The Forkhead Transcription Factor AFX Activates Apoptosis by Induction of the BCL-6 Transcriptional Repressor

Abstract

The activation of the AKT/protein kinase B kinases by mutation of the PTEN lipid phosphatase results in enhanced survival of a diversity of tumors. This resistance to apoptosis is partly accomplished by the inhibition of genetic programs induced by a subfamily of forkhead transcription factors including AFX. Here we describe an AFX-regulated pathway that appears to account for at least part of this apoptotic regulatory system. Cells induced to synthesize an active form of AFX die by activating the apoptotic death pathway. An analysis of genes regulated by AFX demonstrated that BCL-6, a transcriptional repressor, is up-regulated ϳ4 -7-fold. An examination of the BCL-6 promoter demonstrated that AFX bound to specific target sites that could activate transcription. BCL-X L , an anti-apoptotic protein, contains potential BCL-6 target sites in its promoter. An analysis of endogenous BCL-X L levels in AFX-expressing cells revealed enhanced down-regulation of the transcript (ϳ1.3-1.7-fold) and protein, and BCL-6 directly binds to and suppresses the BCL-X L promoter. Finally, macrophages isolated from BCL-6؊/؊ mice show enhanced survival in vitro. These results suggest that AFX regulates apoptosis in part by suppressing the levels of anti-apoptotic BCL-XL through the transcriptional repressor BCL-6.The resistance of tumors to diverse apoptotic stimuli is of major clinical importance. A large number of recent studies has highlighted the significance of hyperactivation of the PI3K 1 pathway to the insensitivity of late stage tumors to chemotherapy and radiation treatments (1-6). The induction of this pathway by growth factor or oncogenic stimuli results in the production of three phosphorylated phosphatidylinositol lipids, which act as binding sites for the pleckstrin homology domains of the downstream kinases, PDK-1 and AKT/PKB (7-13). The membrane colocalization of these kinases results in the phosphorylation of a specific activation site in AKT/PKB by PDK-1 (7,8,(13)(14)(15). Importantly, this pathway is reversed by the lipid phosphatase activity of PTEN, a tumor suppressor that is homozygously deleted in a variety of late stage tumors (2-6). Whereas a number of phosphorylation targets has been described for the AKT/PKB kinases, both biochemical and genetic evidence support a subset of forkhead transcription factors including FKHR, FKHR-L1, and AFX as being important substrates for phosphorylation mediated by these kinases (16 -20). Phosphorylation of these forkhead transcription factors by AKT/PKB kinases results in their sequestration to the cytoplasm in which they are unable to activate transcription of their nuclear targets (17)(18)(19)(20). This outcome is an important component of cell survival mediated by the PI3K pathway, because nuclear localization of these forkhead family members results in the induction of transcriptional programs that lead to rapid cell death by apoptosis (17)(18)(19)(20).Whereas it is clear that the inhibition of the apoptotic programs activated by these forkhead transcription factors is ...

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