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

  • Karin Reif
    Correspondence
    Supported by a Boehringer Ingelheim Fellowship. To whom correspondence should be addressed. Tel.: 0171-269-3307; Fax: 0171-269-3479;
    Affiliations
    From the Lymphocyte Activation Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom and the
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  • Boudewijn M.T. Burgering
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  • Doreen A. Cantrell
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  • Author Footnotes
    * 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. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    1 The abbreviations used areIL-2Rinterleukin-2 receptor;Erkextracellular-signal regulated kinase;PIphosphatidylinositol;ILinterleukin;MAPmitogen-activated protein;p70S6kp70 S6 kinase;FrapFKBP12-rapamycin-associated protein;PKBprotein kinase B;PdBuphorbol 12,13-dibutyrate;Abantibody;mAbmonoclonal antibody;CATchloramphenicol acetyltransferase;PKCprotein kinase C;MekErk kinase;HAhemagglutinin;rIL-2recombinant IL-2;Mops4-morpholinepropanesulfonic acid;PAGEpolyacrylamide gel electrophoresis;H2Bhistone 2B;rCD2rat CD2.
Open AccessPublished:May 30, 1997DOI:https://doi.org/10.1074/jbc.272.22.14426
      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),

      The abbreviations used are

      IL-2R
      interleukin-2 receptor;
      Erk
      extracellular-signal regulated kinase;
      PI
      phosphatidylinositol;
      IL
      interleukin;
      MAP
      mitogen-activated protein;
      p70S6k
      p70 S6 kinase;
      Frap
      FKBP12-rapamycin-associated protein;
      PKB
      protein kinase B;
      PdBu
      phorbol 12,13-dibutyrate;
      Ab
      antibody;
      mAb
      monoclonal antibody;
      CAT
      chloramphenicol acetyltransferase;
      PKC
      protein kinase C;
      Mek
      Erk kinase;
      HA
      hemagglutinin;
      rIL-2
      recombinant IL-2;
      Mops
      4-morpholinepropanesulfonic acid;
      PAGE
      polyacrylamide gel electrophoresis;
      H2B
      histone 2B;
      rCD2
      rat CD2.
      which comprises α-, β-, and γ-subunits controls G1 to S progression, T cell clonal expansion, and functional differentiation (
      • Nelson B.
      • Lord J.
      • Greenberg P.
      ,
      • Nakamura Y.
      • Russell S.
      • Mess S.
      • Friedmann M.
      • Erdos M.
      • Francois C.
      • Jacques Y.
      • Adelstein S.
      • Leonard W.
      ,
      • Smith K.A.
      ). The IL-2R orchestrates downstream effector pathways by protein tyrosine kinase-dependent activation mechanisms engaging the Src family tyrosine kinases Lck and Fyn (
      • Taniguchi T.
      ) and the Janus kinases 1 and 3 (
      • Miyazaki T.
      • Kawahara A.
      • Fujii H.
      • Nakagawa Y.
      • Minami Y.
      • Liu Z.-J.
      • Oishi I.
      • Silvennoinen O.
      • Witthuhn B.
      • Ihle J.
      • Taniguchi T.
      ,
      • Russell S.
      • Johnston J.
      • Noguchi M.
      • Kawamura M.
      • Bacon C.
      • Friedman M.
      • Berg M.
      • McVicar D.
      • Witthuhn B.
      • Silvennoinen O.
      • Goldman A.
      • Schmalstieg F.
      • Ihle J.
      • O'Shea J.
      • Leonard W.
      ,
      • Beadling C.
      • Guschin D.
      • Witthuhn B.A.
      • Ziemiecki A.
      • Ihle J.N.
      • Kerr I.M.
      • Cantrell D.A.
      ). Signaling cascades integrated by the action of these tyrosine kinases include activation of the Ras/Raf/extracellular-signal regulated kinase (Erk) pathway (
      • Izquierdo M.
      • Downward J.
      • Leonard W.J.
      • Otani H.
      • Cantrell D.A.
      ,
      • Turner B.
      • Rapp U.
      • App H.
      • Greene M.
      • Dobashi K.
      • Reid J.
      ,
      • Karnitz L.M.
      • Burns L.A.
      • Sutor S.L.
      • Blenis J.
      • Abraham R.T.
      ), activation of the transcription factors STAT3 and STAT5 (
      • Schindler C.
      • Darnell J.E.J.
      ), and the regulation of phosphatidylinositol 3-kinase (PI 3-kinase) (
      • Williamson P.
      • Merida I.
      • Gaulton G.
      ).
      PI 3-kinase is a ubiquitously expressed enzyme that catalyzes the phosphorylation of phosphoinositides at the D-3 hydroxyl of themyo-inositol ring generating PI 3-phosphate, PI 3,4-bisphosphate, and PI 3,4,5-trisphosphate (
      • Panayotou G.
      • Waterfield M.D.
      ,
      • Kapeller R.
      • Cantley L.
      ). 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 β-chain (
      • Truitt K.E.
      • Mills G.B.
      • Turck C.W.
      • Imboden J.B.
      ); in addition, interleukin-2 (IL-2) stimulation results in the interaction of PI 3-kinase with the Src family kinases Fyn (
      • Karnitz L.
      • Sutor S.
      • Abraham R.
      ) and Lck (
      • Taichman R.
      • Merida I.
      • Torigoe T.
      • Gaulton G.N.
      • Reed J.C.
      ).
      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 (
      • Gold M.R.
      • Duronio V.
      • Saxena S.P.
      • Schrader J.W.
      • Aebersold R.
      ,
      • Venkitaraman A.R.
      • Cowling R.J.
      ). It is also clear that PI 3-kinase activation is necessary for the growth- and differentiation-inducing properties of these cytokines (
      • Monfar M.
      • Lemon K.P.
      • Grammer T.C.
      • Cheatham L.
      • Chung J.K.
      • Vlahos C.J.
      • Blenis J.
      ,
      • Mills G.B.
      • Schmandt R.
      • Gibson S.
      • Leung B.
      • Hill M.
      • May C.
      • Shi Y.F.
      • Branch D.R.
      • Radvanyi L.
      • Truitt K.E.
      • Imboden J.B.
      ,
      • Myers Jr., M.G.
      • Grammer T.C.
      • Wang L.-M.
      • Sun X.J.
      • Pierce J.H.
      • Blenis J.
      • White M.F.
      ,
      • Corcoran A.E.
      • Smart F.M.
      • Cowling R.J.
      • Crompton T.
      • Owen M.J.
      • Venkitaraman A.R.
      ). 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 (
      • Karnitz L.M.
      • Burns L.A.
      • Sutor S.L.
      • Blenis J.
      • Abraham R.T.
      ,
      • Monfar M.
      • Lemon K.P.
      • Grammer T.C.
      • Cheatham L.
      • Chung J.K.
      • Vlahos C.J.
      • Blenis J.
      ). Hence, IL-2 activation of the mitogen-activated protein (MAP) kinase Erk is sensitive to wortmannin (
      • Karnitz L.M.
      • Burns L.A.
      • Sutor S.L.
      • Blenis J.
      • Abraham R.T.
      ). Similarly, IL-2 activation of the serine/threonine kinase p70 S6 kinase (p70S6k) is prevented by these PI 3-kinase inhibitors (
      • Monfar M.
      • Lemon K.P.
      • Grammer T.C.
      • Cheatham L.
      • Chung J.K.
      • Vlahos C.J.
      • Blenis J.
      ). 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) (
      • Proud C.
      ,
      • Brown E.J.
      • Schreiber S.L.
      ). 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 (
      • Proud C.
      ,
      • Brown E.J.
      • Schreiber S.L.
      ). However, this model has been challenged by a recent study showing that the action of Frap is directly inhibited by wortmannin and LY294002 (
      • Brunn G.J.
      • Williams J.
      • Sabers C.
      • Wiederrecht G.
      • Lawrence J., J.C.
      • Abraham R.T.
      ). 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 (
      • Hu Q.
      • Klippel A.
      • Muslin A.J.
      • Fantl W.J.
      • Williams L.T.
      ), but it would be fallacious to extrapolate data obtained inXenopus 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 cell line (
      • Klippel A.
      • Reinhard C.
      • Kavanaugh W.M.
      • Apell G.
      • Escobedo M.A.
      • Williams L.T.
      ,
      • Reif K.
      • Nobes C.D.
      • Thomas G.
      • Hall A.
      • Cantrell D.A.
      ,
      • Didichenko S.A.
      • Tilton B.
      • Hemmings B.A.
      • Ballmer-Hofer K.
      • Thelen M.
      ,
      • Marte B.M.
      • Rodriguez-Viciane P.
      • Wennström S.
      • Warne P.H.
      • Downward J.
      ). 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 (
      • Klippel A.
      • Reinhard C.
      • Kavanaugh W.M.
      • Apell G.
      • Escobedo M.A.
      • Williams L.T.
      ,
      • Reif K.
      • Nobes C.D.
      • Thomas G.
      • Hall A.
      • Cantrell D.A.
      ,
      • Didichenko S.A.
      • Tilton B.
      • Hemmings B.A.
      • Ballmer-Hofer K.
      • Thelen M.
      ,
      • Marte B.M.
      • Rodriguez-Viciane P.
      • Wennström S.
      • Warne P.H.
      • Downward J.
      ). 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 (
      • Reif K.
      • Nobes C.D.
      • Thomas G.
      • Hall A.
      • Cantrell D.A.
      ) 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 (
      • Burgering B.M.
      • Coffer P.J.
      ,
      • Franke F.T.
      • Yang S.
      • Chan O.T.
      • Datta K.
      • Kazlauskas A.
      • Morrison K.D.
      • Kalpan R.D.
      • Tsichlis N.P.
      ,
      • Jones P.F.
      • Jakubowicz T.
      • Pitossi F.J.
      • Maurer F.
      • Hemmings B.A.
      ). PKB was originally identified as the cellular homologue of the directly transforming oncogene of the murine retrovirus AKT8, which causes thymic lymphomas (
      • Staal S.P.
      • Hartley J.W.
      ). 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.

      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-terminal pleckstrin homology domain that can directly bind D-3 phosphoinositides (
      • Franke F.T.
      • Yang S.
      • Chan O.T.
      • Datta K.
      • Kazlauskas A.
      • Morrison K.D.
      • Kalpan R.D.
      • Tsichlis N.P.
      ,
      • James S.R.
      • Downes C.P.
      • Gigg R.
      • Grove S.J.
      • Holmes A.B.
      • Alessi D.R.
      ,
      • Klippel A.
      • Kavanaugh W.M.
      • Pot D.
      • Williams L.T.
      ), 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 (
      • Bellacosa A.
      • Testa J.R.
      • Staal S.P.
      • Tsichlis P.N.
      ), 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 (
      • Brunn G.J.
      • Williams J.
      • Sabers C.
      • Wiederrecht G.
      • Lawrence J., J.C.
      • Abraham R.T.
      ). 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 (
      • Klippel A.
      • Reinhard C.
      • Kavanaugh W.M.
      • Apell G.
      • Escobedo M.A.
      • Williams L.T.
      ). 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 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 inXenopus oocytes (
      • Hu Q.
      • Klippel A.
      • Muslin A.J.
      • Fantl W.J.
      • Williams L.T.
      ) but not in fibroblasts or monoblasts (
      • Klippel A.
      • Reinhard C.
      • Kavanaugh W.M.
      • Apell G.
      • Escobedo M.A.
      • Williams L.T.
      ,
      • Reif K.
      • Nobes C.D.
      • Thomas G.
      • Hall A.
      • Cantrell D.A.
      ,
      • Didichenko S.A.
      • Tilton B.
      • Hemmings B.A.
      • Ballmer-Hofer K.
      • Thelen M.
      ,
      • Marte B.M.
      • Rodriguez-Viciane P.
      • Wennström S.
      • Warne P.H.
      • Downward J.
      ). 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 (
      • Hill C.S.
      • Wynne J.
      • Treisman R.
      ,
      • Genot E.
      • Cleverley S.
      • Henning S.
      • Cantrell D.A.
      ). 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 (
      • Frost J.A.
      • Xu S.
      • Hutchison M.R.
      • Marcus S.
      • Cobb M.H.
      ). Activation of PI 3-kinase is sufficient to induce cytoskeletal rearrangements mediated by the GTPase Rac and Rho in Swiss 3T3 cells (
      • Reif K.
      • Nobes C.D.
      • Thomas G.
      • Hall A.
      • Cantrell D.A.
      ) 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 candidatein vivo targets for D-3 phosphoinositides have been proposed including members of the novel PKC family (
      • Toker A.
      • Meyer M.
      • Reddy K.K.
      • Falck J.R.
      • Aneja R.
      • Aneja S.
      • Parra A.
      • Burns D.J.
      • Ballas L.M.
      • Cantley L.C.
      ) and the atypical PKC family, PKC-λ (
      • Akimoto K.
      • Takahashi R.
      • Moriya S.
      • Nishioka N.
      • Takayanagi J.
      • Kimura K.
      • Fukui Y.
      • Osada S.
      • Mizuno K.
      • Hirai S.
      • Kazlauskas A.
      • Ohno S.
      ) and PKC-ζ (
      • Nakanishi H.
      • Brewer K.A.
      • Exton J.H.
      ), which has recently been implicated as a regulator of Mek and Erk activity in COS cells (
      • Berra E.
      • Diaz-Meco M.T.
      • Lozano J.
      • Frutos S.
      • Municio M.M.
      • Sanchez P.
      • Sanz L.
      • Moscat J.
      ).
      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 (
      • Proud C.
      ,
      • Brown E.J.
      • Schreiber S.L.
      ,
      • Von Manteuffel S.R.
      • Gingras A.C.
      • Ming X.F.
      • Sonenberg N.
      • Thomas G.
      ). 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 (
      • Abraham A.T.
      ) and plays an established, but poorly defined, role as an upstream regulator of p70S6k (
      • Proud C.
      ,
      • Brown E.J.
      • Schreiber S.L.
      ). 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 (
      • Burgering B.M.
      • Coffer P.J.
      ,
      • Franke F.T.
      • Yang S.
      • Chan O.T.
      • Datta K.
      • Kazlauskas A.
      • Morrison K.D.
      • Kalpan R.D.
      • Tsichlis N.P.
      ), 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 (
      • Pearson R.B.
      • Dennis P.B.
      • Han J.-W.
      • Williamson N.A.
      • Kozma S.C.
      • Wettenhall R.E.H.
      • Thomas G.
      ,
      • Dennis P.
      • Pullen N.
      • Kozma S.C.
      • Thomas G.
      ). 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 (
      • Zha J.
      • Harada H.
      • Yang E.
      • Jockel J.
      • Korsmeyer S.J.
      ,
      • Gajewski T.F.
      • Thompson C.B.
      ) 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 (
      • Dudek H.
      • Datta S.R.
      • Franke T.F.
      • Birnbaum M.J.
      • Yao R.
      • Cooper G.M.
      • Segal R.A.
      • Kaplan D.R.
      • Greenberg M.E.
      ,
      • Kauffmann-Zeh A.
      • Rodriguez-Viciana P.
      • Ulrich E.
      • Gilbert C.
      • Coffer P.
      • Downward J.
      • Evan G.
      ). 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 (
      • Cross D.A.E.
      • Alessi D.R.
      • Cohen P.
      • Andjelkovich M.
      • Hemmings B.A.
      ). 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.

      Note Added in Proof

      Recently, Alessi and colleagues described the purification of two upstream kinases that are likely to mediate PKB activation. The activity of at least one of these upstream kinases, PKD1, is regulated by binding D-3 polyphosphate phosphoinositides (Alessi, D. R., James, S. R., Downes, C. P., Holmes, A. B., Gaffney, P. R. J., Reese, C. B., and Cohen, P. (1997) Curr. Biol. 7,261–269).

      Acknowledgments

      We thank George Thomas and Richard Treisman for reagents.

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