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A New Role for the p85-Phosphatidylinositol 3-Kinase Regulatory Subunit Linking FRAP to p70 S6 Kinase Activation*

Open AccessPublished:October 29, 2001DOI:https://doi.org/10.1074/jbc.M103808200
      The serine/threonine kinase p70 S6 kinase (p70S6K) phosphorylates the 40 S ribosomal protein S6, modulating the translation of an mRNA subset that encodes ribosomal proteins and translation elongation factors. p70S6K is activated in response to mitogenic stimuli and is required for progression through the G1 phase of the cell cycle and for cell growth. Activation of p70S6K is regulated by phosphorylation of seven different residues distributed throughout the protein, a subset of which depends on the activity of p85/p110 phosphatidylinositol 3-kinase (PI3K); in fact, the phosphorylation status of Thr229 and Thr389 is intimately linked to PI3K activity. In the full-length enzyme, however, these sites are also acutely sensitive to the action of FKBP 12-rapamycin-associated protein (FRAP). The mechanism by which PI3K and FRAP cooperate to induce p70S6K activation remains unclear. Here we show that the p85 regulatory subunit of PI3K also controls p70S6K activation by mediating formation of a ternary complex with p70S6K and FRAP. The p85 C-terminal SH2 domain is responsible for p85 coupling to p70S6K and FRAP, because deletion of the C-terminal SH2 domain inhibits complex formation and impairs p70S6K activation by PI3K. Formation of this complex is not required for activation of a FRAP-independent form of p70S6K, however, underscoring the role of p85 in regulating FRAP-dependent p70S6K activation. These studies thus show that, in addition to the contribution of PI3K activity, the p85 regulatory subunit plays a critical role in p70S6K activation.
      p70S6K
      p70 S6 kinase
      PtdIns
      phosphoinositide
      PI3K
      phosphatidylinositol 3-kinase
      SH
      Src homology
      PKC
      protein kinase C
      Ab
      antibody
      IL
      interleukin
      C-SH2
      C-terminal SH2
      wt
      wild type
      FRAP
      FKBP 12-rapamycin-associated protein
      PP2A
      protein phosphatase 2A
      p70S6K1 is a mitogen-induced kinase with an important role in ribosome biogenesis. p70S6K phosphorylates the 40 S ribosomal protein S6, regulating the translation of a set of mRNA transcripts containing a polypyrimidine tract at the 5′ transcriptional start site (
      • Jefferies H.B.
      • Fumagalli S.
      • Dennis P.B.
      • Reinhard C.
      • Pearson R.B.
      • Thomas G.
      ). Recruitment of these mRNAs to translating polysomes may be the mechanism by which p70S6K regulates cell growth (
      • Thomas G.
      ). Inhibition of p70S6K with specific antibodies or with the immunosuppressant rapamycin blocks cell cycle progression through G1, implicating this serine/threonine kinase in the control of cell cycle and cell growth (
      • Chung J.
      • Kuo C.J.
      • Crabtree G.R.
      • Blenis J.
      ,
      • Price D.J.
      • Grove J.R.
      • Calvo V.
      • Avruch J.
      • Bierer B.E.
      ,
      • Lane H.A.
      • Fernández A.
      • Lamb N.J.C.
      • Thomas G.
      ). Recent studies support this idea, because p70S6K-deficientDrosophila and p70S6K knockout mice show a significant reduction in body size (
      • Montagne J.
      • Steward M.J.
      • Stocker H.
      • Hafen E.
      • Kozma S.C.
      • Thomas G.
      ,
      • Shima H.
      • Pende M.
      • Chen Y.
      • Fumagalli S.
      • Thomas G.
      • Kozma S.C.
      ).
      p70S6K activity is tightly regulated by the coordinated phosphorylation of at least seven different residues. Four proline-directed sites have been identified in the autoinhibitory domain at the C terminus of the protein (Ser411, Ser418, Thr421, and Ser424). These residues are rapidly phosphorylated in response to mitogenic stimuli, but the kinase(s) responsible for this modification remains to be identified. Phosphorylation of these sites contributes to but is not sufficient for p70S6K activation; other critical residues, such as Thr229 in the activation loop, Ser371, and Thr389 in the linker region must be phosphorylated for full p70S6K activity (
      • Pullen N.
      • Thomas G.
      ). Phosphorylation of Thr229 and Thr389 is sensitive to wortmannin, an inhibitor of PI3K activity, and p70S6K can be activated by constitutively active mutants of PI3K in the absence of mitogenic signals (
      • Chung J.
      • Grammer T.C.
      • Lemon K.P.
      • Kazlauskas A.
      • Blenis J.
      ,
      • Reif K.
      • Burgering B.M.T.
      • Cantrell D.A.
      ), suggesting a role for PI3K in the regulation of p70S6K activity.
      Whereas PI3K does not appear to phosphorylate p70S6K directly, the generation of 3-phosphoinositide (3-PtdIns) products by PI3K is required for Thr229 and Thr389 phosphorylation (
      • Han J.W.
      • Pearson R.B.
      • Dennis P.B.
      • Thomas G.
      ,
      • Balendran A.
      • Currie R.
      • Armstrong C.G.
      • Avruch J.
      • Alessi D.R.
      ). Recent studies show that Thr229 is phosphorylated by the 3-PtdIns-dependent kinase PDK1 (
      • Balendran A.
      • Currie R.
      • Armstrong C.G.
      • Avruch J.
      • Alessi D.R.
      ,
      • Alessi D.R.
      • Kozlowski M.T.
      • Weng Q.P.
      • Morrice N.
      • Avruch J.
      ,
      • Pullen N.
      • Dennis P.B.
      • Andjelkovic M.
      • Dufner A.
      • Kozma S.C.
      • Hemmings B.A.
      • Thomas G.
      ), whereas Thr389 is phosphorylated by the NEK6/7 kinases, members of never-in-mitosis-Aspergillus-like family kinases (
      • Belham C.
      • Comb M.J.
      • Avruch J.
      ). Other authors propose that FRAP catalyzes this step, because Thr389 phosphorylation is blocked by rapamycin (
      • Pearson R.B.
      • Dennis P.B.
      • Han J.W.
      • Williamson N.A.
      • Kozma S.C.
      • Wettenhall R.E.
      • Thomas G.
      ). FRAP has also been shown to phosphorylate Thr389in vitro, although this observation has not been reproduced in vivo (
      • Burnett P.E.
      • Barrow R.K.
      • Cohen N.A.
      • Snyder S.H.
      • Sabatini D.M.
      ,
      • Isotani S.
      • Hara K.
      • Tokunaga C.
      • Inoue H.
      • Avruch J.
      • Yonezawa K.
      ). A distinct function for FRAP, consistent with its ability to regulate phosphorylation of multiple p70S6K residues (
      • Dennis P.B.
      • Pullen N.
      • Kozma S.C.
      • Thomas G.
      ), was recently proposed by Peterson et al. (
      • Peterson R.T.
      • Desai B.N.
      • Hardwick J.S.
      • Schreiber S.L.
      ). They show that PP2A associates directly and dephosphorylates p70S6K and that FRAP inhibits PP2A-mediated p70S6K dephosphorylation. Finally, mitogen-regulated phosphorylation of Ser371 by a still uncharacterized kinase also contributes to p70S6K activation (
      • Moser B.A.
      • Dennis P.B.
      • Pullen N.
      • Pearson R.B.
      • Williamson N.A.
      • Wettenhall R.E.
      • Kozma S.C.
      • Thomas G.
      ). The current model for p70S6K activation thus suggests that the coordinated phosphorylation of several residues is required for enzyme activation. According to this model, the phosphorylation of residues lying within the pseudosubstrate domain by proline-directed kinases releases the catalytic domain from the C-terminal autoinhibitory domain; this permits subsequent Thr389 phosphorylation by the NEK6/7 kinases (
      • Belham C.
      • Comb M.J.
      • Avruch J.
      ), further facilitating Thr229 access to PDK1 (
      • Alessi D.R.
      • Kozlowski M.T.
      • Weng Q.P.
      • Morrice N.
      • Avruch J.
      ,
      • Pullen N.
      • Dennis P.B.
      • Andjelkovic M.
      • Dufner A.
      • Kozma S.C.
      • Hemmings B.A.
      • Thomas G.
      ,
      • Mahalingam D.J.
      • Templeton D.J.
      ). In addition, FRAP action would be required to protect p70S6K from PP2A-mediated dephosphorylation (
      • Peterson R.T.
      • Desai B.N.
      • Hardwick J.S.
      • Schreiber S.L.
      ).
      Here we analyzed the contribution of PI3K to p70S6K activation in greater detail. PtdIns 3-kinases are lipid kinases that modify PtdIns at position 3 of the inositol ring (
      • Panayotou G.
      • Waterfield M.D.
      ). The first characterized form of PI3K, class IA PI3K, is a heterodimer composed of a catalytic (p110α, β, or δ) and a regulatory (p85α, β, or p5γ) subunit (reviewed in Refs.
      • Vanhaesebroek B.
      • Waterfield M.D.
      and
      • Fruman D.A.
      • Meyers R.E.
      • Cantley L.A.
      ). The primary sequence of the p110 catalytic subunit comprises several regions, including the p85-binding region, a Ras-binding domain, a region homologous to phosphatidylinositol 4-kinases, and the catalytic core (
      • Vanhaesebroek B.
      • Waterfield M.D.
      ,
      • Fruman D.A.
      • Meyers R.E.
      • Cantley L.A.
      ). The p85 regulatory subunit has an SH3 domain, a Bcr homologous region flanked by two proline-rich regions, and two SH2 domains separated by an inter-SH2 region (
      • Vanhaesebroek B.
      • Waterfield M.D.
      ,
      • Fruman D.A.
      • Meyers R.E.
      • Cantley L.A.
      ). Stimulation of growth factor receptor Tyr kinases induces p85/p110 binding to these receptors via the p85 SH2 domains, resulting in PI3K activation (
      • Skolnik E.Y.
      • Margolis B.
      • Mohammadi M.
      • Lowenstein E.
      • Fischer R.
      • Drepps A.
      • Ullrich A.
      • Schlessinger J.
      ,
      • Kodaki T.
      • Woscholski R.
      • Hallberg B.
      • Rodrı́guez-Viciana P.
      • Downward J.
      • Parker P.J.
      ). Activated mammalian p85/p110 PI3K controls several important cell functions, including cytoskeletal organization, cell growth, cell division, and survival (
      • Leevers S.J.
      • Weinkove D.
      • McDougall L.K.
      • Hafen E.
      • Waterfield M.D.
      ,
      • Wennstrom S.
      • Hawkins P.
      • Cooke F.
      • Hara K.
      • Yonezawa K.
      • Kasuga M.
      • Jackson T.
      • Claesson-Welsh L.
      • Stephens L.
      ,
      • Yao R.
      • Cooper G.M.
      ,
      • Roche S.
      • Koegl M.
      • Courtneidge S.A.
      ). The mechanism by which p85/p110 PI3K regulates such a variety of cellular responses is not fully known but involves the binding of proteins containing SH2 and pleckstrin homology domains to 3-phosphorylated PtdIns on the cell membrane (
      • Rameh L.E.
      • Chen C.S.
      • Cantley L.C.
      ,
      • Klippel A.
      • Kavanaugh W.M.
      • Pot D.
      • Williams L.T.
      ), a feature that contributes to the induction of some PI3K effectors. In addition, p85 contributes to cell stimulation by recruiting signaling molecules to the stimulated receptors via its SH3, SH2, and Bcr homologous domains (
      • Vanhaesebroek B.
      • Waterfield M.D.
      ,
      • Fruman D.A.
      • Meyers R.E.
      • Cantley L.A.
      ). Many cellular effectors of PI3K have been identified and include the small GTP-binding protein Rac, the Ser/Thr kinase AKT/PKB, Bruton tyrosine kinase, PDK1, certain PKC isoforms, and p70S6K (
      • Chung J.
      • Grammer T.C.
      • Lemon K.P.
      • Kazlauskas A.
      • Blenis J.
      ,
      • Hawkins P.T.
      • Eguinoa A.
      • Qiu R.G.
      • Stokoe D.
      • Cooke F.T.
      • Walters R.
      • Wennstrom S.
      • Claesson-Welsh L.
      • Evans T.
      • Symons M.
      • et al.
      ,
      • Chou M.M.
      • Blenis J.
      ,
      • Burgering B.M.T.
      • Coffer P.J.
      ,
      • 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.
      ,
      • Alessi D.R.
      • James S.R.
      • Downes C.P.
      • Holmes A.B.
      • Gaffney P.R.
      • Reese C.B.
      • Cohen P.
      ).
      In this study, we examined the contribution of the p85 regulatory subunit to p70S6K activation. We used a mutant p85 form, p65PI3K, which lacks one of the adapter SH2 domains but binds to p110, exhibiting higher associated lipid kinase activity than p85/p110 and enhancing receptor-stimulated PI3K activation (
      • Jiménez C.
      • Jones D.R.
      • Rodriguez V.P.
      • González G.A.
      • Leonardo E.
      • Wennstrom S.
      • von Kobbe C.
      • Torán J.L.
      • Rodriguez-B L.
      • Calvo V.
      • Copin S.G.
      • Albar J.P.
      • Gaspar M.L.
      • Dı́ez E.
      • Marcos M.A.
      • Downward J.
      • Martı́nez-A C.
      • Mérida I.
      • Carrera A.C.
      ). With this construct, we show that PI3K regulation of p70S6K activation requires not only PI3K enzymatic activity but also the adaptor function of the p85 regulatory subunit of PI3K. In this function, p85 mediates formation of a multimolecular complex that links FRAP to p70S6K. Formation of this complex, mediated by the C-terminal SH2 domain of p85, is required for efficient p70S6K activation. This complex, however, is not necessary for p70S6K activation when PP2A is inhibited nor for activation of a FRAP-independent form of p70S6K that does not bind PP2A. These results show that p85 contributes to p70S6K activation by bringing FRAP into complex with p70S6K, which in turn protects p70S6K from dephosphorylation. Whereas PI3K activity is required for Thr229 and Thr389 phosphorylation, the p85-PI3K regulatory subunit is necessary for p70S6K to retain its active/phosphorylated conformation.

      DISCUSSION

      Here we show that p85/p110 PI3K has a dual role in p70S6K activation. In addition to the requirement for PI3K activity in PDK1-dependent p70S6K phosphorylation (
      • Alessi D.R.
      • Kozlowski M.T.
      • Weng Q.P.
      • Morrice N.
      • Avruch J.
      ,
      • Pullen N.
      • Dennis P.B.
      • Andjelkovic M.
      • Dufner A.
      • Kozma S.C.
      • Hemmings B.A.
      • Thomas G.
      ), we demonstrate that the p85 regulatory subunit of PI3K contributes to p70S6K stimulation by establishing a multimolecular complex that brings p70S6K into proximity with its activators, PI3K and FRAP. Formation of this complex requires the p85 C-SH2 domain, because the p65PI3K mutant, which lacks this domain (
      • Jiménez C.
      • Jones D.R.
      • Rodriguez V.P.
      • González G.A.
      • Leonardo E.
      • Wennstrom S.
      • von Kobbe C.
      • Torán J.L.
      • Rodriguez-B L.
      • Calvo V.
      • Copin S.G.
      • Albar J.P.
      • Gaspar M.L.
      • Dı́ez E.
      • Marcos M.A.
      • Downward J.
      • Martı́nez-A C.
      • Mérida I.
      • Carrera A.C.
      ), associates moderately with p70S6K and very poorly with FRAP. Complex formation is required for p70S6K activation, which is defective in p65PI3K/p110-expressing cells. In accordance with this, p70S6K shows a lower degree of phosphorylation in multiple residues in these cells. p65PI3K/p110 nonetheless activated a FRAP-resistant p70S6K mutant, suggesting that impaired p70S6K activation in these cells is related to the defect in FRAP complex formation. FRAP protects p70S6K from PP2A-mediated dephosphorylation (
      • Peterson R.T.
      • Desai B.N.
      • Hardwick J.S.
      • Schreiber S.L.
      ). We show that inhibition of PP2A restored p70S6K activation in p65PI3K cells, further indicating that the C-SH2 deletion mutant defect is related to impaired FRAP action. These data highlight the essential role of the p85 C-SH2 domain in establishing the formation of a ternary complex of p85, p70S6K, and FRAP, required for efficient p70S6K phosphorylation and activation.
      For these experiments, we used a mutant form of the PI3K regulatory subunit, p65PI3K, which lacks part of the p85 inter-SH2 and C-SH2 domains (
      • Jiménez C.
      • Jones D.R.
      • Rodriguez V.P.
      • González G.A.
      • Leonardo E.
      • Wennstrom S.
      • von Kobbe C.
      • Torán J.L.
      • Rodriguez-B L.
      • Calvo V.
      • Copin S.G.
      • Albar J.P.
      • Gaspar M.L.
      • Dı́ez E.
      • Marcos M.A.
      • Downward J.
      • Martı́nez-A C.
      • Mérida I.
      • Carrera A.C.
      ). p65PI3K/p110 exhibits higher in vivo lipid kinase activity than p85/p110 and enhances receptor-stimulated PI3K activation (
      • Jiménez C.
      • Jones D.R.
      • Rodriguez V.P.
      • González G.A.
      • Leonardo E.
      • Wennstrom S.
      • von Kobbe C.
      • Torán J.L.
      • Rodriguez-B L.
      • Calvo V.
      • Copin S.G.
      • Albar J.P.
      • Gaspar M.L.
      • Dı́ez E.
      • Marcos M.A.
      • Downward J.
      • Martı́nez-A C.
      • Mérida I.
      • Carrera A.C.
      ). The increase in 3-phosphorylated lipids in p65PI3K-expressing cells appears to be sufficient for activation of the downstream effector AKT (
      • Jiménez C.
      • Jones D.R.
      • Rodriguez V.P.
      • González G.A.
      • Leonardo E.
      • Wennstrom S.
      • von Kobbe C.
      • Torán J.L.
      • Rodriguez-B L.
      • Calvo V.
      • Copin S.G.
      • Albar J.P.
      • Gaspar M.L.
      • Dı́ez E.
      • Marcos M.A.
      • Downward J.
      • Martı́nez-A C.
      • Mérida I.
      • Carrera A.C.
      ) but insufficient to activate p70S6K. These results suggest that PI3K contributes differently to the activation of its effectors AKT and p70S6K. Other activation requirements have been shown to differ for these proteins. AKT activation requires phosphorylation of Thr308 and Ser473, which are homologous to p70S6K residues Thr229 and Thr389. Whereas AKT T308 and p70S6K Thr229 are both phosphorylated by PDK1 (
      • Alessi D.R.
      • Kozlowski M.T.
      • Weng Q.P.
      • Morrice N.
      • Avruch J.
      ,
      • Pullen N.
      • Dennis P.B.
      • Andjelkovic M.
      • Dufner A.
      • Kozma S.C.
      • Hemmings B.A.
      • Thomas G.
      ,
      • Alessi D.R.
      • James S.R.
      • Downes C.P.
      • Holmes A.B.
      • Gaffney P.R.
      • Reese C.B.
      • Cohen P.
      ,
      • Stokoe D.
      • Stephens L.R.
      • Copeland T.
      • Gaffney P.R.
      • Reese C.B.
      • Painter G.F.
      • Holmes A.B.
      • McCormick F.
      • Hawkins P.T.
      ), there are differences in the phosphorylation mechanisms of AKT Ser473 and p70S6K Thr389. AKT Ser473 is phosphorylated by PDK1 complexed to a region of protein kinase C-related kinase-2, called the PDK1-interacting fragment, whereas phosphorylation of Thr389 of p70S6K is mediated by NEK6/7 (
      • Balendran A.
      • Currie R.
      • Armstrong C.G.
      • Avruch J.
      • Alessi D.R.
      ,
      • Belham C.
      • Comb M.J.
      • Avruch J.
      ,
      • Balendran A.
      • Casamayor A.
      • Deak M.
      • Paterson A.
      • Gaffney P.
      • Currie R.
      • Downes C.P.
      • Alessi D.R.
      ). Conus et al. (
      • Conus N.M.
      • Hemmings B.A.
      • Pearson R.B.
      ) have also shown that depletion of calcium from intracellular stores blocks p70S6K activation, whereas the AKT response is unaffected, highlighting the differential activation requirements of these two proteins. The present report suggests that additional differences in the activation mechanism of p70S6K and AKT reside in the contribution of the p85 C-terminal region.
      Several lines of evidence suggest that p70S6K is downstream of AKT. Although constitutively active membrane-targeted forms of AKT activate p70S6K, AKT does not appear to phosphorylate p70S6K directly (
      • Alessi D.R.
      • Kozlowski M.T.
      • Weng Q.P.
      • Morrice N.
      • Avruch J.
      ). The finding that p65PI3K/p110 activates AKT and not p70S6K shows that activation of AKT is not sufficient for p70S6K activation. It has also been suggested that AKT regulates FRAP function (
      • Nave B.T.
      • Ouwens M.
      • Withers D.J.
      • Alessi D.R.
      • Shepherd P.R.
      ), and the data presented here do not refute this possibility. Nonetheless, the observation that kinase-dead mutants of AKT do not inhibit FRAP-dependent p70S6K activation by growth factors (
      • Burgering B.M.T.
      • Coffer P.J.
      ,
      • Dufner A.
      • Andjelkovic M.
      • Burgering B.M.
      • Hemmings B.A.
      • Thomas G.
      ) does question the contribution of AKT in the process.
      To study the mechanism by which the p85 C-terminal region affects p70S6K activation, we examined whether impaired p70S6K activation in p65PI3K/p110-expressing cells correlated with an altered p70S6K phosphorylation pattern. Acidic substitutions of residues in the pseudosubstrate domain and in Thr389 did not compensate the activation defect of p70S6K in p65PI3K/p110-expressing cells. This suggests that defects other than phosphorylation of the pseudosubstrate region or Thr389 contribute to impairment of p70S6K activation in these cells. Similarly, although a significant reduction in overall p70S6K phosphorylation was found in p65PI3K/active p110-expressing cells, phosphotryptic peptide maps of p70S6K immunoprecipitated from p85/p110 or from p65PI3K/p110-expressing cells did not reveal a specific phosphorylation site defect. The lower overall phosphorylation may result from an inefficient early step in p70S6K activation or, alternatively, may reflect that p70S6K is dephosphorylated in p65PI3K-expressing cells.
      The observations presented demonstrate that the complex of p85, p70S6K, and FRAP plays an essential role in p70S6K activation. Romanelliet al. (
      • Romanelli A.
      • Martin K.A.
      • Toker A.
      • Blenis J.
      ) reported that p70S6K associates with PKCζ and PDK1 and that formation of this complex is essential for PI3K-dependent p70S6K activation but not for PMA-induced p70S6K activation (
      • Romanelli A.
      • Martin K.A.
      • Toker A.
      • Blenis J.
      ,
      • Akimoto K.
      • Nakaya M.
      • Yamanaka T.
      • Tanaka J.
      • Matsuda S.
      • Weng Q.P.
      • Avruch J.
      • Ohno S.
      ). Although atypical PKCs are PI3K effectors and modulate p70S6K activity (
      • Romanelli A.
      • Martin K.A.
      • Toker A.
      • Blenis J.
      ,
      • Akimoto K.
      • Nakaya M.
      • Yamanaka T.
      • Tanaka J.
      • Matsuda S.
      • Weng Q.P.
      • Avruch J.
      • Ohno S.
      ,
      • Akimoto K.
      • Takahashi R.
      • Moriya S.
      • Nishioka N.
      • Takayanagi J.
      • Kimura K.
      • Fukui Y.
      • Osada S.
      • Mizuno K.
      • Hirai S.
      • Kazlauskas A.
      • Ohno S.
      ,
      • Standaert M.L.
      • Galloway L.
      • Karnam P.
      • Bandyopadhyay G.
      • Moscat J.
      • Farese R.V.
      ), it is unlikely that the inability of p65PI3K/p110 to activate p70S6K depends on an atypical PKC, because constitutively active mutants of this protein failed to rescue p70S6K activation in p65PI3K transfectants (Fig. 5). In contrast, differential association was found of p85 and p65PI3K with p70S6K and even more clearly with FRAP. Whereas p85 associated efficiently with both p70S6K and FRAP, the association of these proteins with the mutant p65PI3K form was significantly weaker and, in the case of FRAP, could be detected only by reprecipitation techniques after prolonged exposure of the gels. We propose that p85 association with p70S6K and FRAP involves the C-SH2 domain, because this is absent in p65PI3K. Furthermore, the finding that the defective activation of p70S6K in p65PI3K cells can be overcome by deletion of the N- and C-terminal domains of p70S6K, conferring FRAP sensitivity, shows that the defective association with FRAP is responsible for the impaired p70S6K activation in p65PI3K cells. This observation underscores the role of the FRAP·p85 complex in regulating the endogenous p70S6K enzyme and supports the hypothesis that p85 links FRAP to p70S6K.
      The use of the immunosuppressant rapamycin has positioned FRAP upstream of p70S6K in the signaling cascade. FRAP has been shown to phosphorylate Thr389in vitro, but this observation has not been reproduced in vivo (
      • Burnett P.E.
      • Barrow R.K.
      • Cohen N.A.
      • Snyder S.H.
      • Sabatini D.M.
      ,
      • Isotani S.
      • Hara K.
      • Tokunaga C.
      • Inoue H.
      • Avruch J.
      • Yonezawa K.
      ). Indeed, the finding that the N- and C-terminally truncated p70S6K mutant is phosphorylated in vivo on Thr389 in the presence of rapamycin (
      • Dennis P.B.
      • Pullen N.
      • Kozma S.C.
      • Thomas G.
      ) suggests that FRAP must regulate p70S6K at a different stage. Concurring with this, whereas deletion of the regions that confer FRAP sensitivity restored p65PI3K/p110 activation of p70S6K, acidic substitution at p70S6K residue Thr389 was unable to rescue p70S6K activation. Belhamet al. (
      • Belham C.
      • Comb M.J.
      • Avruch J.
      ) recently provided evidence suggesting that NEK6 and NEK7 are the Thr389 kinases. A distinct function for FRAP, consistent with its ability to regulate phosphorylation of multiple p70S6K residues (
      • Dennis P.B.
      • Pullen N.
      • Kozma S.C.
      • Thomas G.
      ), was recently proposed by Petersonet al. (
      • Peterson R.T.
      • Desai B.N.
      • Hardwick J.S.
      • Schreiber S.L.
      ). They show that PP2A associates directly and dephosphorylates p70S6K and that FRAP inhibits PP2A-mediated p70S6K dephosphorylation. Moreover, they show that activation of the ΔN54ΔC104-p70S6K deletion mutant is FRAP-independent, because this p70S6K mutant does not bind PP2A (
      • Peterson R.T.
      • Desai B.N.
      • Hardwick J.S.
      • Schreiber S.L.
      ). p65PI3K/p110 activates the ΔN54ΔC104-p70S6K mutant that does not bind PP2A (Fig.8A). In addition, PP2A inhibition restored p70S6K activation in p65PI3K-expressing cells (Fig. 8C). These observations demonstrate that the deficient phosphorylation and activation of p70S6K in p65PI3K-expressing cells is related to inefficient PI3K·p70S6K·FRAP complex formation by p65PI3K, which results in dephosphorylation of p70S6K by PP2A.
      Our results support a specific role for the p85 C-SH2 domain in mediating complex formation with p70S6K and FRAP. It is nevertheless possible that the p65PI3K N-SH2 domain partially compensates for the absence of C-SH2, accounting for the moderate association of p65PI3K with p70S6K and the very low association with FRAP. The differential specificity of the two SH2 domains of p85 (
      • Fruman D.A.
      • Meyers R.E.
      • Cantley L.A.
      ,
      • Rameh L.E.
      • Chen C.S.
      • Cantley L.C.
      ) offers an explanation for the greater efficiency of the C-SH2 compared with the N-SH2 in mediating complex formation. Despite the defective p70S6K activation in p65PI3K cells, these cells grow efficiently. Activation of the other p70S6K isoform (p70S6K-II) (
      • Shima H.
      • Pende M.
      • Chen Y.
      • Fumagalli S.
      • Thomas G.
      • Kozma S.C.
      ,
      • Gout I.
      • Minami T.
      • Hara K.
      • Tsujishita Y.
      • Filonenko V.
      • Waterfield M.D.
      • Yonezawa K.
      ,
      • Lee-Fruman K.K.
      • Kuo C.J.
      • Lippincott J.
      • Terada N.
      • Blenis J.
      ), which is less dependent on PI3K and FRAP (
      • Gout I.
      • Minami T.
      • Hara K.
      • Tsujishita Y.
      • Filonenko V.
      • Waterfield M.D.
      • Yonezawa K.
      ), or activation of p90rsk(
      • Templeton D.J.
      ) may compensate for the p70S6K-I activation defect in these cells.
      Our results highlight the importance of the PI3K regulatory subunit in stabilizing a protein complex required for p70S6K activation. This complex includes p70S6K, PI3K, FRAP, and probably PDK1 and PKCζ, bringing p70S6K into the proximity of its activators and thereby mediating p70S6K activation. The studies presented here show that PI3K exerts two independent actions on p70S6K activation. The first, described previously, is dependent on PI3K enzymatic activity implicated in phosphorylation of Thr229 and Thr389 residues (
      • Chung J.
      • Grammer T.C.
      • Lemon K.P.
      • Kazlauskas A.
      • Blenis J.
      ,
      • Reif K.
      • Burgering B.M.T.
      • Cantrell D.A.
      ,
      • Han J.W.
      • Pearson R.B.
      • Dennis P.B.
      • Thomas G.
      ,
      • Balendran A.
      • Currie R.
      • Armstrong C.G.
      • Avruch J.
      • Alessi D.R.
      ); the other, described here, depends on the p85-PI3K regulatory subunit that mediates the formation of a multimolecular complex involving p85, FRAP, and p70S6K. This complex is required for efficient p70S6K activation.

      Acknowledgments

      We thank Drs. D. M. Sabatini for the FRAP-expressing plasmids, J. Downward for pMT2-wt-p110, G. Thomas for wild type p70S6K, D3Ep70S6K, and E389D3Ep70S6K cDNAs and for comments on the manuscript, P. J. Parker for plasmids encoding PKC mutants, and D. J. Templeton for EE-p70S6K vector. We also thank Drs. I. Mérida, V. Calvo, A. Jaeschke, and P. Dennis for technical help and comments on the manuscript.

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