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Akt Is a Direct Target of the Phosphatidylinositol 3-Kinase

ACTIVATION BY GROWTH FACTORS, v-src and v-Ha-ras, IN Sf9 AND MAMMALIAN CELLS*
  • Ketaki Datta
    Affiliations
    Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111
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  • Alfonso Bellacosa
    Footnotes
    Affiliations
    Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111
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  • Tung O. Chan
    Affiliations
    Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111
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  • Philip N. Tsichlis
    Correspondence
    To whom correspondence should be addressed:
    Affiliations
    Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111
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  • Author Footnotes
    * This work was supported in part by United States Public Health Service Grant CA57436. Additional support was provided by United States Public Health Service Grant CA06927 and by an appropriation from the Commonwealth of Pennsylvania to the Fox Chase Cancer Center. 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.
    Supported in part by the Scientific Exchange Program of the National Cancer Institute and the Consiglio Nazionale delle Ricerche-Italy.
Open AccessPublished:November 29, 1996DOI:https://doi.org/10.1074/jbc.271.48.30835
      The Akt protooncogene encodes a serine-threonine protein kinase which is activated by growth factor-generated signals that are transduced via the phosphatidylinositol 3′-kinase (PI3-K). Earlier studies suggested that the activation of Akt by PI3-K may be mediated by the binding of D3-phosphorylated phosphoinositides to the Akt pleckstrin homology (PH) domain. On the basis of these studies, it was hypothesized that Akt is a direct PI3-K target. To test this hypothesis, we reconstituted the pathway of Akt activation in baculovirus-infected Sf9 cells. The results showed that Akt, which is normally catalytically inactive in these cells, was activated when coexpressed with the activated PI3-K. Moreover, they showed that activated forms of c-Ha-ras (v-Ha-ras) and c-src (v-src or srcY527F), two molecules that transduce growth factor-generated signals, also activate Akt in a PI3-K-dependent manner in Sf9 as well as NIH 3T3 cells. The activation of Akt by both growth factors and v-ras and v-src (or srcY527F) depends on the integrity of the Akt PH domain and carboxyl-terminal tail. These results show that Akt activation via the PI3-K can be faithfully reproduced in baculovirus-infected Sf9 cells. The same results support the hypothesis that Akt is a direct target of the PI3-K and identify cytoplasmic signaling molecules that may contribute to the transduction of PI3-K/Akt activation signals.

      INTRODUCTION

      c-akt, the cellular homolog of the viral protooncogene v-akt (
      • Bellacosa A.
      • Testa J.R.
      • Staal S.
      • Tsichlis P.N.
      ), encodes a serine-threonine protein kinase which is highly related to protein kinase C and contains an amino-terminal PH
      The abbreviations used are: PH
      pleckstrin homology
      PI3-K
      phosphatidylinositol 3-kinase
      PDGF
      platelet-derived growth factor
      PPI
      phosphorylated phosphoinositides
      PDGFRβ
      platelet-derived growth factor receptor β
      D-PBS
      Dulbecco's phosphate-buffered saline
      HA
      hemagglutinin
      DMEM
      Dulbecco's modified Eagle's medium.
      domain (
      • Bellacosa A.
      • Testa J.R.
      • Staal S.
      • Tsichlis P.N.
      ,
      • Haslam R.J.
      • Koide H.B.
      • Hemmings B.A.
      ,
      • Mayer B.J.
      • Ren R.
      • Clark K.L.
      • Baltimore D.
      ). Akt is activated by serum and a variety of growth factors sharing the ability to activate the PI3-K, such as PDGF, epidermal growth factor, bovine fibroblast growth factor, insulin (
      • Andjelkovic M.
      • Jakubowicz T.
      • Cron P.
      • Ming X.F.
      • Han J.W.
      • Hemmings B.A.
      ,
      • Burgering B.M.T.
      • Coffer P.J.
      ,
      • Franke T.F.
      • Yang S.-I.
      • Chan T.O.
      • Datta K.
      • Kazlauskas A.
      • Morrison D.K.
      • Kaplan D.R.
      • Tsichlis P.N.
      ,
      • Kohn A.D.
      • Kovacina K.S.
      • Roth R.A.
      ), insulin-like growth factor-1,
      A. Bellacosa and P. N. Tsichlis, unpublished data.
      and interleukin-2.
      N. N. Ahmed, T. O. Chan, A. Bellacosa, and P. N. Tsichlis manuscript in preparation.
      Activation of Akt by growth factors depends on the integrity of the PH domain (
      • Franke T.F.
      • Yang S.-I.
      • Chan T.O.
      • Datta K.
      • Kazlauskas A.
      • Morrison D.K.
      • Kaplan D.R.
      • Tsichlis P.N.
      ) and is blocked by wortmannin (
      • Andjelkovic M.
      • Jakubowicz T.
      • Cron P.
      • Ming X.F.
      • Han J.W.
      • Hemmings B.A.
      ,
      • Burgering B.M.T.
      • Coffer P.J.
      ,
      • Franke T.F.
      • Yang S.-I.
      • Chan T.O.
      • Datta K.
      • Kazlauskas A.
      • Morrison D.K.
      • Kaplan D.R.
      • Tsichlis P.N.
      ,
      • Kohn A.D.
      • Kovacina K.S.
      • Roth R.A.
      ), a powerful PI3-K inhibitor. In vitro incubation of inactive Akt immunoprecipitated from serum-starved NIH 3T3 cells, with enzymatically synthesized D3-phosphorylated phosphoinositides (PPIs), activated Akt in a dose-dependent manner (
      • Franke T.F.
      • Yang S.-I.
      • Chan T.O.
      • Datta K.
      • Kazlauskas A.
      • Morrison D.K.
      • Kaplan D.R.
      • Tsichlis P.N.
      ). This suggested that the modulation of the Akt catalytic activity is mediated by binding of PI3-K-generated PPIs to the Akt PH domain. The preceding results were interpreted to indicate that Akt is a direct target of the PI3-K. However, this conclusion was challenged by a report showing that the activation of Akt by insulin, contrary to its activation by PDGF, was unaffected by PH domain mutations (
      • Kohn A.D.
      • Kovacina K.S.
      • Roth R.A.
      ). To test the hypothesis that Akt is a direct target of the activated PI3-K, we reconstituted the pathway of Akt activation by infecting Sf9 cells with combinations of Akt-, p85-, p110-, and PDGFRβ-expressing baculoviruses. The results reported here support the hypothesis that the Akt activation by PI3-K is direct.
      The activation of the PI3-K by growth factor-generated signals is mediated by intracellular signaling molecules. One such molecule, the cytoplasmic tyrosine kinase c-src binds p85, the regulatory subunit of the PI3-K (
      • Fukui Y.
      • Hanafusa H.
      ), while another, c-Ha-ras, binds and activates p110, the PI3-K catalytic subunit (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Dhand R.
      • Vanhaesebroeck B.
      • Gout I.
      • Fry M.J.
      • Waterfield M.D.
      • Downward J.
      ). Our earlier studies had indeed shown that the activation of Akt by PDGF was inhibited by the dominant negative mutant RasN17 suggesting that Ras contributes to the transduction of PDGF-induced signals that activate Akt (
      • Franke T.F.
      • Yang S.-I.
      • Chan T.O.
      • Datta K.
      • Kazlauskas A.
      • Morrison D.K.
      • Kaplan D.R.
      • Tsichlis P.N.
      ). To determine whether activated forms of c-src and c-Ha-ras activate Akt and whether their potential contributions to the activation of Akt were dependent on the PI3-K, we examined their role in Akt activation in Sf9 and NIH 3T3 cells. The data presented in this report show that v-src (or srcY527F) and v-Ha-ras or the combination of the two activate Akt in a PI3-K-dependent manner in both Sf9 and mammalian cells. The activation of Akt by both growth factors and v-src (or srcY527F) and/or v-Ha-ras depends on the Akt PH domain and carboxyl-terminal tail.

      DISCUSSION

      The data presented in this manuscript show that the activation of Akt via the PI3-K can be reproduced faithfully in Sf9 cells and provide support to the hypothesis that Akt is a direct target of the PI3-K. Moreover, they show that the activation of the PI3-K by both growth factors and intracellular signaling molecules is sufficient to activate Akt in both Sf9 and mammalian cells.
      Earlier studies had shown that Akt activation by PDGF requires that the Akt protein has an intact PH domain (
      • Franke T.F.
      • Yang S.-I.
      • Chan T.O.
      • Datta K.
      • Kazlauskas A.
      • Morrison D.K.
      • Kaplan D.R.
      • Tsichlis P.N.
      ). This observation combined with the in vitro activation of Akt by enzymatically synthesized PPIs was interpreted to suggest that Akt activation depends on the direct interaction between the Akt PH domain and PI3-K-generated PPIs (
      • Franke T.F.
      • Yang S.-I.
      • Chan T.O.
      • Datta K.
      • Kazlauskas A.
      • Morrison D.K.
      • Kaplan D.R.
      • Tsichlis P.N.
      ). The validity of this hypothesis, however, was questioned because of independent studies showing that the PH domain is dispensable for Akt activation by insulin (
      • Kohn A.D.
      • Kovacina K.S.
      • Roth R.A.
      ). Due to these conflicting data, we proceeded to re-evaluate the role of the PH domain in the PDGF-induced activation of Akt. The results reported here confirmed that only Akt with an intact PH domain can be fully activated and provided support to the hypothesis that Akt activation is induced by binding of PI3-K-generated PPIs to the PH domain. Alternatively, these mutations could lock Akt into a nonactivatable conformation. Such a conformational change could interfere with the ability of Akt to form dimers (
      • Datta K.
      • Franke T.F.
      • Chan T.O.
      • Makris A.
      • Yang S.-I.
      • Kaplan D.
      • Morrison D.K.
      • Golemis E.A.
      • Tsichlis P.N.
      ) or to interact with other proteins or it could block its stimulation-dependent translocation to the cell membrane. The differences between PDGF and insulin regarding the role of the PH domain in Akt activation could perhaps be due to differences in signaling between these factors.
      Among the Akt PH domain mutants, particularly interesting was the mutant AktΔ11-60 which showed high constitutive kinase activity but failed to respond to PDGFRβ-generated signals. The high basal kinase activity of the AktΔ11-60 mutant suggests that mutations in the PH domain may either increase the affinity of the PH domain to activating PPIs or abrogate the binding of inhibitory PPIs. The former possibility appears to be supported by the fact that the kinase activity of this mutant is partially inhibited by wortmannin. Alternatively, such mutations may lock the Akt protein in an active conformation perhaps because they may affect its ability to form dimers or to interact with other proteins.
      The carboxyl-terminal tails of several kinases appear to play important regulatory roles. Thus, truncation of the carboxyl-terminal tail of c-src (
      • Cooper J.A.
      • Gould K.L.
      • Cartwright C.A.
      • Hunter T.
      ) and Tpl-24 (
      • Patriotis C.
      • Makris A.
      • Bear S.E.
      • Tsichlis P.N.
      ) or mutation of Tyr-527 of c-src into phenylalanine (
      • Cartwright C.A.
      • Eckhart W.
      • Simon S.
      • Kaplan P.L.
      ) activate these kinases constitutively. The data presented in this report show that deletion of the carboxyl-terminal tail of Akt gives rise to a protein that is catalytically inactive and fails to respond to the activated PI3-K. Therefore, the Akt carboxyl-terminal tail may also play a regulatory role which is, however, qualitatively different from that of the c-src and Tpl-2 tails. The tail contribution to the regulation of the Akt kinase activity could be mediated through its interaction with residues in the kinase domain. Such interactions could maintain the protein in an activable conformation. A proline-rich region in the tail (amino acids 423-427) may provide the flexibility required for this function. Since the tail contains several potential phosphorylation sites, the proposed interaction between the tail and the kinase domain of Akt could be regulated by phosphorylation. Such carboxyl-terminal tail phosphorylation events have been proposed to also contribute to the regulation of other kinases such as Raf-1 (
      • Morrison D.K.
      • Heidecker G.
      • Rapp U.R.
      • Copeland T.D.
      ), p70S6K (
      • Han J-W.
      • Pearson R.B.
      • Dennis P.B.
      • Thomas G.
      ), and PKCβII (
      • Flint A.J.
      • Paladini R.D.
      • Koshland Jr., D.E.
      ).
      The finding that Akt is activated directly by the PI3-K suggested that all growth factors and intracellular signaling molecules that activate the PI3-K may also activate Akt. Here we examined the potential role of two such signaling molecules, Ha-ras and Src, in Akt activation. c-Ha-ras is known to interact with, and to activate p110, the catalytic subunit of the PI3-K (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Dhand R.
      • Vanhaesebroeck B.
      • Gout I.
      • Fry M.J.
      • Waterfield M.D.
      • Downward J.
      ). Moreover, our earlier studies had shown that the PDGF-mediated activation of Akt is inhibited by the dominant negative mutant RasN17 transiently transfected into NIH 3T3 cells (
      • Franke T.F.
      • Yang S.-I.
      • Chan T.O.
      • Datta K.
      • Kazlauskas A.
      • Morrison D.K.
      • Kaplan D.R.
      • Tsichlis P.N.
      ). Src and other src-related kinases, on the other hand, are known to interact through their SH3 domains with a proline-rich region (amino acids 84-99) in the regulatory subunit of the PI3-K (
      • Pleiman C.M.
      • Hertz W.M.
      • Cambier J.C.
      ). Through this interaction, p85 appears to be phosphorylated on tyrosine residues and contributes to the activation of the PI3-K (
      • Pleiman C.M.
      • Hertz W.M.
      • Cambier J.C.
      ). The results of the experiments reported here confirmed that both v-src (or srcY527F) and v-Ha-ras, as well as the combination of the two, activate Akt in Sf9 and mammalian cells in a PI3-K-dependent manner.
      In summary, the data presented in this report showed that the activation of Akt by the PI3-K can be reproduced in baculovirus-infected Sf9 cells and provided additional support to the hypothesis that Akt is a direct target of the PI3-K. The same data showed that Akt is activated by both growth factors and intracellular signaling molecules that transduce growth factor-generated signals that activate the PI3-K. Finally, both the PH domain and the carboxyl-terminal tail of Akt play critical roles in regulating its activity.

      Acknowledgments

      We thank Dr. George Panayotou for baculovirus constructs of p85, p110, and PDGFRβ, Dr. Roger Davis for the srcY527F construct, Dr. Jonathan Chernoff for baculovirus constructs of v-Ha-ras and v-src, Drs. Erica Golemis and Susan Bear for critical reading of the manuscript, and Pat Bateman for secretarial assistance.

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