PMC:3585731 / 17563-19834 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3585731","sourcedb":"PMC","sourceid":"3585731","source_url":"http://www.ncbi.nlm.nih.gov/pmc/3585731","text":"Late Increase in Akt Thr308 Phosphorylation Contributes to the Induction of Necroptotic Cell Death\nWe next investigated if the delayed RIP1 kinase-dependent increase in Akt Thr308 phosphorylation functionally contributes to the execution of necroptotic cell death. Firstly, PDGF/zVAD.fmk, which cannot induce necroptosis (Fig. 2A), triggered only the initial, rapid Akt and JNK phosphorylation changes and not the delayed activation (Fig. 4A), indicating that late, rather than early Akt phosphorylation correlates with necroptosis. Secondly, we saw that the ability of the Akt inhibitor to protect cells from necroptosis rapidly declined after 6 hrs of stimulation with zVAD.fmk, TNFα or bFGF/zVAD.fmk and no protection was observed when the inhibitor was added at 9 hrs (Fig. 4B,C). This time frame coincides with the timing of the secondary Akt Thr308 phosphorylation. Finally, we terminated the bFGF signal one hour after addition of bFGF by the addition of PD173074. This allowed us to retain early Akt activation, but to suppress the secondary increase (Fig. 4D). Both pre-addition and delayed addition of PD173074 fully prevented necroptosis (Fig. 4E). Overall, these data, while correlative, indicate that early Akt activation is insufficient to promote necroptosis and are strongly supportive of an important role for the delayed activation of Akt in the induction of necroptotic cell death.\n10.1371/journal.pone.0056576.g004 Figure 4 Late Thr308 phosphorylation of Akt contributes to necroptosis.\n(A) L929 cells were treated with zVAD.fmk and bFGF or PDGF, with or without Nec-1, for the indicated periods of time. (B,C) L929 cells were stimulated by zVAD.fmk or TNFα (B) or bFGF/zVAD.fmk under serum free conditions (C). Akt inh. VIII was added 15 min before necroptotic stimulation (Pre) or at indicated times after stimulation. Viability was measured 24 hr after activation of necroptosis. (D) L929 cells were stimulated with bFGF/zVAD under serum free conditions. PD173074 was added 15 min before or 1 hr after FGF/zVAD. Samples for western blot were collected at 15 min and 9 hr time points. (E) Cells were pretreated with PD173074 or it was added 1 hr after bFGF/zVAD.fmk, followed by viability assessment at 24 hr. 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