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Anti-apoptotic Role of Focal Adhesion Kinase (FAK)

INDUCTION OF INHIBITOR-OF-APOPTOSIS PROTEINS AND APOPTOSIS SUPPRESSION BY THE OVEREXPRESSION OF FAK IN A HUMAN LEUKEMIC CELL LINE, HL-60*
Open AccessPublished:May 26, 2000DOI:https://doi.org/10.1074/jbc.275.21.16309
      Focal adhesion kinase (FAK) has an anti-apoptotic role in anchorage-dependent cells via an unknown mechanism. To elucidate the role of FAK in anti-apoptosis, we have established several FAK cDNA-transfected HL-60 cell lines and examined whether FAK-transfected cells have resistance to apoptotic stimuli. FAK-transfected HL-60 (HL-60/FAK) cells were highly resistant to apoptosis induced with hydrogen peroxide (1 mm) and etoposide (50 μg/ml) compared with the parental HL-60 cells or the vector-transfected cells, when determined using viability assay, DNA fragmentation, and flow cytometry analysis. Because no proteolytic cleavage of pro-caspase 3 to mature caspase 3 fragment was observed in HL-60/FAK cells, FAK was presumed to inhibit an upstream signal pathway leading to the activation of caspase 3. HL-60/FAK activated the phosphatidylinositide 3′-OH-kinase-Akt survival pathway and exhibited significant activation of NF-κB with marked induction of inhibitor-of-apoptosis proteins (IAPs: cIAP-1, cIAP-2, XIAP), regardless of the hydrogen peroxide-treated or untreated conditions, whereas no significant IAPs were detected in the parental or vector-transfected HL-60 cells. Apoptotic agents induced higher NF-κB activation in HL-60/FAK cells than in HL-60/Vect cells, and it appeared that sustained NF-κB activation is critical to the anti-apoptotic states in HL-60/FAK cells. Mutagenesis of FAK cDNA revealed that Y397 and Y925, which are involved in the tyrosine-phosphorylation sites, were prerequisite for the anti-apoptotic activity as well as induction of IAPs, and that K454, which is involved in the kinase activity, was also required for the full anti-apoptotic activity of FAK. Taken together, we have demonstrated definitively that FAK-transfected HL-60 cells, otherwise sensitive to apoptosis, become resistant to the apoptotic stimuli. We conclude that FAK activates the phosphatidylinositide 3′-OH-kinase-Akt survival pathway with the concomitant activation of NF-kB and induction of IAPs, which ultimately inhibit apoptosis by inhibiting caspase-3 cascade.
      CAD
      caspase-activated DNase
      FAK
      focal adhesion kinase
      PI3-kinase
      phosphatidylinositide 3′-OH-kinase
      PDTC
      pyrrolidine dithiocarbamate
      Ac-YVAD-AMC
      N-acetyl-Tyr-Val-Ala-Asp-7-amino-4-methylcoumarin
      Ac-DEVD-AMC
      N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin
      ICE
      interleukin-1β converting enzyme
      FACS
      fluorescence-activated cell sorter
      IAP
      inhibitor-of-apoptosis protein
      ROS
      reactive oxygen species
      TNF
      tumor necrosis factor
      mAb
      monoclonal antibody
      RT-PCR
      reverse transcription-polymerase chain reaction
      PMA
      phorbol 12-myristate 13-acetate
      Apoptosis (programmed cell death) contributes to the normal development and tissue remodeling of multicellular organisms (
      • Wyllie A.H.
      • Kerr J.F.R.
      • Currie A.R.
      ). The responsible molecules exerting or regulating apoptosis identified so far include the caspase family (
      • Nicholson D.W.
      • Ali A.
      • Thornberry N.A.
      • Vaillancount J.P.
      • Ding C.K.
      • Gallant M.
      • Gareau Y.
      • Griffin P.R.
      • Labelle M.
      • Lazebnik Y.A.
      • Munday N.A.
      • Raju S.M.
      • Smulson M.E.
      • Yamin T-T., Yu, V.L.
      • Miller D.K.
      ,
      • Tewari M.
      • Quan L.T.
      • O'Rounke K.
      • Desnoyers S.
      • Zeng Z.
      • Beidler D.R.
      • Poirier G.G.
      • Salvesen G.S.
      • Dixit V.M.
      ,
      • Ibrado A.M.
      • Huang H.
      • Fang G.
      • Liu L.
      • Bhalla K.
      ), the Bcl-2 family (
      • 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.
      ), caspase-activated DNase (CAD)1 (
      • Enari M.
      • Sakahira H.
      • Yokoyama H.
      • Okawa K.
      • Iwamatsu A.
      • Nagata S.
      ), and inhibitor of CAD (
      • Sakahira H.
      • Enari M.
      • Nagata S.
      ).
      Reactive oxygen species (ROS) are presumed to be important regulators of apoptosis. Production of ROS is found to be stimulated by tumor necrosis factor-α (TNF-α) (
      • Albrecht H.
      • Tschopp J.
      • Jongeneel C.V.
      ), lipopolysaccharide (
      • Wang J.H.
      • Redmond H.P.
      • Watson R.W.
      • Bouchier-Hayes D.
      ), ceramide (
      • Garcia-Ruiz C.
      • Colell A.
      • Mari M.
      • Morales A.
      • Fernandez-Checa J.C.
      ), growth factor withdrawal (
      • Atabay C.
      • Cagnoli C.M.
      • Kharlamov E.
      • Ikonomovic M.D.
      • Nanev H.
      ), human immunodeficiency virus infection (
      • Dobmeyer T.S.
      • Findhammer S.
      • Dobmeyer J.M.
      • Klein S.A.
      • Raffel B.
      • Hoelzer D.
      • Helm E.B.
      • Kabelitz D.
      • Rossol R.
      ), or p53-induced apoptosis (
      • Polyak K.
      • Xia Y.
      • Zweier J.L.
      • Kinzler K.W.
      • Voglestein B.
      ,
      • Johnson T.M., Yu, Z.X.
      • Ferrans V.J.
      • Lowenstein R.A.
      • Finkel T.
      ). In contrast, overexpression of thioredoxin (
      • Iwata S.
      • Hori T.
      • Sata N.
      • Hirota K.
      • Sasada T.
      • Mitsui A.
      • Hirakawa T.
      • Yodoi J.
      ), manganese superoxide dismutase (
      • Wong G.H.
      • Elwell J.H.
      • Oberley L.W.
      • Goeddel D.V.
      ), or Bcl-2 (
      • Vaux D.L.
      • Cory S.
      • Adams J.M.
      ,
      • Tsujimoto Y.
      ) can delay apoptosis. The mechanism of these antioxidant molecules to suppress apoptosis has not fully been elucidated.
      Focal adhesion kinase (FAK) has been implicated in the integration of signals from integrins, oncogenes, and neuropeptides (
      • Schaller M.D.
      ,
      • Burridge K.
      • Chrzanowska-Wodnika M.
      ). FAK has also been shown to play an important role in the cell survival of anchorage-dependent cells (
      • Frisch S.M.
      • Vuori K.
      • Ruoslahti E.
      • Chan-Hui P.-Y.
      ). Proteolytic cleavage of FAK by caspase-3 has been reported during growth factor deprivation-induced apoptosis in human umbilical vein endothelial cells (
      • Levkau B.
      • Herren B.
      • Koyama H.
      • Ross R.
      • Raines E.W.
      ), which implies an association between FAK and apoptosis.
      We found that FAK was tyrosine-phosphorylated by oxidative stress before apoptosis occurred (
      • Sonoda Y.
      • Kasahara T.
      • Yokota-Aizu E.
      • Ueno M.
      • Watanabe S.
      ). Furthermore, protein kinase B/Akt which has been implicated in the pathway of survival signal, was serine-phosphorylated following tyrosine phosphorylation of FAK. This proposed that FAK has an anti-apoptotic role in oxidative stress-induced apoptosis in the human glioblastoma cell line T98G (
      • Sonoda Y.
      • Watanabe S.
      • Matsumoto Y.
      • Aizu-Yokota E.
      • Kasahara T.
      ). In this paper, we attempted to elucidate the possibility that FAK may also have an anti-apoptotic function in HL-60 cells. We observed that naive HL-60 cells are not resistant to various apoptosis-inducing reagents, whereas FAK-overexpressed cells acquire resistance to oxidative stress, etoposide-induced apoptosis, with a concomitant inhibition of caspase-3 proteases. We further found that the phosphatidylinositide 3′-OH-kinase (PI3-kinase)-Akt survival pathway, NF-κB activation, and increase of inhibitory apoptosis proteins (IAPs) are involved in the FAK-induced resistance to apoptosis. Thus, FAK was found to activate a signal linking NF-κB and IAPs.

      DISCUSSION

      In this study, we demonstrated that overexpression of FAK endowed HL-60 cells to protect against apoptosis otherwise induced by two representative apoptosis-inducers, an oxidative stress or an anticancer drug, etoposide. In addition to hydrogen peroxide and etoposide, FAK-overexpressed cells were also found to be resistant to C2-ceramide-induced apoptosis (data not shown).
      It has been widely recognized that HL-60 cells do not adhere, but 10 nm PMA-treated cells adhered mildly after 1 day of treatment with PMA and differentiated to macrophage-like cells. HL-60/FAK cells adhered mildly similarly to PMA-treated HL-60 cells (data not shown). It is unknown whether the expression of FAK is prerequisite for or merely concomitant with the differentiation into macrophage form. It should be noted that PMA-treated cells exhibited enhanced FAK expression and acquired the resistance to the hydrogen peroxide-induced apoptosis similar to the HL-60/FAK, strongly suggesting that FAK plays a role in the anti-apoptosis during the differentiation into macrophages. There have been several reports describing the anti-apoptotic roles of FAK in various apoptosis-inducing system. Hungerford et al. (
      • Hungerford J.E.
      • Compton M.T.
      • Matter M.L.
      • Hoffstrom B.G.
      • Otey C.
      ) reported on anchorage-dependent cells that became apoptotic when cells were microinjected with anti-FAK antibody, or with a peptide corresponding to the portion of the β1-integrin cytoplasmic domain presumed to be required for the β1-integrin-FAK interaction. In another study, Frischet al. (
      • Frisch S.M.
      • Vuori K.
      • Ruoslahti E.
      • Chan-Hui P.-Y.
      ) reported that constitutively activated FAK protected MDCK cells from apoptosis consequent to the loss of matrix contact. Furthermore, Xu et al. (
      • Xu L.
      • Owens G.C.
      • Sturge X.
      • Yang E.T.
      • Liu R.J.
      • Craven R.J.
      • Cance W.G.
      ) reported that attenuation of FAK expression leads to apoptosis in some tumor cells. Interestingly, Ilic et al. (
      • Ilic D.
      • Almeida E.A.C.
      • Schlaepfer D.D.
      • Dazin P.
      • Aizawa S.
      • Damsky C.H.
      ) reported that the extracellular matrix survival signals transduced by FAK suppressed a p53-regulated apoptosis by serum withdrawal in anchorage-dependent cells. Whether FAK has an anti-apoptotic effect on other stress or drug-induced apoptosis has not been explored, and the mechanism of FAK anti-apoptosis has not been elucidated so far. In this study, we showed evidences that demonstrate that FAK has an anti-apoptotic role in the apoptosis induced by oxidative stress as well as etoposide in anchorage-independent HL-60 cells. Etoposide and hydrogen peroxide are known to produce reactive oxygen species (ROS) (
      • Amarante-Mendes G.P.
      • Naekyung K.C.
      • Liu L.
      • Huang Y.
      • Perkins C.L.
      • Green D.R.
      • Bhalla K.
      ). The conditions used in this study induced apoptosis in parental HL-60 and HL-60/Vect cells during a 4-h incubation period. In contrast, HL-60/FAK cells did not result in apoptosis by these stimuli during the 4-h incubation period. The anti-apoptotic effect by FAK continued at 24 h (data not shown). So far, there has been no reports on the anti-apoptotic role of FAK in ROS-producing stimuli.
      Although the mechanism of etoposide-induced apoptosis remains largely unknown, one possible mechanism is that ROS produced by etoposide induce apoptosis. ROS are presumed to destroy mitochondria function and induce apoptosis by activating caspase-3 (
      • Wong G.H.
      • Elwell J.H.
      • Oberley L.W.
      • Goeddel D.V.
      ). Caspase-3 is a critical downstream protease in the caspase cascade (
      • Xue D.
      • Shaham S.
      • Horvitz H.R.
      ), responsible for the cleavage of important substrates such as poly(adenosine diphosphate ribose) polymerase (
      • Nicholson D.W.
      • Thornberry N.A.
      ) and inhibitor of caspase-activated deoxyribonuclease (
      • Liu X.
      • Zou H.
      • Slaughter C.
      • Wang X.
      ). The caspase family controls apoptosis by multiple stimuli, including Fas ligand and TNF-α (
      • Martin S.J.
      • Green D.R.
      ,
      • Nagata S.
      ). We found HL-60/FAK blocked the caspase-3 activation by hydrogen peroxide or etoposide, which indicated that FAK regulation occurs upstream of caspase-3 activation.
      Fas-associated death domain-like ICE inhibitory protein (
      • Tschopp J.
      • Irmler M.
      • Thome M.
      ) is known as an inhibitor of Fas- and TNF-mediated apoptosis. The anti-apoptotic proteins, CrmA and p35, have also been shown to inhibit apoptosis by directly inhibiting ICE family proteases (
      • Tewari M.
      • Quan L.T.
      • O'Rounke K.
      • Desnoyers S.
      • Zeng Z.
      • Beidler D.R.
      • Poirier G.G.
      • Salvesen G.S.
      • Dixit V.M.
      ), most likely by functioning as substrates for and as competitive inhibitors of ICE family proteases. In contrast, IAPs bind to caspases and inhibit its activity. Expression of the IAP mRNAs (c-IAP1,2 and XIAP) was minimal in HL-60/Vect cells but remarkable in HL-60/FAK cells during untreated conditions, indicating FAK augmented the expression of these genes. HL-60/FAK showed higher basal NF-κB activation than did HL-60/Vect cells. High constitutive NF-κB activation may be critical to the antiapoptotic states induced by FAK cDNA transfection, which results in sustained expression of IAPs. It is reasonable to link FAK to the IAP, because IAP genes are the NF-κB-regulated genes. We showed here that the FAK-PI3-kinase-Akt survival pathway is constitutively activated in HL-60/FAK cells. Recently, it is reported that Akt mediates IκBkinase phosphorylation followed by NF-κB activation (
      • Owen J.D.
      • Ruest P.J.
      • Fry D.W.
      • Hanks S.K.
      ,
      • Ozes O.N.
      • Mayo L.D.
      • Gustin D.A.
      • Pfeffer S.R.
      • Pfeffer L.M.
      • Donner D.B.
      ). We should also take into consideration whether this pathway functions in HL-60/FAK cells.
      FAK Tyr-397 is an autophosphorylation site and a high-affinity binding site for Src homology 2 domains of Src family kinases (
      • Owen J.D.
      • Ruest P.J.
      • Fry D.W.
      • Hanks S.K.
      ). PI3-kinase and phospholipase Cγ also interact with this site. FAK Lys-454 is essential for kinase activity. FAK Tyr-925 is a binding site for the Grb2 Src homology 2 domain, and this interaction contributes to integrin-stimulated activation of Ras. Transfection with kinase-inactive FAK (K454R) indicated that the catalytic activity of FAK might be necessary for the full anti-apoptosis effect of FAK. Transfection with FAK mutants (Y397F and Y925F) indicated that tyrosine residues of 397 and 925 were essential for the anti-apoptotic effect. These results suggested that the signals through Src family kinase, PI3-kinase and/or Grb2, link to NF-κB activation and induction of IAPs. Further signal analysis of NF-κB activation by FAK should provide more information about the role of FAK in apoptosis. Recently, Chan et al. (
      • Chan P.-C.
      • Lai J.-F.
      • Cheng C.-H.
      • Tang M.-J.
      • Chiu C.-C.
      • Chen H.-C.
      ) reported that overexpression of FAK in Madin-Darby canine kidney cells suppressed the UV-induced apoptosis. Furthermore, they compared the anti-apoptotic activity of wild type FAK with those of FAK mutants (D395A, Y397F, p712/715A, and Y925F). These mutants failed to promote cell survival upon UV irradiation. The interaction of FAK with these proteins might lead to survival signals from FAK, proposing that the binding of PI3-kinase and p130cas with FAK is required for the anti-apoptotic function of FAK. We demonstrated here that FAK protects against oxidative stress-induced apoptosis in HL-60 cells as well as the glioblastoma cell line T98G (
      • Sonoda Y.
      • Watanabe S.
      • Matsumoto Y.
      • Aizu-Yokota E.
      • Kasahara T.
      ). In addition, Chan et al. (
      • Chan P.-C.
      • Lai J.-F.
      • Cheng C.-H.
      • Tang M.-J.
      • Chiu C.-C.
      • Chen H.-C.
      ) indicated that FAK protects against the UV-induced apoptosis in Madin-Darby canine kidney cells, supporting the notion of the general feature of FAK as a role of anti-apoptosis in various cells. Particularly, the involvement of NF-κB and IAPs and the resulting abrogation of caspase-3 activation was evidenced in the suppression of apoptosis in this study. To our knowledge, the data presented here provide the first line of evidence for the regulation of IAPs through NF-κB by FAK.

      Acknowledgments

      We are grateful to Kiyoe Sakairi, Hiromi Yamada, Youko Makuta, Shiro Watanabe, Masaya Ueno, and Shinya Sakurai for their technical help. We also thank Dr. Eriko Aizu-Yokota for her kind advice throughout the study.

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