Posttranslational modifications of Forkhead box O transcription factors

Abstract

FOXO transcription factors play an important role in essential biological processes such as differentiation, proliferation, apoptosis, DNA repair, metabolism and stress resistance. Phosphorylation is the modification that was first found on FOXOs and much of the subsequent studies focused on this type of modification, studying the roles of kinases like PKB, SGK and JNK in the regulation of FOXO. However, proteins can also be modified and regulated by other posttranslational modifications like acetylation and ubiquitination. Only little is known about such modifications on FOXO. This thesis describes our studies regarding posttranslational modifications of FOXO transcription factors and the consequences thereof at both the cellular and organismal level. In Chapter 2 we provide an explanation for the increase in C.elegans lifespan by the longevity protein Sir2, which requires the worm FOXO-homologue DAF-16. We show that mammalian Sir2, also called SIRT1, directly binds to and deacetylates FOXO4 and thereby activates FOXO transcriptional activity both towards cell cycle regulation and stress resistance. Furthermore, we show that FOXOs are acetylated in response to oxidative stress by the acetyltransferase CBP (Chapter 2). Sir2 activity is inhibited by nicotinamide, a product of the Sir2-mediated deacetylation reaction. Organisms from bacteria to humans have a so-called NAD+-salvage pathway in which nicotinamide is converted. In C.elegans this job likely is performed by a nicotinamidase named PNC1, which converts nicotinamide into nicotinic acid. Potentially, by lowering the levels of nicotinamide, PNC1 increases organismal lifespan. Indeed, we found that RNAi-mediated knockdown of PNC1 decreases worm lifespan. On the contrary, overexpression of PNC1 enhanced the worm's oxidative stress resistance, which has been implicated in increasing lifespan (Chapter 3).In Chapter 4 we show that oxidative stress also induces monoubiquitination of FOXO4. This monoubiquitination results in nuclear retention and increased transcriptional activity of FOXO4. The ubiquitin-moiety is cleaved from FOXO4 by the DUB USP7. As expected from the observed monoubiquitination neither oxidative stress nor USP7 affects FOXO4 half-life, which is about 9 hours.cAMP has long been appreciated as a second messenger that can induce cell cycle arrest. Several models, concerning both MAPK and PI3K/PKB signaling, have been proposed to explain this phenomenon. In Chapter 5 we demonstrate that in MEFs FOXO transcription factors, that are downstream of PI3K signaling, are required for the cAMP-mediated growth arrest and propose that this also applies to many other cell types.To exert their diverse array of effects, FOXOs interact with other proteins. A screen to identify novel FOXO interacting transcription factors identified interferon (IFN) consensus sequence binding protein (ICSBP), nuclear factor of activated T-cells (NFATc), nuclear respiratory factor 1 (NRF1), Transcriptional Intermediary Factor 1 (TIF1), Sp1 and Homeobox D9/10 (HOXD9/10)(Chapter 6). By coimmunoprecipitations we confirmed binding of ICSBP to FOXO4, which is enhanced both by oxidative stress and NFATc, another possible interactor of FOXO. NFATc and ICSBP enhance expression of interleukin 12 (IL-12) and we demonstrate that FOXO4 acts synergistically with NFATc and ICSBP to induce IL-12. IL-12 regulates TH1 cell proliferation, so we hypothesise a novel role for FOXO4 in the adaptive immune response.The consequences of the findings described in this thesis for our comprehension of the role of posttranslational modifications in the regulation of FOXO function in cellular and organismal behaviour are discussed in Chapter 7. We also propose an intriguing model in which FOXOs and p53 are compared regarding their roles in tumour suppression and aging.