Protein phosphorylation in a human glioblastoma cell line, T98G, was examined after exposure to oxidative stress in 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.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 p77 btk , p72 syk , p56/59 hck , and p56 lck (1-4). Many growth factors and cytokines promote cell survival, including insulin-like growth factor 1 (5) and platelet-derived growth factor (6). Phosphatidylinositide 3Ј-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 receptorassociated adaptor proteins. One target of PI 3-kinase is the serine-threonine kinase Akt, also named PKB␣ (5). Akt is a general mediator of growth factor-induced survival and has been shown to suppress apoptotic death by a variety of stimuli (5). Signaling via growth factor receptor activation leads to the sequential activation of PI 3-kinase and Akt. Recently, Datta et al. (7) reported that Akt phosphorylates BAD in vitro and in 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 (BclXs, BAD, Bax, Bak) promote cell death (8 -13). 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 (14), 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 phosphorylat...
