RESUMEN
Circadian oscillations emerge from transcriptional and post-translational feedback loops. An important step in generating rhythmicity is the translocation of clock components into the nucleus, which is regulated in many cases by kinases. In mammals, the kinase promoting the nuclear import of the key clock component Period 2 (PER2) is unknown. Here, we show that the cyclin-dependent kinase 5 (CDK5) regulates the mammalian circadian clock involving phosphorylation of PER2. Knock-down of Cdk5 in the suprachiasmatic nuclei (SCN), the main coordinator site of the mammalian circadian system, shortened the free-running period in mice. CDK5 phosphorylated PER2 at serine residue 394 (S394) in a diurnal fashion. This phosphorylation facilitated interaction with Cryptochrome 1 (CRY1) and nuclear entry of the PER2-CRY1 complex. Taken together, we found that CDK5 drives nuclear entry of PER2, which is critical for establishing an adequate circadian period of the molecular circadian cycle. Of note is that CDK5 may not exclusively phosphorylate PER2, but in addition may regulate other proteins that are involved in the clock mechanism. Taken together, it appears that CDK5 is critically involved in the regulation of the circadian clock and may represent a link to various diseases affected by a derailed circadian clock.
Asunto(s)
Relojes Circadianos , Quinasa 5 Dependiente de la Ciclina/metabolismo , Animales , Núcleo Celular/metabolismo , Ritmo Circadiano , Epistasis Genética , Ratones , Células 3T3 NIH , Proteínas Circadianas Period/química , Proteínas Circadianas Period/metabolismo , Fosforilación , Fosfoserina/metabolismo , Estabilidad Proteica , ARN Interferente Pequeño/metabolismo , Saccharomyces cerevisiae/metabolismo , Núcleo Supraquiasmático/fisiología , Factores de TiempoRESUMEN
Living in the earth's oxygenated environment forced organisms to develop strategies to cope with the damaging effects of molecular oxygen known as reactive oxygen species (ROS). Here, we show that Per2, a molecular component of the mammalian circadian clock, is involved in regulating a cell's response to oxidative stress. Mouse embryonic fibroblasts (MEFs) containing a mutation in the Per2 gene are more resistant to cytotoxic effects mediated by ROS than wild-type cells, which is paralleled by an altered regulation of bcl-2 expression in Per2 mutant MEFs. The elevated survival rate and alteration of NADH/NAD(+) ratio in the mutant cells is reversed by introduction of the wild-type Per2 gene. Interestingly, clock synchronized cells display a time dependent sensitivity to paraquat, a ROS inducing agent. Our observations indicate that the circadian clock is involved in regulating the fate of a cell to survive or to die in response to oxidative stress, which could have implications for cancer development and the aging process.
RESUMEN
The second messenger cGMP controls cardiovascular and gastrointestinal homeostasis in mammals. However, its physiological relevance in the nervous system is poorly understood.1 Now, we have reported that the cGMP-dependent protein kinase type I (PRKG1) is implicated in the regulation of the timing and quality of sleep and wakefulness.2Prkg1 mutant mice showed altered distribution of sleep and wakefulness as well as reduction in rapid-eye-movement sleep (REMS) duration and in non-REMS consolidation. Furthermore, the ability to sustain waking episodes was compromised. These observations were also reflected in wheel-running and drinking activity. A decrease in electroencephalogram power in the delta frequency range (1-4 Hz) under baseline conditions was observed, which was normalized after sleep deprivation. Together with the finding that circadian clock amplitude is reduced in Prkg1 mutants these results indicate a decrease of the wake-promoting output of the circadian system affecting sleep. Because quality of sleep might affect learning we tested Prkg1 mutants in several learning tasks and find normal spatial learning but impaired object recognition memory in these animals. Our findings indicate that Prkg1 impinges on circadian rhythms, sleep and distinct aspects of learning.
RESUMEN
Many effects of nitric oxide (NO) are mediated by the activation of guanylyl cyclases and subsequent production of the second messenger cyclic guanosine-3',5'-monophosphate (cGMP). cGMP activates cGMP-dependent protein kinases (PRKGs), which can therefore be considered downstream effectors of NO signaling. Since NO is thought to be involved in the regulation of both sleep and circadian rhythms, we analyzed these two processes in mice deficient for cGMP-dependent protein kinase type I (PRKG1) in the brain. Prkg1 mutant mice showed a strikingly altered distribution of sleep and wakefulness over the 24 hours of a day as well as reductions in rapid-eye-movement sleep (REMS) duration and in non-REM sleep (NREMS) consolidation, and their ability to sustain waking episodes was compromised. Furthermore, they displayed a drastic decrease in electroencephalogram (EEG) power in the delta frequency range (1-4 Hz) under baseline conditions, which could be normalized after sleep deprivation. In line with the re-distribution of sleep and wakefulness, the analysis of wheel-running and drinking activity revealed more rest bouts during the activity phase and a higher percentage of daytime activity in mutant animals. No changes were observed in internal period length and phase-shifting properties of the circadian clock while chi-squared periodogram amplitude was significantly reduced, hinting at a less robust oscillator. These results indicate that PRKG1 might be involved in the stabilization and output strength of the circadian oscillator in mice. Moreover, PRKG1 deficiency results in an aberrant pattern, and consequently a reduced quality, of sleep and wakefulness, possibly due to a decreased wake-promoting output of the circadian system impinging upon sleep.
Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico/genética , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Sueño/fisiología , Vigilia/fisiología , Animales , Encéfalo/metabolismo , Ritmo Circadiano , GMP Cíclico/metabolismo , Electroencefalografía/métodos , Electromiografía/métodos , Ratones , Ratones Noqueados , Mutación , Óxido Nítrico/metabolismo , Oscilometría , Privación de SueñoRESUMEN
BACKGROUND: Circadian oscillation of clock-controlled gene expression is mainly regulated at the transcriptional level. Heterodimers of CLOCK and BMAL1 act as activators of target gene transcription; however, interactions of PER and CRY proteins with the heterodimer abolish its transcriptional activation capacity. PER and CRY are therefore referred to as negative regulators of the circadian clock. To further elucidate the mechanism how positive and negative components of the clock interplay, we characterized the interactions of PER2, CRY1 and CRY2 with BMAL1 and CLOCK using a mammalian two-hybrid system and co-immunoprecipitation assays. RESULTS: Both PER2 and the CRY proteins were found to interact with BMAL1 whereas only PER2 interacts with CLOCK. CRY proteins seem to have a higher affinity to BMAL1 than PER2. Moreover, we provide evidence that PER2, CRY1 and CRY2 bind to different domains in the BMAL1 protein. CONCLUSION: The regulators of clock-controlled transcription PER2, CRY1 and CRY2 differ in their capacity to interact with each single component of the BMAL1-CLOCK heterodimer and, in the case of BMAL1, also in their interaction sites. Our data supports the hypothesis that CRY proteins, especially CRY1, are stronger repressors than PER proteins.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ritmo Circadiano , Flavoproteínas/metabolismo , Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción ARNTL , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/química , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Sitios de Unión , Proteínas CLOCK , Células COS , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Chlorocebus aethiops , Criptocromos , Flavoproteínas/genética , Regulación de la Expresión Génica , Humanos , Ratones , Modelos Biológicos , Proteínas Nucleares/genética , Proteínas Circadianas Period , Unión Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transactivadores/genética , Factores de Transcripción/genéticaRESUMEN
Cholecystokinin (CCK) is one of the most abundant neuropeptides in the central nervous system (CNS) where it promotes important functions by activation of receptors CCK1 and CCK2. Our aim was to investigate CCK receptors expression and their downstream intracellular signaling in immortalized rat brain neuroblasts. Results show that CCK1 and CCK2 receptor mRNAs and CCK2 receptor protein are expressed in neuroblasts. CCK incubation of neuroblasts leads to stimulation in a time-dependent manner of several signaling pathways, such as tyrosine phosphorylation of adaptor proteins paxillin and p130(Cas), phosphorylation of p44/p42 ERKs as well as PKB (Ser473). Moreover, CCK-8 stimulates the DNA-binding activity of the transcription factor AP-1. The CCK2 receptor agonist gastrin stimulates ERK1/2 phosphorylation in a comparable degree as CCK does. ERK1/2 phosphorylation activated by CCK-8 was markedly inhibited by the CCK2 receptor antagonist CR2945. Incubation for 48 h with CCK-8 increases neuroblasts viability in a similar degree as EGF. In summary, our data clearly identify CCK1 and CCK2 receptor mRNAs and CCK2 receptor protein in brain neuroblasts and show that incubation with CCK promotes cell proliferation and activates the phosphorylation of survival transduction pathways. Stimulation of ERK1/2 phosphorylation by CCK is mainly mediated by the CCK2 receptor. Moreover, this work might provide a novel model of proliferating neuronal cells to further study the biochemical mechanisms by which the neuropeptide CCK exerts its actions in the CNS.
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Encéfalo/metabolismo , Colecistoquinina/farmacología , Neuronas/metabolismo , Receptor de Colecistoquinina A/genética , Receptor de Colecistoquinina B/genética , Animales , Western Blotting , Encéfalo/citología , Encéfalo/efectos de los fármacos , Línea Celular Transformada , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Proteína Sustrato Asociada a CrK/metabolismo , ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Inmunoprecipitación , Neuronas/citología , Neuronas/efectos de los fármacos , Paxillin/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Ratas , Receptor de Colecistoquinina A/metabolismo , Receptor de Colecistoquinina B/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Factor de Transcripción AP-1/metabolismoRESUMEN
A functional circadian clock has long been considered a selective advantage. Accumulating evidence shows that the clock coordinates a variety of physiological processes in order to schedule them to the optimal time of day and thus to synchronize metabolism to changes in external conditions. In mitochondria, both metabolic and cellular defense mechanisms are carefully regulated. Abnormal clock function, might influence mitochondrial function, resulting in decreased fitness of an organism.
Asunto(s)
Ritmo Circadiano/fisiología , Mitocondrias/fisiología , Envejecimiento , Animales , Apoptosis , Transporte de Electrón , Humanos , Ratones , Mitocondrias/metabolismo , Enfermedades Neurodegenerativas/patología , Oxígeno/metabolismo , Fosforilación , Factores de Riesgo , Factores de Tiempo , Transcripción GenéticaRESUMEN
We have previously shown that lovastatin induces apoptosis in spontaneously immortalized rat brain neuroblasts. Focal adhesion proteins and protein kinase Cdelta (PKCdelta) have been implicated in the regulation of apoptosis. We found that lovastatin exposure induced focal adhesion kinase, Crk-associated substrate (p130(Cas)), PKCdelta cleavage and caspase-3 activation in a concentration-dependent manner. Lovastatin effects were fully prevented by mevalonate. The cleavage of p130(Cas) was almost completely inhibited by z-DEVD-fmk, a specific caspase-3 inhibitor, and z-VAD-fmk, a broad spectrum caspase inhibitor, indicating that cleavage is mediated by caspase-3. In contrast, the lovastatin-induced cleavage of PKCdelta was only blocked by z-VAD-fmk suggesting that PKCdelta cleavage is caspase-dependent but caspase-3-independent. Additionally, z-VAD-fmk partially prevented lovastatin-induced neuroblast apoptosis. The present data show that lovastatin may induce neuroblast apoptosis by both caspase-dependent and independent pathways. These findings may suggest that the caspase-dependent component leading to the neuroblast cell death is likely to involve the cleavage of focal adhesion proteins and PKCdelta, which may be partially responsible for some biochemical features of neuroblast apoptosis induced by lovastatin.
Asunto(s)
Apoptosis/fisiología , Encéfalo/fisiología , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Lovastatina/farmacología , Proteína Quinasa C-delta/metabolismo , Ratas/metabolismo , Animales , Encéfalo/efectos de los fármacos , Encéfalo/enzimología , Caspasa 3 , Inhibidores de Caspasas , Caspasas/metabolismo , Línea Celular , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Citometría de Flujo , Ácido Mevalónico/farmacología , Factores de TiempoRESUMEN
Schwann cells lacking the tumor-suppressor-protein merlin tend in man to build benign tumors (schwannoma). We observed that characteristic features of these cells which are relevant to tumorigenicity resemble those described in cells with high Rac activity. Moreover this small GTPase also phosphorylates merlin via PAK activation. We hypothesized that merlin deficiency might cause an activation of Rac and its dependent signaling pathways, in particular the pro-tumorigenic JNK pathway. We show an enhanced activation of Rac1 in primary human schwannoma cells, find both Rac and its effector PAK at the membrane where they colocalize, and describe increased levels of phosphorylated JNK in the nucleus of these cells. Further we describe regulation at post-transcriptional level with upregulated protein, but not mRNA levels for Rac1, and JNK1/2. We conclude that merlin regulates Rac activation, and suggest that this is important for human schwannoma cell dedifferentiation.