Expression of Constitutively Active Akt, Rescues Necroptosis Under Serum Free Conditions
We used L929 cells stably expressing constitutively active wild type Akt1 (Myr-Akt) or the catalytically inactive mutant K179M in order to further understand the contribution of growth factors and RIP1 kinase to Akt activation during necroptosis. Constitutively active Akt1 (Myr-Akt) was generated as previously described [37] by the addition of a myristoylation signal which provides constitutive localization to the plasma membrane and by the deletion of the auto-inhibitory PH domain (Fig. 7A) resulting in an Akt that is active under serum free. It is important to note that the cells expressing Myr-Akt were viable, grew in a manner indistinguishable from the empty vector control cells, and were not triggered to induce necroptosis by serum starvation (Fig. 7B). This indicates that active Akt alone is not sufficient to induce necroptotic cell death. Under serum free conditions Myr-Akt, but not the K179M mutant, fully restored zVAD.fmk-induced necroptosis (Fig. 7A,B). Nec-1 prevented both Myr-Akt dependent cell death and the necroptosis-specific delayed increase in Akt Thr308 phosphorylation (Fig. 7B,C). Myr-Akt also allowed other zVAD.fmk-dependent events, including activation of JNK and c-Jun phosphorylation (Fig. 7C) and upregulation of TNFα mRNA (Fig. 7D) to occur under serum free conditions, confirming an important role for Akt at the apex of necroptotic signaling. These data demonstrated that the presence of active and membrane localized Akt is sufficient to uncouple Akt activation during necroptosis from growth factor signaling. RIP1 kinase was still able to regulate Akt activation during necroptosis, suggesting that growth factors and RIP1 kinase provide two independent inputs required for Akt changes during necroptosis.
10.1371/journal.pone.0056576.g007 Figure 7  Over expression of constitutively active Akt restores necroptosis under serum free conditions.
(A,B) L929 cells were stably infected with empty MSCV retrovirus or viruses encoding Myr-Akt or the catalytically inactive Myr-Akt K179M. Necroptosis was induced by the addition of zVAD.fmk under serum free conditions (A) or serum or serum free conditions with Nec-1 (B). Viability assays were performed after 24 hr. (C) Myr-Akt and Myr-Akt K179M cells were treated with zVAD.fmk and/or Nec-1 under serum free conditions for 9 hr, followed by western blot using the indicated antibodies. Endogenous Akt (∼) and Myr-Akt (*) bands are indicated. (D) L929 cells, stably infected with Myr-Akt and Myr-Akt K179KM, were stimulated with zVAD.fmk for 9 hr under serum free conditions. TNFα mRNA levels were determined by qRT-PCR and normalized using mouse 18S RNA. (E-G) L929 cells expressing Myr-Akt and Ala and Asp mutants of Thr308 and Ser473 were treated with zVAD.fmk under serum free conditions, followed by viability assay at 24 hr (E), western blot at 9 hr (F), or evaluation of TNFα mRNA levels by qRT-PCR at 9 hrs (G). In all graphs, average±SD was plotted. RIP1 kinase-dependent Thr308 phosphorylation of Myr-Akt during necroptosis increased Myr-Akt activity as it did with endogenous Akt (Fig. 5). Phosphorylation of many previously described Akt substrates was increased upon the expression of Myr-Akt, but not the K179M mutant, confirming that these molecules are Akt substrates in L929 cells (Fig. 7C). The effect of zVAD.fmk on their phosphorylation varied, likely due to the increased basal activity of Myr-Akt. Some substrates, including p70S6K, S6, GSK-3 and FoxO4, were fully phosphorylated even in the absence of zVAD.fmk. On the other hand, phosphorylation of FoxO1 and MDM2 was significantly increased in the presence of zVAD.fmk, indicating that necroptotic Thr308 phosphorylation of Myr-Akt still promoted its activity.
Under serum free conditions all zVAD.fmk-induced downstream events (cell death, JNK activation, TNFα production) were dependent on the over expressed Myr-Akt. This allowed us to examine the effects of other Akt mutations on necroptosis. First, we found that membrane localization of Akt is required. Full length Akt or a mutant lacking both the PH domain and the Myr tag did not support the activation of cell death or increased Thr308 phosphorylation following zVAD.fmk addition under serum free conditions (Fig. S8A,B). Second, we found a specific and critical role for Thr308 phosphorylation in the regulation of the necroptotic functions of Akt. It has been reported that Ala mutations at Thr308 and Ser473 cause a reduction in the catalytic activity of Akt, while Asp mutants increase activity [38]. We examined the effect of Ala and Asp mutants at both sites during necroptosis. In our hands, both Asp mutants displayed activity comparable to wild type Akt, while both Ala mutants displayed comparable decreases in activity (Fig. S8C). Despite similar catalytic activities, Thr308 and Ser473 mutants displayed major differences in their ability to promote necroptotic changes (Fig. 7E,F,G). As expected, the S473D mutant, which was phosphorylated on Thr308 after the addition of zVAD, displayed only slightly reduced activity, while S473A was significantly less active in all aspects of necroptosis. S473A was unable to be efficiently phosphorylated on Thr308 possibly due to the inability of the Ala mutated 473 site to be phosphorylated and provide a docking site for PDK1 to phosphorylate Thr308 [39]. Strikingly, both Ala and Asp mutants of Thr308 were significantly less active in promoting cell death, phosphorylation of JNK and c-Jun, and TNFα mRNA. This suggests that T308D, in spite of being an active Akt construct, may not be a perfect mimic of phosphorylation and this mutant form of the kinase may not have sufficient activity to phosphorylate the entire repertoire of substrates in the cells. When tested, T308D did not support the downstream phosphorylation of several substrates that were phosphorylated by the Myr-Akt construct in the presence of zVAD including FoxO1, Foxo4, MDM2, and p70S6K (Fig. S8D). Our model, based on these results, is that necroptosis-specific Thr308 phosphorylation provides a critical link between necroptotic machinery and Akt kinase, allowing Akt to phosphorylate substrates during necroptosis, promote TNFα synthesis, JNK activation and eventual cell death.