ABSTRACT
FOXO (Forkhead box O) transcription factors are important regulators of cellular metabolism, cell-cycle progression and cell death. FOXO activity is regulated by multiple post-translational modifications, including phosphorylation, acetylation and polyubiquitination. Here, we show that FOXO becomes monoubiquitinated in response to increased cellular oxidative stress, resulting in its re-localization to the nucleus and an increase in its transcriptional activity. Deubiquitination of FOXO requires the deubiquitinating enzyme USP7/HAUSP (herpesvirus-associated ubiquitin-specific protease), which interacts with and deubiquitinates FOXO in response to oxidative stress. Oxidative stress-induced ubiquitination and deubiquitination by USP7 do not influence FOXO protein half-life. However, USP7 does negatively regulate FOXO transcriptional activity towards endogenous promoters. Our results demonstrate a novel mechanism of FOXO regulation and indicate that USP7 has an important role in regulating FOXO-mediated stress responses.
Subject(s)
Endopeptidases/metabolism , Gene Expression Regulation/physiology , Transcription Factors/genetics , Ubiquitin/metabolism , Animals , Cell Cycle Proteins , Cells, Cultured , Forkhead Transcription Factors , Humans , Hydrogen Peroxide/pharmacology , Kidney/metabolism , Lung Neoplasms/metabolism , Mice , NIH 3T3 Cells , Oxidants/pharmacology , Oxidative Stress , Protein Processing, Post-Translational , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription Factors/metabolism , Transcription, Genetic , Transfection , Ubiquitin Thiolesterase , Ubiquitin-Specific Peptidase 7 , Ubiquitin-Specific ProteasesABSTRACT
Forkhead transcription factors of the FOXO class are negatively regulated by PKB/c-Akt in response to insulin/IGF signalling, and are involved in regulating cell cycle progression and cell death. Here we show that, in contrast to insulin signalling, low levels of oxidative stress generated by treatment with H2O2 induce the activation of FOXO4. Upon treatment of cells with H2O2, the small GTPase Ral is activated and this results in a JNK-dependent phosphorylation of FOXO4 on threonine 447 and threonine 451. This Ral-mediated, JNK-dependent phosphorylation is involved in the nuclear translocation and transcriptional activation of FOXO4 after H2O2 treatment. In addition, we show that this signalling pathway is also employed by tumor necrosis factor alpha to activate FOXO4 transcriptional activity. FOXO members have been implicated in cellular protection against oxidative stress via the transcriptional regulation of manganese superoxide dismutase and catalase gene expression. The results reported here, therefore, outline a homeostasis mechanism for sustaining cellular reactive oxygen species that is controlled by signalling pathways that can convey both negative (PI-3K/PKB) and positive (Ras/Ral) inputs.
Subject(s)
Homeostasis , JNK Mitogen-Activated Protein Kinases/metabolism , Oxidative Stress , Signal Transduction/physiology , Transcription Factors/metabolism , ral GTP-Binding Proteins/metabolism , 14-3-3 Proteins/metabolism , Animals , Cell Cycle Proteins , Cell Line , Enzyme Activation , Forkhead Transcription Factors , Humans , Hydrogen Peroxide/metabolism , Insulin/metabolism , JNK Mitogen-Activated Protein Kinases/genetics , Mice , Mice, Knockout , Oxidants/metabolism , Phosphorylation , Reactive Oxygen Species/metabolism , Threonine/metabolism , Transcription Factors/genetics , Transcription, Genetic , Tumor Necrosis Factor-alpha/metabolism , ral GTP-Binding Proteins/geneticsABSTRACT
The serine/threonine kinase protein kinase B (PKB/c-Akt) acts downstream of the lipid kinase phosphoinositide 3-kinase (PI3K) and functions as an essential mediator in many growth-factor-induced cellular responses such as cell cycle regulation, cell survival and transcriptional regulation. PI3K activation generates 3'-phosphorylated phosphatidylinositol lipids (PtdIns3P) and PKB activation requires PtdIns3P-dependent membrane translocation and phosphorylation by upstream kinases. However PKB activation and function is also regulated by interaction with other proteins. Here we show binding of PKB to periplakin, a member of the plakin family of cytolinker proteins. Interaction between PKB and periplakin was mapped to part of the pleckstrin homology (PH) domain of PKB, which is probably not involved in lipid binding, and indeed binding to periplakin did not affect PKB activation. We therefore investigated the possibility that periplakin may act as a scaffold or localization signal for PKB. In cells endogenous periplakin localizes to different cellular compartments, including plasma membrane, intermediate filament structures, the nucleus and mitochondria. Overexpression of the C-terminal part of periplakin, encompassing the PKB binding region, results in predominant intermediate filament localization and little nuclear staining. This also resulted in inhibition of nuclear PKB signalling as indicated by inhibition of PKB-dependent Forkhead transcription factor regulation. These results suggest a possible role for periplakin as a localization signal in PKB-mediated signalling.
