Serine 574 phosphorylation alters transcriptional programming of FOXO3 by selectively enhancing apoptotic gene expression

Abstract

Forkhead box O3 (FOXO3) is a multispecific transcription factor that is responsible for multiple and conflicting transcriptional programs such as cell survival and apoptosis. The protein is heavily post-translationally modified and there is considerable evidence that post-transcriptional modifications (PTMs) regulate protein stability and nuclear-cytosolic translocation. Much less is known about how FOXO3 PTMs determine the specificity of its transcriptional program. In this study we demonstrate that exposure of hepatocytes to ethanol or exposure of macrophages to lipopolysaccharide (LPS) induces the c-Jun N-terminal kinase (JNK)-dependent phosphorylation of FOXO3 at serine-574. Chromatin immunoprecipitation (ChIP), mRNA and protein measurements demonstrate that p-574-FOXO3 selectively binds to promoters of pro-apoptotic genes but not to other well-described FOXO3 targets. Both unphosphorylated and p-574-FOXO3 bound to the B-cell lymphoma 2 (Bcl-2) promoter, but the unphosphorylated form was a transcriptional activator, whereas p-574-FOXO3 was a transcriptional repressor. The combination of increased TRAIL (TNF-related apoptosis-inducing ligand) and decreased Bcl-2 was both necessary and sufficient to induce apoptosis. LPS treatment of a human monocyte cell line (THP-1) induced FOXO3 S-574 phosphorylation and apoptosis. LPS-induced apoptosis was prevented by knockdown of FOXO3. It was restored by overexpressing wild-type FOXO3 but not by overexpressing a nonphosphorylatable S-574A FOXO3. Expression of an S-574D phosphomimetic form of FOXO3 induced apoptosis even in the absence of LPS. A similar result was obtained with mouse peritoneal macrophages where LPS treatment increased TRAIL, decreased Bcl-2 and induced apoptosis in wild-type but not FOXO3 −/− cells. This work thus demonstrates that S-574 phosphorylation generates a specifically apoptotic form of FOXO3 with decreased transcriptional activity for other well-described FOXO3 functions. Forkhead box O3 (FOXO3) is a multispecific transcription factor that serves as a longevity factor 1,2 and tumor suppressor and is critically involved in multiple seemingly independent biological process including cell-cycle arrest, 3 DNA repair, 4 antioxidant and stress responses, 5 apoptosis, 6 autophagy, glucose metabolism and aging. 7 Its many transcriptional programs are frequently in opposition to each other as it induces apoptosis, yet it is also critical for cell survival and longevity. Although there is considerable information regarding the mechanisms that regulate the nuclear/cytosolic distribution and protein stability of FOXO3, the control mechanisms that regulate transcriptional specificity are poorly understood.FOXO3 function is tightly regulated by multiple posttranslational modifications (PTMs) including phosphorylation, acetylation, ubiquitination and arginine and lysine methylation. Specific PTMs regulate the partitioning of FOXO3 between the cytosol and the nucleus 8-15 and its stability and degradation, 16 but the links between specific PTMs and FOXO3 tran...

Mechanisms of doxorubicin resistance in hepatocellular carcinoma

“…Doxorubicin increases nuclear accumulation of FOXO3 in breast cancer [69,70], lung cancer, neuroblastoma [71] and osteosarcoma cells [72], and pharmacological approaches that inhibit Akt or otherwise increase FOXO3 nuclear accumulation work synergistically with doxorubicin to enhance apoptosis [73,74]. The mechanisms by which FOXO3 mediates doxorubicin-induced apoptosis include its transcriptional repression of miR-21 which represses translation of Fas-L [71], transcriptional upregulation of Bim, a pro-apoptotic Bcl-2 homolog [75], and transcriptional repression of Bcl-2 [76] and Survivin, an anti-apoptotic Bcl-2 family member [77]. …”

“…Phosphorylation of FOXO3 at serine-7 by p38 causes it to translocate to the nucleus in response to doxorubicin [70] and phosphorylation at serine-574 causes it to selectively bind to pro-apoptotic promoters and induces cell death. In the absence of this phosphorylation, FOXO3 initiates an antioxidant and cell protective transcriptional program [76]. Thus, whether FOXO3 serves as a pro-apoptotic or pro-survival factor likely depends on the state of its modification by upstream enzymes.…”

JNK Signaling: Regulation and Functions Based on Complex Protein-Protein Partnerships

“…These JNK substrates encompass members of many different families, including those of the bZIP (Jun, ATF2, Myc [164,224,[229][230][231]), bHLH (Hes1, Twist1 [232,233]), Zinc finger (Sp1 [234]), Forkhead (FOXO4, FOXO3 [235][236][237]), and RUNT (p53, p73 [238,239]) families, as well as proteins of the nuclear receptor family (androgen receptor, glucocorticoid receptor, peroxisome proliferator-activated receptors, RAR␣, and RXR␣ [240][241][242][243][244][245]). Coactivators and corepressors lacking a direct DNA-binding capacity (such as YAP1 [246]) can also be targeted by JNK.…”

Targeting AMPK Signaling as a Neuroprotective Strategy in Parkinson’s Disease

“…FOXO3 mediates multiple conflicting transcriptional programs, many of which promote cell survival and are associated with increased cell viability and lifespan [149-152]. However, during cellular stress, c-Jun N-terminal kinase (JNK) can phosphorylate FOXO3 at serine 574 producing a proapoptotic transcriptional program [153]. Specifically, FOXO3 can increase transcription of Bim, facilitating formation of the BAX/BAK mediated mitochondrial outer membrane pore and release of apoptotic initiators cytochrome c and apoptosis-inducing factor (AIF), and this has been linked to prolonged AMPK activation [154,155].…”