RESUMEN
The activity-dependent plasticity of synapses is believed to be the cellular basis of learning. These synaptic changes are mediated through the coordination of local biochemical reactions in synapses and changes in gene transcription in the nucleus to modulate neuronal circuits and behavior. The protein kinase C (PKC) family of isozymes has long been established as critical for synaptic plasticity. However, because of a lack of suitable isozyme-specific tools, the role of the novel subfamily of PKC isozymes is largely unknown. Here, through the development of fluorescence lifetime imaging-fluorescence resonance energy transfer activity sensors, we investigate novel PKC isozymes in synaptic plasticity in CA1 pyramidal neurons of mice of either sex. We find that PKCδ is activated downstream of TrkB and DAG production, and that the spatiotemporal nature of its activation depends on the plasticity stimulation. In response to single-spine plasticity, PKCδ is activated primarily in the stimulated spine and is required for local expression of plasticity. However, in response to multispine stimulation, a long-lasting and spreading activation of PKCδ scales with the number of spines stimulated and, by regulating cAMP response-element binding protein activity, couples spine plasticity to transcription in the nucleus. Thus, PKCδ plays a dual functional role in facilitating synaptic plasticity.SIGNIFICANCE STATEMENT Synaptic plasticity, or the ability to change the strength of the connections between neurons, underlies learning and memory and is critical for brain health. The protein kinase C (PKC) family is central to this process. However, understanding how these kinases work to mediate plasticity has been limited by a lack of tools to visualize and perturb their activity. Here, we introduce and use new tools to reveal a dual role for PKCδ in facilitating local synaptic plasticity and stabilizing this plasticity through spine-to-nucleus signaling to regulate transcription. This work provides new tools to overcome limitations in studying isozyme-specific PKC function and provides insight into molecular mechanisms of synaptic plasticity.
Asunto(s)
Isoenzimas , Transducción de Señal , Animales , Ratones , Transducción de Señal/fisiología , Sinapsis/fisiología , Plasticidad Neuronal/fisiología , Proteína Quinasa C/metabolismoRESUMEN
In mammalian females, germ cells remain arrested as primordial follicles. Resumption of meiosis is heralded by germinal vesicle breakdown, condensation of chromosomes, and their eventual alignment on metaphase plates. At the first meiotic division, anaphase-promoting complex/cyclosome associated with Cdc20 (APC/CCdc20) activates separase and thereby destroys cohesion along chromosome arms. Because cohesion around centromeres is protected by shugoshin-2, sister chromatids remain attached through centromeric/pericentromeric cohesin. We show here that, by promoting proteolysis of cyclins and Cdc25B at the germinal vesicle (GV) stage, APC/C associated with the Cdh1 protein (APC/CCdh1) delays the increase in Cdk1 activity, leading to germinal vesicle breakdown (GVBD). More surprisingly, by moderating the rate at which Cdk1 is activated following GVBD, APC/CCdh1 creates conditions necessary for the removal of shugoshin-2 from chromosome arms by the Aurora B/C kinase, an event crucial for the efficient resolution of chiasmata.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromosomas , Meiosis , Animales , Subunidad Apc2 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Aurora Quinasa B/metabolismo , Aurora Quinasa C/metabolismo , Proteína Quinasa CDC2/metabolismo , Proteínas Cdc20/fisiología , Proteínas Cdh1/metabolismo , Centrómero , Proteínas Cromosómicas no Histona/metabolismo , Femenino , Centro Germinal , Masculino , Ratones , Ratones Noqueados , Modelos Teóricos , Separasa/metabolismo , Fosfatasas cdc25/fisiología , CohesinasRESUMEN
Whereas proliferating cells enter M phase shortly after DNA replication, the first M phase of meiosis is preceded by an extended prophase in which homologous chromosomes undergo recombination. Exit from prophase I is controlled by the recombination checkpoint (RC), which, in yeast, represses the meiosis-specific transcription factor Ndt80 required for the expression of B-type cyclins and other M phase regulators. We show that an extended prophase I additionally requires the suppression of latent, mitotic cell-cycle controls by the anaphase-promoting complex (APC/C) and its meiosis-specific activator Ama1, which trigger the degradation of M phase regulators and Ndd1, a subunit of a mitotic transcription factor. ama1Δ mutants exit from prophase I prematurely and independently of the RC, which results in recombination defects and chromosome missegregation. Thus, control of prophase I by meiotic mechanisms depends on the suppression of the alternative, mitotic mechanisms by a meiosis-specific form of the APC/C.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Meiosis , Profase , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ciclosoma-Complejo Promotor de la Anafase , Proteínas Cdc20 , Segregación Cromosómica , Cromosomas Fúngicos/metabolismo , Proteínas de Unión al ADN/metabolismo , Metafase , Proteínas Serina-Treonina Quinasas/metabolismo , Proteolisis , Huso Acromático , Factores de Transcripción/metabolismoRESUMEN
During meiosis, two rounds of chromosome segregation after a single round of DNA replication produce haploid gametes from diploid precursors. At meiosis I, maternal and paternal kinetochores are pulled toward opposite poles, and chiasmata holding bivalent chromosomes together are resolved by cleavage of cohesin's alpha-kleisin subunit (Rec8) along chromosome arms. This creates dyad chromosomes containing a pair of chromatids joined solely by cohesin at centromeres that had resisted cleavage. The discovery that centromeric Rec8 is protected from separase during meiosis I by shugoshin/MEI-S332 proteins that bind PP2A phosphatase suggests that phosphorylation either of separase or cohesin may be necessary for Rec8 cleavage. We show here that multiple phosphorylation sites within Rec8 as well as two different kinases, casein kinase 1delta/epsilon (CK1delta/epsilon) and Dbf4-dependent Cdc7 kinase (DDK), are required for Rec8 cleavage and meiosis I nuclear division. Rec8 with phosphomimetic mutations is no longer protected from separase at centromeres and is cleaved even when the two kinases are inhibited. Our data suggest that PP2A protects centromeric cohesion by opposing CK1delta/epsilon- and DDK-dependent phosphorylation of Rec8.
Asunto(s)
Caseína Cinasa 1 épsilon/metabolismo , Quinasa Idelta de la Caseína/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Endopeptidasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sitios de Unión , Caseína Cinasa 1 épsilon/genética , Quinasa de la Caseína I/genética , Quinasa de la Caseína I/metabolismo , Quinasa Idelta de la Caseína/genética , Proteínas de Ciclo Celular/genética , Centrómero/metabolismo , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Endopeptidasas/genética , Genes Fúngicos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Meiosis/genética , Meiosis/fisiología , Modelos Biológicos , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilación , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Separasa , CohesinasRESUMEN
Meiosis differs from mitosis in that DNA replication is followed by the segregation of homologous chromosomes but not sister chromatids. This depends on the formation of interhomolog connections through crossover recombination and on the attachment of sister kinetochores to microtubules emanating from the same spindle pole. We show that in yeast, the Dbf4-dependent Cdc7 kinase (DDK) provides a link between premeiotic S phase, recombination, and monopolar attachment. Independently from its established role in initiating DNA replication, DDK promotes double-strand break formation, the first step of recombination, and the recruitment of the monopolin complex to kinetochores, which is essential for monopolar attachment. DDK regulates monopolin localization together with the polo-kinase Cdc5 bound to Spo13, probably through phosphorylation of the monopolin subunit Lrs4. Thus, activation of DDK both initiates DNA replication and commits meiotic cells to reductional chromosome segregation in the first division of meiosis.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiología , Meiosis , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Ciclo Celular , Cromosomas/ultraestructura , Replicación del ADN , Eliminación de Gen , Cinetocoros/metabolismo , Cinetocoros/ultraestructura , Microtúbulos/metabolismo , Modelos Biológicos , Modelos Genéticos , Proteínas Nucleares/metabolismo , Proteínas Quinasas/metabolismo , Saccharomyces cerevisiaeRESUMEN
Hepatoma-derived growth factor is a nuclear targeted mitogen containing a PWWP domain that mediates binding to DNA. To date, almost nothing is known about the molecular mechanisms of the functions of hepatoma-derived growth factor, its routes of secretion and internalization or post-translational modifications. In the present study, we show for the first time that hepatoma-derived growth factor is modified by the covalent attachment of small ubiquitin-related modifier 1 (SUMO-1), a post-translational modification with regulatory functions for an increasing number of proteins. Using a basal SUMOylation system in Escherichia coli followed by a MALDI-TOF-MS based peptide analysis, we identified the lysine residue SUMOylated located in the N-terminal part of the protein adjacent to the PWWP domain. Surprisingly, this lysine residue is not part of the consensus motif described for SUMOylation. With a series of hepatoma-derived growth factor mutants, we then confirmed that this unusual location is also used in mammalian cells and that SUMOylation of hepatoma-derived growth factor takes place in the nucleus. Finally, we demonstrate that SUMOylated hepatoma-derived growth factor is not binding to chromatin, in contrast to its unSUMOylated form. These observations potentially provide new perspectives for a better understanding of the functions of hepatoma-derived growth factor.