ABSTRACT
For many years, a connection between circadian clocks and cancer has been postulated. Here we describe an unexpected function for the circadian repressor CRY2 as a component of an FBXL3-containing E3 ligase that recruits T58-phosphorylated c-MYC for ubiquitylation. c-MYC is a critical regulator of cell proliferation; T58 is central in a phosphodegron long recognized as a hotspot for mutation in cancer. This site is also targeted by FBXW7, although the full machinery responsible for its turnover has remained obscure. CRY1 cannot substitute for CRY2 in promoting c-MYC degradation. Their unique functions may explain prior conflicting reports that have fueled uncertainty about the relationship between clocks and cancer. We demonstrate that c-MYC is a target of CRY2-dependent protein turnover, suggesting a molecular mechanism for circadian control of cell growth and a new paradigm for circadian protein degradation.
Subject(s)
Cell Transformation, Neoplastic/genetics , Circadian Clocks/genetics , Cryptochromes/genetics , F-Box Proteins/genetics , Gene Expression Regulation, Neoplastic , Lymphoma/genetics , Proto-Oncogene Proteins c-myc/genetics , Animals , Carrier Proteins/chemistry , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Circadian Rhythm/genetics , Cryptochromes/chemistry , Cryptochromes/metabolism , Cullin Proteins/chemistry , Cullin Proteins/genetics , Cullin Proteins/metabolism , F-Box Proteins/chemistry , F-Box Proteins/metabolism , Fibroblasts , HEK293 Cells , Humans , Lymphoma/metabolism , Lymphoma/mortality , Lymphoma/pathology , Mice , Mice, Knockout , Models, Molecular , Protein Stability , Protein Structure, Secondary , Proteolysis , Proto-Oncogene Proteins c-myc/chemistry , Proto-Oncogene Proteins c-myc/metabolism , S-Phase Kinase-Associated Proteins/chemistry , S-Phase Kinase-Associated Proteins/genetics , S-Phase Kinase-Associated Proteins/metabolism , Signal Transduction , Survival AnalysisABSTRACT
Nuclear hormone receptors (NRs) regulate physiology by sensing lipophilic ligands and adapting cellular transcription appropriately. A growing understanding of the impact of circadian clocks on mammalian transcription has sparked interest in the interregulation of transcriptional programs. Mammalian clocks are based on a transcriptional feedback loop featuring the transcriptional activators circadian locomotor output cycles kaput (CLOCK) and brain and muscle ARNT-like 1 (BMAL1), and transcriptional repressors cryptochrome (CRY) and period (PER). CRY1 and CRY2 bind independently of other core clock factors to many genomic sites, which are enriched for NR recognition motifs. Here we report that CRY1/2 serve as corepressors for many NRs, indicating a new facet of circadian control of NR-mediated regulation of metabolism and physiology, and specifically contribute to diurnal modulation of drug metabolism.
Subject(s)
CLOCK Proteins/metabolism , Circadian Rhythm/physiology , Cryptochromes/metabolism , Period Circadian Proteins/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Transcription, Genetic/physiology , ARNTL Transcription Factors/metabolism , Animals , Cell Line , Cell Line, Tumor , Circadian Clocks/physiology , Feedback, Physiological/physiology , Female , Gene Expression Regulation/physiology , HEK293 Cells , Hep G2 Cells , Humans , Male , Mice , Nuclear Proteins/metabolism , Trans-Activators/metabolismABSTRACT
Cellular senescence, a state of sustained cell cycle arrest, has been identified as an important anti-tumor barrier. Senescent cells secrete various growth factors and cytokines, such as IL6 and IL8, which collectively constitute the senescence-associated secretory phenotype (SASP). The SASP can signal to the tumor environment and elicit the immune-mediated clearance of tumor cells or, depending on the context, could potentially promote tumor progression. Despite the importance of the SASP to tumor biology, its regulation remains relatively unknown. Here, we show that IκBζ, an atypical member of the inhibitor of NFκB proteins and selective coactivator of particular NFκB target genes, is an important regulator of SASP expression. Several models of DNA damage- and oncogene-induced senescence revealed a robust induction of IκBζ expression. RNAi-mediated knockdown of IκBζ impaired IL6 and IL8 expression, whereas transgenic IκBζ expression resulted in enhanced SASP cytokine expression. Importantly, during senescence of IκBζ knockout cells induction of IL6 and IL8, but not of the cell cycle inhibitor p21(WAF/CIP1), was completely abolished. Thus, we propose an important and hitherto unappreciated role of IκBζ in SASP formation in both DNA damage- and oncogene-induced senescence.
Subject(s)
DNA Damage , I-kappa B Proteins/metabolism , Cell Cycle Checkpoints , Cell Line, Tumor , Cellular Senescence/genetics , Cellular Senescence/physiology , Cytokines/metabolism , Humans , I-kappa B Proteins/biosynthesis , I-kappa B Proteins/genetics , MCF-7 Cells , Oncogenes , Phenotype , Signal TransductionSubject(s)
Aging , Blood Platelets , Blood Platelets/metabolism , Blood Platelets/physiology , Aging/physiology , Humans , Cell Differentiation , Animals , MiceSubject(s)
Terminal Care , Humans , Terminal Care/psychology , Terminal Care/ethics , Quality of Life/psychology , Social SupportSubject(s)
Cardiomyopathies , Peripartum Period , Pre-Eclampsia , Female , Pregnancy , Pre-Eclampsia/physiopathology , HumansABSTRACT
We recently demonstrated that the circadian clock component CRY2 is an essential cofactor in the SCFFBXL3-mediated ubiquitination of c-MYC. Because our demonstration that CRY2 recruits phosphorylated substrates to SCFFBXL3 was unexpected, we investigated the scope of this role by searching for additional substrates of FBXL3 that require CRY1 or CRY2 as cofactors. Here, we describe an affinity purification mass spectrometry (APMS) screen through which we identified more than one hundred potential substrates of SCFFBXL3+CRY1/2, including the cell cycle regulated Tousled-like kinase, TLK2. Both CRY1 and CRY2 recruit TLK2 to SCFFBXL3, and TLK2 kinase activity is required for this interaction. Overexpression or genetic deletion of CRY1 and/or CRY2 decreases or enhances TLK2 protein abundance, respectively. These findings reinforce the idea that CRYs function as co-factors for SCFFBXL3, provide a resource of potential substrates, and establish a molecular connection between the circadian and cell cycle oscillators via CRY-modulated turnover of TLK2.
Subject(s)
Cryptochromes/metabolism , Protein Serine-Threonine Kinases/metabolism , Ubiquitin-Protein Ligase Complexes/metabolism , Animals , Cells, Cultured , Circadian Clocks , Cryptochromes/genetics , F-Box Proteins/metabolism , Humans , Mice , Protein Serine-Threonine Kinases/drug effects , Stem Cell Factor/metabolism , Ubiquitin-Protein LigasesABSTRACT
Cellular metabolite balance and mitochondrial function are under circadian control, but the pathways connecting the molecular clock to these functions are unclear. Peroxisome proliferator-activated receptor delta (PPARδ) enables preferential utilization of lipids as fuel during exercise and is a major driver of exercise endurance. We show here that the circadian repressors CRY1 and CRY2 function as co-repressors for PPARδ. Cry1-/-;Cry2-/- myotubes and muscles exhibit elevated expression of PPARδ target genes, particularly in the context of exercise. Notably, CRY1/2 seem to repress a distinct subset of PPARδ target genes in muscle compared to the co-repressor NCOR1. In vivo, genetic disruption of Cry1 and Cry2 enhances sprint exercise performance in mice. Collectively, our data demonstrate that CRY1 and CRY2 modulate exercise physiology by altering the activity of several transcription factors, including CLOCK/BMAL1 and PPARδ, and thereby alter energy storage and substrate selection for energy production.