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PTEN Interactions with Focal Adhesion Kinase and Suppression of the Extracellular Matrix-dependent Phosphatidylinositol 3-Kinase/Akt Cell Survival Pathway*

  • Masahito Tamura
    Affiliations
    From the Craniofacial Developmental Biology and Regeneration Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892-4370
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  • Jianguo Gu
    Affiliations
    From the Craniofacial Developmental Biology and Regeneration Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892-4370
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  • Author Footnotes
    ‡ Present address: Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
    ,
    Author Footnotes
    § Supported by a fellowship from the Dutch Cancer Society.
    Erik H.J. Danen
    Footnotes
    ‡ Present address: Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
    § Supported by a fellowship from the Dutch Cancer Society.
    Affiliations
    From the Craniofacial Developmental Biology and Regeneration Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892-4370
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  • Author Footnotes
    ¶ Supported by a fellowship from the Japan Society for the Promotion of Science.
    Takahisa Takino
    Footnotes
    ¶ Supported by a fellowship from the Japan Society for the Promotion of Science.
    Affiliations
    From the Craniofacial Developmental Biology and Regeneration Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892-4370
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  • Author Footnotes
    ‖ Present address: National Kyushu Cancer Center, Notame 3-1-1, Minami-ku, Fukuoka 815, Japan.
    Shingo Miyamoto
    Footnotes
    ‖ Present address: National Kyushu Cancer Center, Notame 3-1-1, Minami-ku, Fukuoka 815, Japan.
    Affiliations
    From the Craniofacial Developmental Biology and Regeneration Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892-4370
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  • Kenneth M. Yamada
    Correspondence
    To whom correspondence should be addressed: CDBRB, NIDCR, NIH, Bldg. 30, Rm. 421, 30 Convent Dr. MSC 4370, Bethesda, MD 20892-4370. Tel.: 301-496-9124; Fax: 301-402-0897;
    Affiliations
    From the Craniofacial Developmental Biology and Regeneration Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892-4370
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  • Author Footnotes
    * 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.
    ‡ Present address: Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
    § Supported by a fellowship from the Dutch Cancer Society.
    ¶ Supported by a fellowship from the Japan Society for the Promotion of Science.
    ‖ Present address: National Kyushu Cancer Center, Notame 3-1-1, Minami-ku, Fukuoka 815, Japan.
Open AccessPublished:July 16, 1999DOI:https://doi.org/10.1074/jbc.274.29.20693
      The tumor suppressor PTEN is a phosphatase with sequence homology to tensin. PTEN dephosphorylates phosphatidylinositol 3,4,5-trisphosphate (PIP3) and focal adhesion kinase (FAK), and it can inhibit cell growth, invasion, migration, and focal adhesions. We investigated molecular interactions of PTEN and FAK in glioblastoma and breast cancer cells lacking PTEN. The PTEN trapping mutant D92A bound wild-type FAK, requiring FAK autophosphorylation site Tyr397. In PTEN-mutated cancer cells, FAK phosphorylation was retained even in suspension after detachment from extracellular matrix, accompanied by enhanced PI 3-K association with FAK and sustained PI 3-K activity, PIP3 levels, and Akt phosphorylation; expression of exogenous PTEN suppressed all five properties. PTEN-mutated cells were resistant to apoptosis in suspension, but most of the cells entered apoptosis after expression of exogenous PTEN or wortmannin treatment. Moreover, overexpression of FAK in PTEN-transfected cells reversed the decreased FAK phosphorylation and PI 3-K activity, and it partially rescued PIP3 levels, Akt phosphorylation, and PTEN-induced apoptosis. Our results show that FAK Tyr397 is important in PTEN interactions with FAK, that PTEN regulates FAK phosphorylation and molecular associations after detachment from matrix, and that PTEN negatively regulates the extracellular matrix-dependent PI 3-K/Akt cell survival pathway in a process that can include FAK.
      PTEN (phosphatase and tensin homologue deleted on chromosome 10, also called MMAC1 or TEP1) is a tumor suppressor gene identified on human chromosome 10q23 (
      • Li J.
      • Yen C.
      • Liaw D.
      • Podsypanina K.
      • Bose S.
      • Wang S.I.
      • Puc J.
      • Miliaresis C.
      • Rodgers L.
      • McCombie R.
      • Bigner S.H.
      • Giovanella B.C.
      • Ittmann M.
      • Tycko B.
      • Hibshoosh H.
      • Wigler M.H.
      • Parsons R.
      ,
      • Steck P.A.
      • Pershouse M.A.
      • Jasser S.A.
      • Yung W.K.
      • Lin H.
      • Ligon A.H.
      • Langford L.A.
      • Baumgard M.L.
      • Hattier T.
      • Davis T.
      • Frye C.
      • Hu R.
      • Swedlund B.
      • Teng D.H.
      • Tavtigian S.V.
      ,
      • Li D.M.
      • Sun H.
      ).PTEN is frequently deleted or mutated in a wide range of human cancers, including glioblastoma (
      • Wang S.I.
      • Puc J.
      • Li J.
      • Bruce J.N.
      • Cairns P.
      • Sidransky D.
      • Parsons R.
      ), melanoma (
      • Guldberg P.
      • thor Straten P.
      • Birck A.
      • Ahrenkiel V.
      • Kirkin A.F.
      • Zeuthen J.
      ), and prostate (
      • Cairns P.
      • Okami K.
      • Halachmi S.
      • Halachmi N.
      • Esteller M.
      • Herman J.G.
      • Jen J.
      • Isaacs W.B.
      • Bova G.S.
      • Sidransky D.
      ), breast (
      • Rhei E.
      • Kang L.
      • Bogomolniy F.
      • Federici M.G.
      • Borgen P.I.
      • Boyd J.
      ), and endometrial cancers (
      • Tashiro H.
      • Blazes M.S.
      • Wu R.
      • Cho K.R.
      • Bose S.
      • Wang S.I.
      • Li J.
      • Parsons R.
      • Ellenson L.H.
      ). Germ line PTENmutations are present in patients with Cowden disease and Bannayan-Zonana syndrome (
      • Liaw D.
      • Marsh D.J.
      • Li J.
      • Dahia P.L.
      • Wang S.I.
      • Zheng Z.
      • Bose S.
      • Call K.M.
      • Tsou H.C.
      • Peacocke M.
      • Eng C.
      • Parsons R.
      ,
      • Marsh D.J.
      • Dahia P.L.
      • Zheng Z.
      • Liaw D.
      • Parsons R.
      • Gorlin R.J.
      • Eng C.
      ). Besides functioning as a tumor suppressor, PTEN is also essential for embryonic development (
      • Di Cristofano A.
      • Pesce B.
      • Cordon-Cardo C.
      • Pandolfi P.P.
      ,
      • Suzuki A.
      • de la Pompa J.L.
      • Stambolic V.
      • Elia A.J.
      • Sasaki T.
      • Barrantes I.B.
      • Ho A.
      • Wakeham A.
      • Itie A.
      • Khoo W.
      • Fukumoto M.
      • Mak T.W.
      ,
      • Podsypanina K.
      • Ellenson L.H.
      • Nemes A.
      • Gu J.
      • Tamura M.
      • Yamada K.M.
      • Cordon-Cardo C.
      • Catoretti G.
      • Fisher P.E.
      • Parsons R.
      ).
      Domains of PTEN share a high degree of homology with the family of protein-tyrosine phosphatases and the cytoskeletal protein tensin (
      • Li J.
      • Yen C.
      • Liaw D.
      • Podsypanina K.
      • Bose S.
      • Wang S.I.
      • Puc J.
      • Miliaresis C.
      • Rodgers L.
      • McCombie R.
      • Bigner S.H.
      • Giovanella B.C.
      • Ittmann M.
      • Tycko B.
      • Hibshoosh H.
      • Wigler M.H.
      • Parsons R.
      ,
      • Steck P.A.
      • Pershouse M.A.
      • Jasser S.A.
      • Yung W.K.
      • Lin H.
      • Ligon A.H.
      • Langford L.A.
      • Baumgard M.L.
      • Hattier T.
      • Davis T.
      • Frye C.
      • Hu R.
      • Swedlund B.
      • Teng D.H.
      • Tavtigian S.V.
      ). PTEN functions as a dual specificity phosphatase and lipid phosphatase in vitro (
      • Myers M.P.
      • Stolarov J.P.
      • Eng C.
      • Li J.
      • Wang S.I.
      • Wigler M.H.
      • Parsons R.
      • Tonks N.K.
      ,
      • Myers M.P.
      • Pass I.
      • Batty I.H.
      • Van der Kaay J.
      • Stolarov J.P.
      • Hemmings B.A.
      • Wigler M.H.
      • Downes C.P.
      • Tonks N.K.
      ). Specific substrates include phosphatidylinositol 3,4,5-trisphosphate (PIP3)
      The abbreviations PIP3
      phosphatidylinositol 3,4,5-trisphosphate
      FAK
      focal adhesion kinase
      p130Cas
      p130 Crk-associated substrate
      GFP
      green fluorescent protein
      HA
      hemagglutinin
      PI 3-K
      phosphatidylinositol 3-kinase
      MAP
      mitogen-activated protein
      JNK
      c-Jun amino-terminal kinase
      IL-2R
      interleukin-2 receptor
      TUNEL
      TdT-mediated dUTP nick end labeling
      PTP1B
      protein-tyrosine phosphatase 1B
      PBS
      phosphate-buffered saline
      PIPES
      1,4-piperazinediethanesulfonic acid
      PDGF
      platelet-derived growth factor
      1The abbreviations PIP3
      phosphatidylinositol 3,4,5-trisphosphate
      FAK
      focal adhesion kinase
      p130Cas
      p130 Crk-associated substrate
      GFP
      green fluorescent protein
      HA
      hemagglutinin
      PI 3-K
      phosphatidylinositol 3-kinase
      MAP
      mitogen-activated protein
      JNK
      c-Jun amino-terminal kinase
      IL-2R
      interleukin-2 receptor
      TUNEL
      TdT-mediated dUTP nick end labeling
      PTP1B
      protein-tyrosine phosphatase 1B
      PBS
      phosphate-buffered saline
      PIPES
      1,4-piperazinediethanesulfonic acid
      PDGF
      platelet-derived growth factor
      and focal adhesion kinase (FAK) (
      • Maehama T.
      • Dixon J.E.
      ,
      • Stambolic V.
      • Suzuki A.
      • de la Pompa J.L.
      • Brothers G.M.
      • Mirtsos C.
      • Sasaki T.
      • Ruland J.
      • Penninger J.M.
      • Siderovski D.P.
      • Mak T.W.
      ,
      • Tamura M.
      • Gu J.
      • Matsumoto K.
      • Aota S.
      • Parsons R.
      • Yamada K.M.
      ). Many tumor-associated missense mutations cluster around the phosphatase domain, and most remaining mutations are predicted to truncate the protein due to nonsense or frameshift mutations (
      • Li J.
      • Yen C.
      • Liaw D.
      • Podsypanina K.
      • Bose S.
      • Wang S.I.
      • Puc J.
      • Miliaresis C.
      • Rodgers L.
      • McCombie R.
      • Bigner S.H.
      • Giovanella B.C.
      • Ittmann M.
      • Tycko B.
      • Hibshoosh H.
      • Wigler M.H.
      • Parsons R.
      ,
      • Steck P.A.
      • Pershouse M.A.
      • Jasser S.A.
      • Yung W.K.
      • Lin H.
      • Ligon A.H.
      • Langford L.A.
      • Baumgard M.L.
      • Hattier T.
      • Davis T.
      • Frye C.
      • Hu R.
      • Swedlund B.
      • Teng D.H.
      • Tavtigian S.V.
      ,
      • Teng D.H.-F.
      • Hu R.
      • Lin H.
      • Davis T.
      • Iliev D.
      • Frye C.
      • Swedlund B.
      • Hansen K.L.
      • Vinson V.L.
      • Gumpper K.L.
      • Ellis L.
      • El-Naggar A.
      • Frazier M.
      • Jasser S.
      • Langford L.A.
      • Lee J.
      • Mills G.B.
      • Pershouse M.A.
      • Pollack R.E.
      • Tornos C.
      • Troncoso P.
      • Yung W.K.A.
      • Fujii G.
      • Berson A.
      • Bookstein R.
      • Bolen J.B.
      • Tavtigian S.V.
      • Steck P.A.
      ), suggesting that the phosphatase activity of PTEN plays important roles in PTEN function. In fact, suppression of cell growth (
      • Furnari F.B.
      • Lin H.
      • Huang H.S.
      • Cavenee W.K.
      ), focal adhesion formation (
      • Tamura M.
      • Gu J.
      • Matsumoto K.
      • Aota S.
      • Parsons R.
      • Yamada K.M.
      ), and cell migration and invasion (
      • Tamura M.
      • Gu J.
      • Takino T.
      • Yamada K.M.
      ) in PTEN-deficient glioblastoma cells by PTEN cDNA expression requires a functional phosphatase catalytic domain.
      The cellular mechanisms of PTEN function are still not completely understood. Recent evidence demonstrates the ability of PTEN to directly dephosphorylate position D3 of PIP3, a product of PI 3-K (
      • Maehama T.
      • Dixon J.E.
      ). In PTEN-mutated glioblastoma cells and mouse embryonic fibroblasts, the activity of Akt (also called protein kinase B) is constitutively elevated (
      • Stambolic V.
      • Suzuki A.
      • de la Pompa J.L.
      • Brothers G.M.
      • Mirtsos C.
      • Sasaki T.
      • Ruland J.
      • Penninger J.M.
      • Siderovski D.P.
      • Mak T.W.
      ,
      • Haas-Kogan D.
      • Shalev N.
      • Wong M.
      • Mills G.
      • Yount G.
      • Stokoe D.
      ). Akt is a survival-promoting serine-threonine protein kinase regulated by PIP3 that is implicated in survival signaling in a wide variety of cells, including fibroblastic, epithelial, and neuronal cells (
      • Kulik G.
      • Weber M.J.
      ). PTEN increases sensitivity to cell death in response to several apoptotic stimuli, including UV irradiation and treatment with tumor necrosis factor α, by negatively regulating the PI 3-K/Akt pathway (
      • Stambolic V.
      • Suzuki A.
      • de la Pompa J.L.
      • Brothers G.M.
      • Mirtsos C.
      • Sasaki T.
      • Ruland J.
      • Penninger J.M.
      • Siderovski D.P.
      • Mak T.W.
      ). In addition to its role in regulating the PI 3-K/Akt cell survival pathway, PTEN also inhibits growth factor-induced Shc phosphorylation and suppresses the mitogen-activated protein (MAP) kinase signaling pathway (
      • Gu J.
      • Tamura M.
      • Yamada K.M.
      ), suggesting that PTEN has roles in independent signaling pathways.
      PTEN also interacts with FAK, a key molecule implicated in integrin signaling pathways, and it directly dephosphorylates tyrosine-phosphorylated FAK (
      • Tamura M.
      • Gu J.
      • Matsumoto K.
      • Aota S.
      • Parsons R.
      • Yamada K.M.
      ). The activation of integrins by cell binding to extracellular matrix leads to increases in FAK tyrosine phosphorylation levels and enhances kinase activity (
      • Hanks S.K.
      • Polte T.R.
      ,
      • Schlaepfer D.D.
      • Hunter T.
      ,
      • Guan J.L.
      ,
      • Clark E.A.
      • Brugge J.S.
      ,
      • Yamada K.M.
      • Geiger B.
      ). Activation of FAK leads to its association with several kinases, signal transduction molecules, and cytoskeletal proteins including PI 3-K, Src, Grb2, and paxillin. Binding is mediated by specific tyrosine-phosphorylated residues within FAK and is followed by activation of downstream signaling pathways including extracellular signal-regulated kinase/MAP kinase and PI 3-K/Akt cell survival pathways (
      • Schlaepfer D.D.
      • Hanks S.K.
      • Hunter T.
      • van der Geer P.
      ,
      • Schaller M.D.
      • Hildebrand J.D.
      • Shannon J.D.
      • Fox J.W.
      • Vines R.R.
      • Parsons J.T.
      ,
      • Schlaepfer D.D.
      • Hunter T.
      ,
      • Schlaepfer D.D.
      • Jones K.C.
      • Hunter T.
      ,
      • Bellis S.L.
      • Miller J.T.
      • Turner C.E.
      ,
      • Guan J.L.
      • Shalloway D.
      ). In fact, the integrin-mediated MAP kinase signaling pathway is also suppressed by PTEN (
      • Gu J.
      • Tamura M.
      • Yamada K.M.
      ).
      Many mammalian cell types are dependent on adhesion to the extracellular matrix for their continued survival. When the signals from matrix are interrupted, normal cells may undergo apoptosis in a process termed anoikis (
      • Frisch S.M.
      • Francis H.
      ). In contrast, the ability of malignant cells to proliferate in the absence of adhesion, termed anchorage independence of growth, correlates closely with tumorigenicity. In a study published while this paper was under review, Davies et al. (
      • Davies M.A.
      • Lu Y.
      • Sano T.
      • Fang X.
      • Tang P.
      • LaPushin R.
      • Koul D.
      • Bookstein R.
      • Stokoe D.
      • Yung W.K.
      • Mills G.B.
      • Steck P.A.
      ) reported that PTEN expression in a cell line lacking PTEN increases the rate of apoptosis approximately 2-fold both before and especially after detachment from extracellular matrix. Another recent study reported that PTEN overexpression in human breast cancer cells induces apoptosis, even while the cells were substrate-attached and regardless of the presence of endogenous PTEN, and Akt was identified as a key molecule in this effect (
      • Li J.
      • Simpson L.
      • Takahashi M.
      • Miliaresis C.
      • Myers M.P.
      • Tonks N.
      • Parsons R.
      ).
      Other studies have implicated FAK in the general process of anoikis,i.e. apoptosis after loss of matrix interactions. Inhibition of FAK activity in fibroblasts or attenuation of FAK expression in tumor cells leads to apoptosis (
      • Hungerford J.E.
      • Compton M.T.
      • Matter M.L.
      • Hoffstrom B.G.
      • Otey C.A.
      ,
      • Xu L.H.
      • Owens L.V.
      • Sturge G.C.
      • Yang X.
      • Liu E.T.
      • Craven R.J.
      • Cance W.G.
      ). Constitutively activated FAK protects Madin-Darby canine kidney cells from apoptosis caused by loss of matrix contact, and Tyr397 of FAK is required for this effect (
      • Frisch S.M.
      • Vuori K.
      • Ruoslahti E.
      • Chan-Hui P.Y.
      ). Association of the p85 subunit of PI 3-K with Tyr397 in FAK is induced by the attachment of cells to matrix (
      • Chen H.C.
      • Guan J.L.
      ,
      • Chen H.-C.
      • Appeddu P.A.
      • Isoda H.
      • Guan J.-L.
      ). PI 3-K is required for integrin-stimulated Akt activation (
      • King W.G.
      • Mattaliano M.D.
      • Chan T.O.
      • Tsichlis P.N.
      • Brugge J.S.
      ). These results provide evidence that FAK is an important mediator of integrin-mediated survival signals upstream of the PI 3-K/Akt cell survival pathway. Several studies have also established that levels of FAK expression are often increased in proliferating cells or advanced cancers (
      • Weiner T.M.
      • Liu E.T.
      • Craven R.J.
      • Cance W.G.
      ,
      • Owens L.V.
      • Xu L.
      • Craven R.J.
      • Dent G.A.
      • Weiner T.M.
      • Kornberg L.
      • Liu E.T.
      • Cance W.G.
      ,
      • Tremblay L.
      • Hauck W.
      • Nguyen L.T.
      • Allard P.
      • Landry F.
      • Chapdelaine A.
      • Chevalier S.
      ).
      In the present study, we have investigated further the interactions between PTEN and FAK in trying to determine whether PTEN dephosphorylation of FAK is involved in processes related to cancer progression. Our results suggest that the major autophosphorylation site of FAK (Tyr397) is responsible for the initialin vivo association of PTEN with FAK, a prerequisite for FAK dephosphorylation by PTEN. A trapping mutant of PTEN (D92A) competed for the binding of Src and PI 3-K, which also bind to Tyr397 of FAK, without effects on binding to other sites. In order to explore PTEN signaling pathways, we also tested whether FAK dephosphorylation by PTEN was associated with effects on PI 3-K and downstream Akt cell survival signaling. In PTEN-mutated cancer cells, FAK phosphorylation was retained even in the absence of extracellular matrix contact, accompanied by sustained PI 3-K binding to FAK, activity of PI 3-K, levels of PIP3, and phosphorylation of Akt. PTEN-mutated cells were markedly resistant to apoptosis triggered by detachment from extracellular matrix. Expression of exogenous PTEN in PTEN-mutated cells inhibited FAK phosphorylation, and it restored a normal pattern of FAK/PI 3-K association, PI 3-K activity, PIP3 levels, Akt phosphorylation, and apoptosis in response to detachment from matrix. Furthermore, overexpression of FAK could effectively inhibit these effects of PTEN on PI 3-K activity and partially inhibited its effects on PIP3 levels, Akt phosphorylation, and apoptosis. Our results suggest that PTEN interactions with FAK may lead to inhibition of the PI 3-K/Akt cell survival pathway in parallel with its direct effects on PIP3, thereby promoting apoptosis in response to detachment from matrix.

      DISCUSSION

      Many recent lines of evidence have implicated functional inactivation of the PTEN gene in the pathogenesis of tumors of various tissues, indicating that PTEN acts as a tumor suppressor gene. In fact, recent reports that re-expression of PTEN in human glioma cell lines with mutated PTEN alleles suppresses cell growth and tumorigenicity further establish PTEN as a tumor suppressor (
      • Furnari F.B.
      • Lin H.
      • Huang H.S.
      • Cavenee W.K.
      ,
      • Cheney I.W.
      • Johnson D.E.
      • Vaillancourt M.T.
      • Avanzini J.
      • Morimoto A.
      • Demers G.W.
      • Wills K.N.
      • Shabram P.W.
      • Bolen J.B.
      • Tavtigian S.V.
      • Bookstein R.
      ). Increased proliferation in PTEN mutant embryos also suggests that PTEN plays roles in regulating cell proliferation (
      • Stambolic V.
      • Suzuki A.
      • de la Pompa J.L.
      • Brothers G.M.
      • Mirtsos C.
      • Sasaki T.
      • Ruland J.
      • Penninger J.M.
      • Siderovski D.P.
      • Mak T.W.
      ). It is important to elucidate the cellular functions of PTEN in order to understand how PTEN regulates normal cell behavior and acts as a tumor suppressor in vivo. Recently, both lipid and protein candidate substrates for PTEN have been identified, including PIP3, FAK, and Shc (
      • Maehama T.
      • Dixon J.E.
      ,
      • Tamura M.
      • Gu J.
      • Matsumoto K.
      • Aota S.
      • Parsons R.
      • Yamada K.M.
      ,
      • Gu J.
      • Tamura M.
      • Yamada K.M.
      ). In this study, we have (a) established that Tyr397 in FAK is required for the interaction between PTEN and FAK; (b) found that the interaction between FAK and the molecules bound to Tyr397 in FAK including PI 3-K is inhibited by PTEN trapping mutant binding to FAK; (c) established that FAK tyrosine phosphorylation is maintained in PTEN-mutated cells even after detachment from matrix substrates, which is accompanied by sustained FAK and PI 3-K association, PI 3-K activity, PIP3levels, Akt phosphorylation, and resistance to apoptosis triggered by loss of matrix contact; (d) shown that expression of exogenous PTEN in mutant cells restores both their sensitivity to matrix-dependent apoptosis and normal patterns of FAK and Akt phosphorylation, association of FAK and PI 3-K, and levels of PI 3-K activity and PIP3; (e) found similar effects of PTEN on FAK and Akt phosphorylation in three glioblastoma and breast cancer cell lines, whereas another nonreceptor protein-tyrosine phosphatase, PTP1B, had no effects similar to PTEN; (f) established that the ability to maintain Akt phosphorylation and to protect cells from apoptosis was inhibited by a PI 3-K inhibitor but not by a MEK1 inhibitor; and (g) demonstrated that FAK overexpression could manipulate PI 3-K activity, PIP3levels, and Akt phosphorylation and partially rescue suspended cells from PTEN-induced apoptosis. These results indicate that PTEN interacts with FAK through residue Tyr397 in the FAK molecule and suggest that it may down-regulate the downstream PI 3-K/Akt cell survival pathway not only by direct dephosphorylation of PIP3 but also by inhibition of upstream FAK.
      We have recently reported that PTEN inhibits cell migration, invasion, spreading, and focal adhesions (
      • Tamura M.
      • Gu J.
      • Matsumoto K.
      • Aota S.
      • Parsons R.
      • Yamada K.M.
      ,
      • Tamura M.
      • Gu J.
      • Takino T.
      • Yamada K.M.
      ). PTEN directly interacts with FAK and reduces its tyrosine phosphorylation as well as that of a potential downstream effector p130Cas. FAK is an important regulator of integrin-mediated focal adhesion assembly, cell adhesion, and cell migration. Overexpression of dominant negative FAK causes a transient reduction in cell spreading (
      • Richardson A.
      • Parsons J.T.
      ). FAK overexpression stimulates cell migration (
      • Cary L.A.
      • Chang J.F.
      • Guan J.-L.
      ), while cells in which FAK is inhibited exhibit decreased cell migration (
      • Romer L.H.
      • McLean N.
      • Turner C.E.
      • Burridge K.
      ,
      • Ilic D.
      • Furuta Y.
      • Kanazawa S.
      • Takeda N.
      • Sobue K.
      • Nakatsuji N.
      • Nomura S.
      • Fujimoto J.
      • Okada M.
      • Yamamoto T.
      ,
      • Gilmore A.P.
      • Romer L.H.
      ). Recently, p130Cas has been reported to be a mediator of FAK-promoted cell migration (
      • Cary L.A.
      • Han D.C.
      • Polte T.R.
      • Hanks S.K.
      • Guan J.L.
      ,
      • Klemke R.L.
      • Leng J.
      • Molander R.
      • Brooks P.C.
      • Vuori K.
      • Cheresh D.A.
      ). In fact, PTEN down-regulation of p130Cas through FAK results in inhibition of cell migration (
      • Tamura M.
      • Gu J.
      • Takino T.
      • Yamada K.M.
      ). Activation and autophosphorylation of FAK in response to integrin binding to extracellular matrix also leads to its binding to a number of intracellular signaling molecules besides p130Cas, including Grb2 (
      • Schlaepfer D.D.
      • Hanks S.K.
      • Hunter T.
      • van der Geer P.
      ,
      • Schaller M.D.
      • Hildebrand J.D.
      • Shannon J.D.
      • Fox J.W.
      • Vines R.R.
      • Parsons J.T.
      ). FAK/Src association can lead to activation of the MAP kinase pathway through Grb2 binding to FAK (
      • Schlaepfer D.D.
      • Hanks S.K.
      • Hunter T.
      • van der Geer P.
      ,
      • Schlaepfer D.D.
      • Hunter T.
      ), although other mechanisms of integrin-mediated MAP kinase activation also exist (
      • Wary K.K.
      • Mainiero F.
      • Isakoff S.J.
      • Marcantonio E.E.
      • Giancotti F.G.
      ,
      • Lin T.H.
      • Aplin A.E.
      • Shen Y.
      • Chen Q.
      • Schaller M.
      • Romer L.
      • Aukhil I.
      • Juliano R.L.
      ). A recent report shows that integrin-mediated MAP kinase activation is also inhibited by PTEN (
      • Gu J.
      • Tamura M.
      • Yamada K.M.
      ), suggesting that PTEN inhibition of FAK leads to several downstream signaling pathways.
      In this study, we demonstrated that Tyr397 in FAK is crucial for the initial association of FAK and PTEN; PTEN interaction with FAK is a necessary prelude to PTEN-mediated dephosphorylation. Integrin-stimulated FAK tyrosine phosphorylation is complex and occurs at least at six sites in vivo (
      • Hanks S.K.
      • Polte T.R.
      ). Two sites within the N terminus of FAK (Tyr397 and Tyr407), two sites within the kinase domain (Tyr576 and Tyr577), one site within the C terminus domain (Tyr861), and one site within the focal adhesion targeting domain (Tyr925) are phosphorylated in vivo (Fig. 1 A). Tyr397 is a major autophosphorylation site that is phosphorylated upon integrin activation and is a binding site for Src family protein-tyrosine kinases and PI 3-K (
      • Schaller M.D.
      • Hildebrand J.D.
      • Shannon J.D.
      • Fox J.W.
      • Vines R.R.
      • Parsons J.T.
      ,
      • Chen H.-C.
      • Appeddu P.A.
      • Isoda H.
      • Guan J.-L.
      ). Mutation of Tyr397 to F inhibited the formation of stable complexes with the D92A trapping mutant of PTEN, which can bind but not dephosphorylate FAK. Furthermore, D92A PTEN binding to endogenous wild-type FAK disrupted the interactions of FAK with the Tyr397 FAK binding molecules Src and PI 3-K, indicating that Tyr397 in FAK is important for the association of FAK with PTEN in vivo. However, wild-type PTEN did not form stable complexes with FAK, consistent with dissociation after dephosphorylation. Further experiments are needed to establish whether PTEN dephosphorylates only phosphorylated Tyr397 in FAK, because phosphorylation at Tyr397 is thought to be an initial step in integrin-mediated FAK activation, and phosphorylation of Tyr397 promotes transphosphorylation of other tyrosine residues in FAK in concert with activated Src.
      Our data also provide interesting insights into FAK regulation upon detachment. It has been thought that FAK may be negatively regulated by putative tyrosine phosphatases that dephosphorylate Tyr397of FAK in response to the detachment from matrix (
      • Guan J.L.
      ). InPTEN-mutated cells, we found that FAK remained abnormally phosphorylated even in suspension but could be almost completely dephosphorylated after the expression of PTEN. These findings are consistent with a role for PTEN in matrix-dependent regulation of FAK phosphorylation.
      FAK also binds to PI 3-K and increases its activity in response to integrin-mediated cell adhesion (
      • Chen H.C.
      • Guan J.L.
      ). In this study, we showed that the association of PI 3-K with FAK and Akt phosphorylation are closely correlated. Furthermore, Akt phosphorylation was down-regulated when the cells were detached from matrix and lost FAK phosphorylation by PTEN expression in PTEN-mutated cells, suggesting that Akt might be partially regulated by integrin-mediated FAK activation. The demonstration that inhibition of PI 3-K completely suppressed Akt phosphorylation suggests that PI 3-K activation is necessary for the FAK-mediated Akt activation. These results are consistent with previous reports that PI 3-K is required for integrin-stimulated Akt activation (
      • King W.G.
      • Mattaliano M.D.
      • Chan T.O.
      • Tsichlis P.N.
      • Brugge J.S.
      ,
      • Khwaja A.
      • Rodriguez-Viciana P.
      • Wennstrom S.
      • Warne P.H.
      • Downward J.
      ). This sequence of signaling events was further confirmed by our finding that PTEN down-regulated both PI 3-K activity and PIP3 levels and that PTEN inhibition of Akt could be partially reversed by intracellular overexpression of FAK. We speculate that overexpressing FAK may enhance its phosphorylation by increasing the total amounts of FAK available for phosphorylation and out-competing the phosphatase activity of PTEN. PTEN also dephosphorylates PIP3 (
      • Maehama T.
      • Dixon J.E.
      ) and decreases Shc tyrosine phosphorylation levels (
      • Gu J.
      • Tamura M.
      • Yamada K.M.
      ). It is therefore also possible that overexpression of FAK might play a dominant negative role by substrate competition. However, the finding that FAK overexpression could totally overcome the effects of PTEN on PI 3-K p85 phosphorylation and PI 3-K activity while it only partially rescued PIP3 levels suggests that PTEN can affect the PI 3-K/Akt pathway at more than one stage of signaling, i.e. at both the level of PIP3 and at an upstream FAK-dependent point.
      Activation of Akt has been implicated in protection from apoptosis in response to several signals including growth factors (
      • Kulik G.
      • Weber M.J.
      ,
      • Ulrich E.
      • Duwel A.
      • Kauffmann-Zeh A.
      • Gilbert C.
      • Lyon D.
      • Rudkin B.
      • Evan G.
      • Martin-Zanca D.
      ), cytokines (
      • del Peso L.
      • Gonzalez-Garcia M.
      • Page C.
      • Herrera R.
      • Nunez G.
      ), c-myc overexpression (
      • Kauffmann-Zeh A.
      • Rodriguez-Viciana P.
      • Ulrich E.
      • Gilbert C.
      • Coffer P.
      • Downward J.
      • Evan G.
      ) UV irradiation (
      • Kulik G.
      • Weber M.J.
      ), and matrix detachment (
      • Khwaja A.
      • Rodriguez-Viciana P.
      • Wennstrom S.
      • Warne P.H.
      • Downward J.
      ,
      • Xiong W.
      • Parsons J.T.
      ). Activation of Akt leads to phosphorylation of the Bcl-2 family member Bad, thereby suppressing apoptosis and promoting cell survival (
      • Datta S.R.
      • Dudek H.
      • Tao X.
      • Masters S.
      • Fu H.
      • Gotoh Y.
      • Greenberg M.E.
      ). Loss of PTEN in mouse embryonic fibroblasts results in decreased sensitivity to cell death in response to various apoptotic stimuli by increasing basal Akt activity (
      • Stambolic V.
      • Suzuki A.
      • de la Pompa J.L.
      • Brothers G.M.
      • Mirtsos C.
      • Sasaki T.
      • Ruland J.
      • Penninger J.M.
      • Siderovski D.P.
      • Mak T.W.
      ). Constitutively increased Akt phosphorylation levels are also reported in PTEN-mutated tumor cells (
      • Myers M.P.
      • Pass I.
      • Batty I.H.
      • Van der Kaay J.
      • Stolarov J.P.
      • Hemmings B.A.
      • Wigler M.H.
      • Downes C.P.
      • Tonks N.K.
      ), consistent with our data indicating that Akt phosphorylation levels were decreased in PTEN-expressing cells plated on matrix. The acquisition of anchorage independence and apoptosis resistance are critical for tumor malignancy (
      • Frisch S.M.
      • Francis H.
      ). PTEN-mutated U-87MG glioblastoma cells that we used in this study also have the ability to grow in suspension (
      • Cheney I.W.
      • Johnson D.E.
      • Vaillancourt M.T.
      • Avanzini J.
      • Morimoto A.
      • Demers G.W.
      • Wills K.N.
      • Shabram P.W.
      • Bolen J.B.
      • Tavtigian S.V.
      • Bookstein R.
      ). Recent studies have implicated FAK in this type of cell survival (reviewed in Ref.
      • Meredith J.E.
      • Schwartz M.A.
      ). Inhibition of FAK in several cell types results in growth suppression (
      • Gilmore A.P.
      • Romer L.H.
      ) and apoptosis (
      • Hungerford J.E.
      • Compton M.T.
      • Matter M.L.
      • Hoffstrom B.G.
      • Otey C.A.
      ,
      • Sonoda Y.
      • Watanabe S.
      • Matsumoto Y.
      • Aizu-Yokota E.
      • Hasahara T.
      ), although FAK may not mediate survival in all cases (
      • Xu L.H.
      • Owens L.V.
      • Sturge G.C.
      • Yang X.
      • Liu E.T.
      • Craven R.J.
      • Cance W.G.
      ). Conversely, overexpression of activated FAK can rescue Madin-Darby canine kidney cells from anoikis (
      • Frisch S.M.
      • Vuori K.
      • Ruoslahti E.
      • Chan-Hui P.Y.
      ). These results suggest that FAK is an important mediator of integrin-mediated survival signals. In fact, several studies have established that levels of FAK expression are often increased in proliferating cells or advanced cancers (
      • Weiner T.M.
      • Liu E.T.
      • Craven R.J.
      • Cance W.G.
      ,
      • Owens L.V.
      • Xu L.
      • Craven R.J.
      • Dent G.A.
      • Weiner T.M.
      • Kornberg L.
      • Liu E.T.
      • Cance W.G.
      ,
      • Tremblay L.
      • Hauck W.
      • Nguyen L.T.
      • Allard P.
      • Landry F.
      • Chapdelaine A.
      • Chevalier S.
      ).
      In this study, we demonstrated that loss of PTEN protected cells from apoptosis triggered by matrix detachment and that re-expression of PTEN in PTEN-mutated cells caused apoptosis in cells in suspension. Our studies combined with those of Davieset al. (
      • Davies M.A.
      • Lu Y.
      • Sano T.
      • Fang X.
      • Tang P.
      • LaPushin R.
      • Koul D.
      • Bookstein R.
      • Stokoe D.
      • Yung W.K.
      • Mills G.B.
      • Steck P.A.
      ) establish that PTEN plays important roles in anchorage-dependent cell survival. Sustained association of FAK with PI 3-K and Akt phosphorylation levels were closely correlated with the ability to survive in suspension. Furthermore, inhibition of PI 3-K suppressed Akt phosphorylation and resulted in apoptosis, suggesting that the ability to survive in suspension inPTEN-mutated cells is dependent on a PI 3-K/Akt pathway. In addition, however, FAK overexpression could manipulate PI 3-K activity and PIP3 levels, suggesting a level of PTEN action beyond a simple effect directly on PIP3.
      Death signals activated in the absence of integrin-mediated adhesion may also include the JNK pathway, although its function in induction and protection from anoikis remains controversial (
      • Frisch S.M.
      • Vuori K.
      • Kelaita D.
      • Sicks S.
      ,
      • Nishina H.
      • Fischer K.D.
      • Radvanyi L.
      • Shahinian A.
      • Hakem R.
      • Rubie E.A.
      • Bernstein A.
      • Mak T.W.
      • Woodgett J.R.
      • Penninger J.M.
      ,
      • Kyriakis J.M.
      • Avruch J.
      ). JNK phosphorylation levels showed normal reactions in our cells in response to UV irradiation consistent with previous reports (
      • Stambolic V.
      • Suzuki A.
      • de la Pompa J.L.
      • Brothers G.M.
      • Mirtsos C.
      • Sasaki T.
      • Ruland J.
      • Penninger J.M.
      • Siderovski D.P.
      • Mak T.W.
      ,
      • Gu J.
      • Tamura M.
      • Yamada K.M.
      ), but no significant changes could be observed in response to detachment from matrix, indicating that this pathway is not affected by PTEN in our cells. A recent report showed that FAK-transduced matrix survival signal signals suppress p53-mediated apoptosis under serum-depleted conditions (
      • Ilic D.
      • Almeida E.A.C.
      • Schlaepfer D.D.
      • Dazin P.
      • Aizawa S.
      • Damsky C.H.
      ), but we could not detect the involvement of p53 in PTEN-mediated apoptosis under our culture conditions in regular serum-containing medium.
      In summary, we have elucidated interactions and signaling processes involving PTEN and FAK. We demonstrated that Tyr397 in FAK is important for PTEN-FAK interaction. PTEN restoration in tumor cells with mutated PTEN alleles results in inhibition of FAK phosphorylation (enhanced dephosphorylation) after detachment from matrix and results in inhibition of PI 3-K association with FAK. PTEN reconstitution also restores matrix-dependent regulation of FAK and Akt phosphorylation and apoptosis in cells in suspension. Overexpression of FAK antagonized the effects of PTEN. Our data demonstrate that PTEN functions to suppress the ability of cells to survive in the absence of matrix attachment. We suggest that PTEN has at least two potential targets in the PI 3-K/Akt pathway: direct dephosphorylation of PIP3 and FAK down-regulation of the upstream PI 3-K pathway.

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