RESUMO
DNA interstrand crosslinks (ICLs), highly toxic lesions that covalently link the Watson and Crick strands of the double helix, are repaired by a complex, replication-coupled pathway in higher eukaryotes. The earliest DNA processing event in ICL repair is the incision of parental DNA on either side of the ICL ("unhooking"), which allows lesion bypass. Incisions depend critically on the Fanconi anemia pathway, whose activation involves ubiquitylation of the FANCD2 protein. Using Xenopus egg extracts, which support replication-coupled ICL repair, we show that the 3' flap endonuclease XPF-ERCC1 cooperates with SLX4/FANCP to carry out the unhooking incisions. Efficient recruitment of XPF-ERCC1 and SLX4 to the ICL depends on FANCD2 and its ubiquitylation. These data help define the molecular mechanism by which the Fanconi anemia pathway promotes a key event in replication-coupled ICL repair.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Recombinases/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Clivagem do DNA , Dano ao DNA , Proteínas de Ligação a DNA/química , Endodesoxirribonucleases , Endonucleases/química , Exodesoxirribonucleases/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/química , Humanos , Cinética , Enzimas Multifuncionais , Ligação Proteica , Recombinases/química , Ubiquitinação , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
Recent compelling data show that reactive oxygen species (ROS) not only are a harmful by-product of aerobic metabolism, but also are used as signaling molecules to regulate various cellular processes. In mammalian cells, ROS are produced transiently in response to many extracellular stimuli, including insulin, and specific inhibition of the ROS suppresses insulin-dependent signaling. Initially, this finding rationalized the concept of ROS acting as insulin mimetics. However, it is becoming evident that ROS are also causal to diabetes, a metabolic disorder characterized by insufficiency of secretion of, or receptor insensitivity to, endogenous insulin. This notion underlines a dual role for ROS in insulin signaling as both deleterious and beneficiary. Moreover, it strongly suggests that a delicate redox balance is required for insulin signaling to remain "healthy" for an organism.
Assuntos
Insulina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Humanos , Modelos BiológicosRESUMO
Forkhead box O (FOXO) transcription factors are involved in various cellular processes, including cell proliferation, stress resistance, metabolism, and longevity. Regulation of FOXO transcriptional activity occurs mainly through a variety of post-translational modifications, including phosphorylation, acetylation, and ubiquitination. Here we describe nemo-like kinase (NLK) as a novel regulator of FOXOs. NLK binds to and phosphorylates FOXO1, FOXO3a, and FOXO4 on multiple residues. NLK acts as a negative regulator of FOXO transcriptional activity. For FOXO4 we show that NLK-mediated loss of FOXO4 activity co-occurs with inhibition of FOXO4 monoubiquitination. Previously, we have shown that oxidative stress-induced monoubiquitination of FOXO4 stimulates its transactivation, which leads to activation of an antioxidant defensive program. Conversely, NLK-dependent inhibition of FOXO4 activity can provide a means to downregulate this defensive program, when oxidative stress reaches a level beyond which repair is no longer feasible and cells need to undergo apoptosis.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Estresse Oxidativo/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ciclo Celular , Fatores de Transcrição Forkhead , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Estresse Oxidativo/genética , Proteínas Serina-Treonina Quinases/genética , Fatores de Transcrição/genética , UbiquitinaçãoRESUMO
Oncogene-induced senescence (OIS) is a potent tumor-suppressive mechanism that is thought to come at the cost of aging. The Forkhead box O (FOXO) transcription factors are regulators of life span and tumor suppression. However, whether and how FOXOs function in OIS have been unclear. Here, we show a role for FOXO4 in mediating senescence by the human BRAF(V600E) oncogene, which arises commonly in melanoma. BRAF(V600E) signaling through mitogen-activated protein kinase/extracellular signal-regulated kinase kinase resulted in increased reactive oxygen species levels and c-Jun NH(2) terminal kinase-mediated activation of FOXO4 via its phosphorylation on Thr(223), Ser(226), Thr(447), and Thr(451). BRAF(V600E)-induced FOXO4 phosphorylation resulted in p21(cip1)-mediated cell senescence independent of p16(ink4a) or p27(kip1). Importantly, melanocyte-specific activation of BRAF(V600E) in vivo resulted in the formation of skin nevi expressing Thr(223)/Ser(226)-phosphorylated FOXO4 and elevated p21(cip1). Together, these findings support a model in which FOXOs mediate a trade-off between cancer and aging.