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FAK Is the Upstream Signal Protein of the Phosphatidylinositol 3-Kinase-Akt Survival Pathway in Hydrogen Peroxide-induced Apoptosis of a Human Glioblastoma Cell Line*

  • Yoshiko Sonoda
    Affiliations
    From the Department of Biochemistry, Kyoritsu College of Pharmacy, Shibakoen 1-5-30, Minato-ku, Tokyo, Japan 105-8512
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  • Shiro Watanabe
    Affiliations
    From the Department of Biochemistry, Kyoritsu College of Pharmacy, Shibakoen 1-5-30, Minato-ku, Tokyo, Japan 105-8512
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  • Yaeko Matsumoto
    Affiliations
    From the Department of Biochemistry, Kyoritsu College of Pharmacy, Shibakoen 1-5-30, Minato-ku, Tokyo, Japan 105-8512
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  • Eriko Aizu-Yokota
    Affiliations
    From the Department of Biochemistry, Kyoritsu College of Pharmacy, Shibakoen 1-5-30, Minato-ku, Tokyo, Japan 105-8512
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  • Tadashi Kasahara
    Correspondence
    To whom correspondence should be addressed: Dept. of Biochemistry, Kyoritsu College of Pharmacy, Shibakoen, Minato-ku, Tokyo, 105, Japan. Tel./Fax: 81-3-5400-2697;
    Affiliations
    From the Department of Biochemistry, Kyoritsu College of Pharmacy, Shibakoen 1-5-30, Minato-ku, Tokyo, Japan 105-8512
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  • Author Footnotes
    * This study was supported in part by grants from the Human Science Project of Japan and from the Science Reserch Promotion Fund, Japan Private School Promotion Foundation.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.
Open AccessPublished:April 09, 1999DOI:https://doi.org/10.1074/jbc.274.15.10566
      Protein phosphorylation in a human glioblastoma cell line, T98G, was examined after exposure to oxidative stressin vitro. Hydrogen peroxide (1 mm) markedly induced tyrosine phosphorylation of focal adhesion kinase (FAK) and serine phosphorylation of Akt at 1 h after stimulation. Concommitantly, the association of FAK with phosphatidylinositide 3′-OH-kinase (PI 3-kinase) was also observed by the hydrogen peroxide stimulation. When T98G cells were incubated with wortmannin, a PI 3-kinase inhibitor, both PI 3-kinase activity and phosphorylation of Akt were inhibited, whereas apoptosis by oxidative stress was accelerated. Concomitant with apoptosis, elevated level of CPP32 protease activity (caspase-3) was observed, with decreases in Bcl-2 protein and increases in Bax protein. These results suggested that in the signal transduction pathway from FAK to PI 3-kinase, Akt promotes survival. Thus, it became apparent that FAK is the upstream signal protein of the PI 3-kinase-Akt survival pathway in hydrogen peroxide-induced apoptosis in T98G cells.
      ROS
      reactive oxygen species
      FAK
      focal adhesion kinase
      PI 3-kinase
      phosphatidylinositide 3′-OH kinase
      PtdIns
      phosphatidylinositol
      Ab
      antibody
      mAb
      monoclonal antibody
      Phosphorylation and dephosphorylation on tyrosine residues play critical roles in the signal transduction pathways that regulate cell activation, proliferation, and differentiation. Reactive oxygen species (ROS)1 have been reported to induce increased tyrosine phosphorylation of several proteins, such as p77btk, p72syk, p56/59hck, and p56lck(
      • Schieven G.L.
      • Kirihara J.M.
      • Burg D.L.
      • Geahlen R.L.
      • Ledbetter J.A.
      ,
      • Qin S.
      • Inazu T.
      • Takata M.
      • Kurosaki T.
      • Homma Y.
      • Yamamura H.
      ,
      • Suzuki Y.
      • Ohsugi K.
      • Ono Y.
      ,
      • Nakamura K.
      • Ori T.
      • Sato N.
      • Sugie K.
      • Kawakami T.
      • Yodoi J.
      ).
      Many growth factors and cytokines promote cell survival, including insulin-like growth factor 1 (
      • 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.
      ) and platelet-derived growth factor (
      • Yao R.
      • Cooper G.M.
      ). Phosphatidylinositide 3′-OH-kinase (PI 3-kinase) has recently been shown to be involved in cell survival. Growth factors activate PI 3-kinase, and p85 subunit of PI 3-kinase associates with specific phosphotyrosine either on the cytoplasmic domain of growth factor receptors or on receptor-associated adaptor proteins. One target of PI 3-kinase is the serine-threonine kinase Akt, also named PKBα (
      • 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.
      ). Akt is a general mediator of growth factor-induced survival and has been shown to suppress apoptotic death by a variety of stimuli (
      • 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.
      ). Signaling via growth factor receptor activation leads to the sequential activation of PI 3-kinase and Akt. Recently, Datta et al.(
      • Datta S.R.
      • Dudek H.
      • Tao X.
      • Masters S.
      • Fu H.
      • Gotoh Y.
      • Greenberg M.E.
      ) reported that Akt phosphorylates BAD in vitro andin vivo and blocks BAD-induced death of primary neurons. In eukaryotes, Bcl-2 family are central to the regulation of cell death. Several members of the Bcl-2 family (Bcl-2, Bcl-XL, MCl-1, and A1) promote survival, whereas other members (Bcl-Xs, BAD, Bax, Bak) promote cell death (
      • Hengartner M.O.
      • Horvitz H.R.
      ,
      • Yang E.
      • Zha J.
      • Jokcel J.
      • Boise L.H.
      • Thompson C.B.
      • Korsmeyer S.J.
      ,
      • Chittenden T.
      • Flemington C.
      • Houghton A.B.
      • Ebb R.G.
      • Elangovan B.
      • Chinnadurai G.
      • Lutz R.J.
      ,
      • Oltvai Z.N.
      • Milliman C.
      • Korsmeyer S.J.
      ,
      • Xu Q.
      • Reed J.C.
      ,
      • Schlesinger P.H.
      • Gross A.
      • Yin X.M.
      • Yamamoto K.
      • Saito M.
      • Waksman G.
      • Korsmeyer S.J.
      ). Bcl-2 family proteins homo- and heterodimerize, and the balance between homo- and heterodimers appears to be critical to the maintenance of cell survival and cell death. The mechanisms of Bcl-2 family members function yet remains to be determined.
      In a previous study (
      • Sonoda Y.
      • Kasahara T.
      • Yokota-Aizu E.
      • Ueno M.
      • Watanabe S.
      ), we reported that hydrogen peroxide markedly induced rapid tyrosine phosphorylation of focal adhesion kinase (FAK) followed by the decrease of phosphorylation concomitant with apoptosis. Further, the inhibition of tyrosine phosphorylation of FAK by herbimycin A, a tyrosine kinase inhibitor, accelerated apoptosis, and antisense oligonucleotides of FAK decreased cell viability. From these studies, we proposed an anti-apoptotic role of FAK in hydrogen peroxide-induced apoptosis. Based on these findings, we hypothesized FAK may be an upstream signal protein in the PI 3-kinase and Akt pathway and promotes cell survival against stresses in some cell types. Therefore, we examined the relationships between FAK, PI 3-kinase, Akt, Bcl-2 family proteins, and CPP32 protease (caspase-3) using a human glioblastoma cell line, T98G.
      To investigate the role of PI 3-kinase and Akt in hydrogen peroxide-induced apoptosis, T98G cells were treated with hydrogen peroxide (1 mm) which caused tyrosine phosphorylation of FAK and serine phosphorylation of Akt. We also found that the association of FAK with PI 3-kinase was stimulated by hydrogen peroxide. Interestingly, the PI 3-kinase inhibitor wortmannin accelerated apoptosis and inhibited serine phosphorylation of Akt. Decreases in Bcl-2 protein and increases in Bax protein and CPP32 protease activity were observed concomitantly with apoptosis. These data suggested that the signal transduction from FAK to PI 3-kinase and Akt exerts an anti-apoptotic effect on apoptosis induced by oxidative stress and FAK locates in the upstream signal of the PI 3-kinase-Akt survival pathway in hydrogen peroxide-induced apoptosis of T98G cells.

      DISCUSSION

      We have reached the following conclusions in this paper. 1) Hydrogen peroxide stimulated the association of FAK with PI 3-kinase. 2) Wortmannin accelerated hydrogen peroxide-induced apoptosis in T98G cells. 3) Hydrogen peroxide stimulated the phosphorylation of Akt. 4) When apoptosis occurred, CPP32 protease was activated, concomitant with the decrease of Bcl-2 protein and increase of Bax protein. 5) Phosphorylation of Akt is inhibited by wortmannin. Recently, we reported the anti-apoptotic role of FAK in hydrogen peroxide-induced apoptosis (
      • Sonoda Y.
      • Kasahara T.
      • Yokota-Aizu E.
      • Ueno M.
      • Watanabe S.
      ). In this study, we demonstrated that tyrosine phosphorylation of FAK, the association of FAK with PI 3-kinase, as well as serine phosphorylation of Akt occur in T98G cells after exposure to hydrogen peroxide. It should be mentioned that in the presence of wortmannin, PI 3-kinase activity and serine phosphorylation of Akt were inhibited with accelerating apoptosis. Putative downstream effectors of PI 3-kinase are the ribosomal protein kinase p70S6K, the Rho family Rac, and the serine/threonine protein kinase Akt/PKB. Akt/PKB, which is a cellular homolog of the retroviral oncogene v-akt, is also homologous to the PKA and PKC families of protein kinases. Akt is involved in the promotion of cell survival through inhibition of apoptosis, possibly playing a role in PI 3-kinase-mediated neuronal cell survival (
      • 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.
      ). As to a mode of survival signaling from Akt to BAD, Datta et al. (
      • Datta S.R.
      • Dudek H.
      • Tao X.
      • Masters S.
      • Fu H.
      • Gotoh Y.
      • Greenberg M.E.
      ) proposed that in the case of insulin-like growth factor 1 stimulation, Akt is activated via PI 3-kinase and activated Akt phosphorylated BAD, which dissociates from Bcl-XL or Bcl-2. Then, phosphorylated BAD is sequestered in the cytosol bound to 14–3-3 (
      • Salvesen G.S.
      • Dixit V.M.
      ). As a result, Bcl-2 homodimer or Bcl-2-Bcl-XL heterodimer was formed, thereby leading to cell survival.
      The above idea appears to be consistent with our observation on the oxidative stress-induced apoptosis as shown in this study. In T98G cells, after stimulation with hydrogen peroxide, FAK was tyrosine-phosphorylated followed by the association and activation of PI 3-kinase. Activation of PI 3-kinase leads to the activation of Akt. Although we could not determine whether the target of Akt in the oxidative stress is BAD or not, Akt might regulate the balance of Bcl-2 family by phosphorylation of apoptosis-related proteins. In this study, the presence of Bcl-2 led to survival, whereas the increase of Bax, a BAD homolog, led to apoptosis. Although the mechanism of the function of Bcl-2 and Bax in apoptosis remains to be determined, Aritomiet al. (
      • Aritomi M.
      • Kunishima N.
      • Inohara N.
      • Ishibashi Y.
      • Ohta S.
      • Morikawa K.
      ) performed crystallographic studies indicating that Bax possesses a greater potential for membrane insertion than either Bcl-2 or Bcl-XL, and thus Bax is likely to form membrane pores. They proposed that the roles of Bcl-XL and Bcl-2 are to inhibit pore formation of Bax or other pore-forming proteins through heterodimerization. Taken together, we speculated that in signal transduction from FAK to PI 3-kinase, Akt plays a survival role by regulating the balance of apoptosis-blocking protein (Bcl-2) and apoptosis-inducing factor (Bax).
      In this study, Akt was translocated to the plasma membrane after stimulation with hydrogen peroxide, and this translocation was sensitive to wortmannin (data not shown). We propose the following model for FAK, PI 3-kinase, Akt, in the apoptosis. After exposure to hydrogen peroxide, FAK is activated by tyrosine phosphorylation, followed by PI 3-kinase activation and translocation of Akt to membrane. Akt is activated by serine phosphorylation and phosphorylates its target proteins in the cytosol, leading to the regulation of the balance of Bcl-2 family. Akt phosphorylation almost disappeared by the transfection of FAK antisense phosphorothioate oligonucleotides as previously employed (
      • Sonoda Y.
      • Kasahara T.
      • Yokota-Aizu E.
      • Ueno M.
      • Watanabe S.
      ) (data not shown). Taken collectively, FAK is an upstream signal protein of the PI 3-kinase-Akt survival pathway in hydrogen peroxide-induced apoptosis. Further analysis of T98G transfectants expressing active FAK or depletion of the FAK gene should provide more information on the role of FAK in apoptosis.

      Acknowledgments

      We are grateful to Masaya Ueno for technical support and are grateful to Dr. Kazushige Yokoyama for the advice on the antisense design. We also thank Dr. Howard Young, NCI-Frederick Cancer Research and Development Center, for kind reviewing of this manuscript.

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