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.
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 microg/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-kappaB 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-kappaB activation in HL-60/FAK cells than in HL-60/Vect cells, and it appeared that sustained NF-kappaB 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.
Sonoda
Y
Y
Department of Biochemistry, Kyoritsu College of Pharmacy, Shibakoen 1-5-30, Minato-ku, Tokyo, 105-8512, Japan.
Matsumoto
Y
Y
Funakoshi
M
M
Yamamoto
D
D
Hanks
S K
SK
Kasahara
T
T
D017209:Apoptosis; D053148:Caspase 3; D061945:Caspase Inhibitors; D002470:Cell Survival / Q000235:genetics; D053938:DNA Fragmentation / Q000187:drug effects; D004789:Enzyme Activation; D005047:Etoposide / Q000494:pharmacology; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D018922:HL-60 Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D016328:NF-kappa B / Q000378:metabolism; D018384:Oxidative Stress; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000032:analysis; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection
D053148:Caspase 3; D061945:Caspase Inhibitors; D005047:Etoposide; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006861:Hydrogen Peroxide; D016328:NF-kappa B; D019869:Phosphatidylinositol 3-Kinases; D019000:Phosphotyrosine; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D019253:Proto-Oncogene Proteins c-bcl-2; C496050:PTK2 protein, human; C494918:AKT1 protein, human; C505407:CASP3 protein, human