were assayed for kinase activity using H2B as a substrate. The constitutively active PI 3-kinase rCD2p110 induced a robust activation of PKB (Fig. 2C). This stimulatory effect of rCD2p110 was dependent on the kinase activity of the chimera, since co-expression of kinase-inactive rCD2p110-R/P did not stimulate PKB activity. As observed previously in other cell

FIG. 3. Co-expression of constitutively active forms of PKB and PI 3-kinase but not of the GTPases Rac and Cdc42 stimulates p70S6k activity in Kit225 cells. A, HA-p70S6k activity was analyzed from extracts of untreated cells (Ϫ) or cells stimulated with 20
ng/ml rIL-2 for 15 min. Before stimulation, Kit225 cells were co-transfected with HA-p70S6k plasmids and vector plasmid (empty) or plasmids encoding for rCD2p110, gagPKB, V12Rac, V12Cdc42, or rCD2p85 as indicated. p70S6k assays were performed as described under "Experimental Procedures." The data were quantified using a PhosphorImager and are presented as the ratio of [ 32 P]phosphate incorporated into S6 to 125 I-conjugated protein A bound to p70S6k (expressed in arbitrary units). B, Kit225 cells were transfected with the LexA operator-controlled CAT reporter plasmid (lexOP-CAT) and the expression plasmid producing the LexA-C2 fusion protein Nlex.C2 together with vector plasmid (empty) or the expression plasmids for V14Rho, V12Rac, and V12Cdc42 as indicated. After 18 h, Kit225 T cells were harvested, and CAT activity was analyzed as described under "Experimental Procedures." The CAT activity is presented as percentage of conversion. The data are from a representative experiment. Similar results were obtained in one further experiment. systems (28,33,48), co-expression of activated Ha-v-ras but not of active V12Rac led to a moderate rise in PKB activity in Kit225 T cells.
Co-expression of an Activated Form of PKB Stimulates p70S6k in Kit225 T Cells-p70S6k is activated by multiple serine/threonine phosphorylation in response to mitogenic stimuli. The retroviral oncogene v-Akt is a chimeric molecule, consisting of the retroviral Gag protein fused to the N terminus of c-Akt, which is myristoylated, and hence v-Akt is predominantly found at the plasma membrane, which may give raise to its oncogenicity (49). The expression of constitutively active PKB, gagPKB, has been described as activating p70S6k in Rat-1 cells (32) and COS1 cells (33). Nevertheless, the ability of phorbol esters to stimulate p70S6k without any discernible activation of PKB indicated that PKB-independent pathways for activation of p70S6k must exist in T cells. To determine the role of PKB in p70S6k activation in T cells, rIL-2-deprived Kit225 cells were co-transfected with a gagPKB expression vector together with an expression vector encoding HA epitopetagged p70S6k. p70S6k activity was analyzed in anti-HA tag immune complexes with S6 ribosomal subunits as a substrate. Co-expression of constitutively active PKB induced a strong activation of p70S6k that was comparable with increases in p70S6k activity seen by co-expression of rCD2p110 (Fig. 3A). A rCD2p85 construct that does not regulate cellular levels of D-3 phosphoinositides (29) did not stimulate p70S6k. In contrast to data described in fibroblasts (48), co-expression of V12Rac and V12Cdc42 had no effect on p70S6k activity in Kit225 cells (Fig.  3A). To confirm that V12Rac and V12Cdc42 are active in Kit225 cells, we tested their ability to activate the stressactivated protein kinases, also known as c-Jun N-terminal kinases (50,51). Stress-activated protein kinase and hence Rac/Cdc42 activity can be measured by the ability of stressactivated protein kinases to phosphorylate the transcription factor ATF-2 (42). To monitor ATF-2 transcriptional activity, a fusion protein comprising the N terminus of ATF-2 (termed C2) linked to the LexA repressor (42) was co-transfected into Kit225 cells together with a LexA operator-controlled chloramphenicol acetyltransferase (CAT) reporter gene. V12Rac and V12Cdc42 potently stimulated ATF-2/LexA-C2 transcriptional activity in Kit225 T cells (Fig. 3B). This response was specific, since the expression of V14Rho, which does not regulate stressactivated protein kinases (50,51), did not induce ATF-2/LexA-C2-controlled gene expression. Therefore, the GTPases V12Rac and V12Cdc42 are active and can stimulate Rac-/Cdc42-regulated signaling pathways in Kit225 T cells.

IL-2 Regulates the Transcription Factor Elk-1 in Kit225 Cells in a PI 3-Kinase-dependent Fashion-In T cells, a PI 3-kinase-
sensitive pathway for regulating the activity of Erk kinase (Mek) and the Erks has been reported to co-exist alongside the PI 3-kinase/p70S6k pathway (10). While p70S6k is thought to exert its mitogenic function by controlling translation initiation and protein synthesis, the MAP kinase Erk is implicated in regulating the phosphorylation and activity of certain transcription factors. One well characterized cellular substrate for Erks in fibroblasts and T cells is the transcription factor Elk-1 (40,52,53). We therefore tested the ability of IL-2 to regulate Elk-1 transcriptional activity and hence Erk in Kit225 cells. To monitor Elk-1 transcriptional activity, a fusion protein comprising the C terminus of Elk-1 linked to the LexA repressor (41) was co-transfected into Kit225 cells with a LexA operatorcontrolled CAT reporter gene. The data in Fig. 4A demonstrate that IL-2 can regulate Elk-1 transcriptional activity in Kit225 cells. To confirm that Elk-1 transactivation is induced by a Mek/Erk-sensitive pathway, we investigated the ability of the well characterized inhibitor of Mek activation, PD098059 (54), to prevent IL-2-mediated activation of Elk-1. Treatment of Kit225 cells with the PD098059 component inhibited stimulation of Elk-1 transcriptional activity triggered by rIL-2 (Fig.  4B). Moreover, rIL-2-induction of Elk-1 activity was prevented by the PI 3-kinase inhibitor wortmannin in a dose-dependent manner (Fig. 4C), which corroborates earlier studies indicating that Erk activation by IL-2 requires PI 3-kinase function (10). Treatment of Kit225 cells with rapamycin did not affect Elk-1 transactivation in Kit225 cells (Fig. 4D). To assess whether constitutively active PI 3-kinase and the in vivo production of D-3 phosphoinositides could induce MAP kinase signaling pathways in T cells, the ability of rCD2p110 to induce transcriptional activation of Elk-1 was analyzed. LexA-Elk-1 transcriptional activity was low in quiescent Kit225 cells but could FIG. 4. Interleukin-2 but not rCD2p110 induces Elk-1-dependent gene expression. A-E, Kit225 cells were transfected with the LexA operator-controlled CAT reporter plasmid (lexOP-CAT) plus the expression plasmid producing the LexA-Elk-1 fusion protein Nlex.Elk. In E the Nlex.Elk/lexOP-CAT reporter plasmids were co-transfected together 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 concentrations of PD098059 (B), 20 ng/ml rIL-2 plus various concentrations of wortmannin (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 experiment. Similar results were obtained in one (A-D) or three (E) further experiments. The CAT activity is presented as percentage of conversion.
be instigated by co-expression of active Ha-v-ras and by stimulation with phorbol esters, whereas expression of rCD2p110 did not stimulate Elk-1 transactivation (Fig. 4E). However, rCD2p110 signals could potentiate phorbol ester induction of the transcriptional activity of Elk-1. This potentiating effect was not observed in cells expressing the kinase-dead rCD2p110-R/P and was thus dependent on the integrity of the lipid kinase and the cellular production of D-3 phosphoinositides. Moreover, gagPKB cannot mimic the effects of PI 3-kinase on the Erk/Elk-1 pathway (Fig. 4E).
PI 3-Kinase Signals Synergize with Phorbol Esters to Induce Erk Activity in Kit225 Cells-To assess the effect of membranelocalized PI 3-kinase on Erk activity directly, rIL-2-deprived Kit225 cells were co-transfected with expression vectors encoding rCD2p110 and HA epitope-tagged p42 Erk2, and cells were stimulated with phorbol esters or left untreated. Co-expression of rCD2p110 did not stimulate Erk2 activity, although Erk2 could be activated by co-expressing the activated Ras, Ha-v-ras (Fig. 5). These results thus confirm the data in Fig. 4E indicating that PI 3-kinase signals are not sufficient to activate the Erk/Elk-1 pathway. The data in Fig. 5 demonstrate that active PI 3-kinase markedly potentiated the level of Erk2 activation triggered by phorbol esters, an effect that was not observed in cells expressing the kinase-dead rCD2p110-R/P. PI 3-kinase signals did not enhance IL-2 activation responses on Erk (data not shown). Taken together, the results in Figs. 4E and 5 clearly demonstrate that although PI 3-kinase signals are not sufficient for Erk/Elk-1 activation, they can synergize with phorbol esters to induce a maximal response. These results are concordant with a model where PI 3-kinase signals bifurcate to activate the PKB/rapamycin-sensitive/p70S6k pathway and independently contribute to the Mek/Erk/Elk-1 pathway via an as yet undefined mechanism (see Fig. 6).

DISCUSSION
The present study has used a membrane-targeted, constitutively active, catalytic subunit of PI 3-kinase as a tool to identify direct targets of PI 3-kinase action in IL-2 signal transduction pathways. We demonstrate that the serine/threonine kinase PKB/Akt can be activated by the cytokine IL-2 via a PI 3-kinase-dependent pathway. Importantly, PI 3-kinase signals alone are sufficient to activate PKB in T cells, demonstrating that PI 3-kinase acts as an upstream regulator of this serine/ threonine kinase in lymphoid cells. PKB contains an N-termi-nal pleckstrin homology domain that can directly bind D-3 phosphoinositides (33,55,56), which may contribute to the regulation of the enzyme. Since PI 3-kinase signals are sufficient to substitute for IL-2 in PKB activation, PKB could be a direct target for PI 3-kinase signals during IL-2 signal transduction. PKB/c-Akt is highly expressed in the thymus (57), and the oncogenic form of this kinase causes thymic malignancies. Therefore, PKB has a pivotal role in controlling T cell proliferation/differentiation. The present data identify one function for PKB in T cells; PKB action is sufficient to stimulate p70S6k. Moreover, PI 3-kinase signals are sufficient for activation of p70S6k, which stresses the close link between PI 3-kinase and PKB in regulating p70S6k activity in T cells. Questions regarding the selectivity of the inhibitors that were first used to define a role for PI 3-kinase in T cell biology have challenged the involvement of this enzyme in the regulation of p70S6k in T cells (26). The present data resolve this controversy and provide unequivocal evidence that PI 3-kinase can function as an upstream regulator of p70S6k in T cells.
Results obtained recently with p110 constructs that were membrane-targeted by myristoylation or farnesylation signals showed that PI 3-kinase signals are sufficient to activate PKB and p70S6k in COS cells (28). It has also been shown in fibroblasts that the GTPases Rac and Cdc42 induce p70S6k activation. We find no evidence for Rac/Cdc42 activation of p70S6k in T cells, indicating that cells of different lineages can differ FIG. 5. PI 3-kinase signals are not sufficient to stimulate Erk2 activity but can synergize with phorbol esters to give an increase in Erk2 activity in Kit225 cells. HA-Erk2 activity was analyzed from extracts of untreated cells (Ϫ) or cells stimulated with 50 ng/ml PdBu for 5 min. Before stimulation, Kit225 cells were co-transfected 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." The data were quantified using a PhosphorImager and are presented as the ratio of [ 32 P]phosphate incorporated into S6 to 125 I-conjugated protein A bound to Erk (expressed in arbitrary units). The data are from a representative experiment. Similar results were obtained in two more experiments.
FIG. 6. A schematic representation of the IL-2-regulated signaling pathways that involve PI 3-kinase. Binding of IL-2 to its receptor activates PI 3-kinase, PKB, p70S6k, and the Ras/Raf/Erk effector pathway. PI 3-kinase signals are sufficient to stimulate PKB and p70S6k. Activated PKB is sufficient to propagate p70S6k activation. Hence, the available evidence suggests that IL-2 activates PI 3-kinase, which subsequently leads to PKB activation, which in turn stimulates p70S6k. Activation of p70S6k by IL-2, PI 3-kinase, and PKB is sensitive to rapamycin, which indicates that the target of rapamycin, the Frap, is required for p70S6k activation either as a downstream target of PKB (1) or in a parallel pathway (2). p70S6k can also be stimulated by phorbol esters via classical or novel PKC isoforms (cPKC), whereas PKB cannot. p70S6k appears to exert its mitogenic function by regulating translation initiation and protein biosynthesis. PI 3-kinase signals are not sufficient to stimulate the MAP kinase Erk and its cellular target, the transcription factor Elk-1. However, PI 3-kinase signals can synergize with phorbol esters to induce Erk or Elk-1 activation. Erk and Elk-1 activity is not inhibited by rapamycin, and activated PKB does not potentiate phorbol ester induction of Elk-1 transcriptional activity. Hence, PI 3-kinase signals bifurcate to activate the PKB/rapamycin-sensitive/p70S6k pathway and independently contribute to the Mek/Erk/Elk-1 pathway. PI 3-kinase signals are implicated to contribute to the Ras/Raf/Mek/Erk pathway at the level of Mek, since IL-2 induction of Mek and Erk but not Ras and Raf are sensitive to wortmannin. Possible mediators of PI 3-kinase action on Mek/Erk are the novel/atypical members of the PKC family (n/aPKC) (1) or the GTPase Rac (2). markedly in their cellular mechanisms for kinase activation. Nevertheless, the present data show a striking conservation of the PI 3-kinase/PKB/p70S6k link in human T cells and simian fibroblasts. The conservation of the PI 3-kinase/PKB/p70S6k signaling cascade in T cells implies a physiological importance of this pathway, which has guaranteed its evolutionary conservation.
The role of PI 3-kinase as an upstream regulator of the Erk kinase pathways can also vary depending on the cell system; expression of an active PI 3-kinase is sufficient for Erk activation in Xenopus oocytes (27) but not in fibroblasts or monoblasts (28 -31). The present data show directly that PI 3-kinase can have a positive regulatory role in Erk activation in T cells (see Fig. 6). However, PI 3-kinase signals alone fail to stimulate Erk signaling pathways but markedly potentiate Erk responses in combination with phorbol esters. Erk regulation of downstream nuclear targets is hereby analyzed using the transactivation capacity of the ternary complex factor Elk-1, a well characterized substrate for Erks in fibroblasts and Jurkat T cells (40,53). We establish that Elk-1 is regulated by IL-2 via Mek-and PI 3-kinase-sensitive pathways. Furthermore, as observed in direct Erk activation assays, PI 3-kinase signals potently enhance phorbol ester induction of Elk-1 transcriptional activity. PI 3-kinase signals may thus be required for activation of MAP kinase pathways in IL-2-dependent T cells, but they are not sufficient and hence are one component of a more complex signaling network. We have not yet explored the PI 3-kinase effector pathways involved in Erk activation, although previous data have excluded the involvement of the Frap/p70S6k pathway, since activation of Erk is not sensitive to rapamycin inhibition. Moreover, PKB, which is a potent activator of p70S6k, cannot mimic the effects of activated PI 3-kinase on the Erk/Elk-1 pathway. These data best fit a model in which PI 3-kinase regulation of MAP kinases and PKB/ p70S6k bifurcate prior to activation of PKB (Fig. 6). Members of the Rho family of GTPases can potentiate Erk activation pathways in fibroblasts (39). Activation of PI 3-kinase is sufficient to induce cytoskeletal rearrangements mediated by the GTPase Rac and Rho in Swiss 3T3 cells (29) and thus has the potential to regulate Rac/Rho signaling pathways in T cells. Accordingly, it is possible that Rac/Rho family GTPases could mediate PI 3-kinase regulation of Erk. However, several other candidate in vivo targets for D-3 phosphoinositides have been proposed including members of the novel PKC family (58) and the atypical PKC family, PKC-(59) and PKC-(60), which has recently been implicated as a regulator of Mek and Erk activity in COS cells (61).
p70S6k plays a key role in cellular growth control mechanisms by coordinating protein biosynthesis via phosphorylation of the S6 subunit of 40 S ribosomes or via regulation of the activity of the eukaryotic initiation factor 4E binding protein, 4E-BP1 (24,25,62). Expression of an activated PKB can stimulate p70S6k activity in T cells, indicating that PKB substrates are part of the p70S6k activation pathways. Moreover, given the ability of PI 3-kinase signals to stimulate PKB and p70S6k, it seems probable that PKB mediates the PI 3-kinase activation of p70S6k in T cells. The immunosuppressive drug rapamycin inhibits the cell cycle progression and proliferation of T lymphocytes and has been shown previously to block IL-2 activation of p70S6k. Rapamycin forms a complex with the intracellular protein FKBP12, which subsequently provides a high affinity inhibitor of Frap. The Frap kinase is a member of the PI 3-kinase family of enzymes (47) and plays an established, but poorly defined, role as an upstream regulator of p70S6k (24,25). Rapamycin prevents the activation of p70S6k induced in T cells by the constitutively active PI 3-kinase, rCD2p110, (data not shown) or by active PKB (32,33), thus indicating that PI 3-kinase or PKB activation signals cannot bypass the role of Frap in p70S6k activation pathways. A simple interpretation of these data is that PI 3-kinase and PKB activation of p70S6k is mediated by Frap, although the possibility cannot be excluded that Frap regulates p70S6k by an essential signaling pathway operating in parallel with PI 3-kinase/PKB signals (Fig. 6). Frap controls p70S6k activation by regulating the phosphorylation of key residues in the enzyme (63,64). Nevertheless, p70S6k is not a direct substrate for Frap, and some intermediate p70S6k kinase(s), as yet uncharacterized, must be invoked to explain the role of Frap in p70S6k activation. Although the evidence that PKB mediates PI 3-kinase effects on p70S6k are compelling, these data do not exclude the possibility that there are PKB-independent mechanisms for p70S6k activation of T cells. In this context, the present data show that activation of PKC by phorbol esters stimulates p70S6k without any discernible stimulatory effect on PKB.
Recent studies showing that cytokine activation of serine kinases is important for the regulation of apoptosis (65,66) have focused attention on cytokine-induced serine kinase cascades. PI 3-kinase and PKB have been implicated in the prevention of apoptosis in other cell systems (67,68). The present study demonstrates that PI 3-kinase can couple the IL-2R to a selective subset of serine/threonine kinase pathways in T cells, and in this respect, the PI 3-kinase/PKB link is intriguing, since PKB mediates activation of the Frap/p70S6k pathway but may also regulate other kinase cascades that bifurcate from the PKB/p70S6k pathway including glycogen synthase kinase-3 (GSK3) signaling pathways (69). Therefore, PI 3-kinase and/or PKB have the potential for pleiotropic functions in T cells, and their downstream effectors may include additional serine/threonine kinases evoked by IL-2R engagement.
Finally, PI 3-kinase is activated by members of the cytokine receptor family such as the IL-2R, the IL-4 receptor, the IL-7 receptor, and the IL-13 receptor. Signaling pathways regulated by PI 3-kinase can hence have an impact on lymphocyte biology at multiple points. Accordingly, it is important to establish the function of this enzyme in lymphoid cells. The IL-2R is a prototypical member of this hematopoietin receptor family. The present results directly define PI 3-kinase function in T cells and position PKB for the first time in a physiologically relevant cytokine-induced signal transduction pathway in lymphoid cells. The model described herein may also be applicable to serine/threonine kinase pathways regulated by other receptors that activate PI 3-kinase in T cells.