RESUMEN
Distal appendages (DAs) of the mother centriole are essential for the initial steps of ciliogenesis in G1/G0 phase of the cell cycle. DAs are released from centrosomes in mitosis by an undefined mechanism. Here, we show that specific DAs lose their centrosomal localization at the G2/M transition in a manner that relies upon Nek2 kinase activity to ensure low DA levels at mitotic centrosomes. Overexpression of active Nek2A, but not kinase-dead Nek2A, prematurely displaced DAs from the interphase centrosomes of immortalized retina pigment epithelial (RPE1) cells. This dramatic impact was also observed in mammary epithelial cells with constitutively high levels of Nek2. Conversely, Nek2 knockout led to incomplete dissociation of DAs and cilia in mitosis. As a consequence, we observed the presence of a cilia remnant that promoted the asymmetric inheritance of ciliary signaling components and supported cilium reassembly after cell division. Together, our data establish Nek2 as an important kinase that regulates DAs before mitosis.
Asunto(s)
Centriolos/enzimología , Cilios/enzimología , Células Epiteliales/enzimología , Mitosis , Quinasas Relacionadas con NIMA/metabolismo , Epitelio Pigmentado de la Retina/enzimología , Animales , Sitios de Unión , Línea Celular , Centriolos/genética , Cilios/genética , Femenino , Puntos de Control de la Fase G2 del Ciclo Celular , Células Madre Hematopoyéticas/enzimología , Humanos , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/enzimología , Ratones , Proteínas de Microtúbulos/genética , Proteínas de Microtúbulos/metabolismo , Quinasas Relacionadas con NIMA/genética , Unión Proteica , Epitelio Pigmentado de la Retina/citología , Transducción de Señal , Factores de TiempoRESUMEN
Polo-like kinase 4 (Plk4) initiates an early step in centriole assembly by phosphorylating Ana2/STIL, a structural component of the procentriole. Here, we show that Plk4 binding to the central coiled-coil (CC) of Ana2 is a conserved event involving Polo-box 3 and a previously unidentified putative CC located adjacent to the kinase domain. Ana2 is then phosphorylated along its length. Previous studies showed that Plk4 phosphorylates the C-terminal STil/ANa2 (STAN) domain of Ana2/STIL, triggering binding and recruitment of the cartwheel protein Sas6 to the procentriole assembly site. However, the physiological relevance of N-terminal phosphorylation was unknown. We found that Plk4 first phosphorylates the extreme N terminus of Ana2, which is critical for subsequent STAN domain modification. Phosphorylation of the central region then breaks the Plk4-Ana2 interaction. This phosphorylation pattern is important for centriole assembly and integrity because replacement of endogenous Ana2 with phospho-Ana2 mutants disrupts distinct steps in Ana2 function and inhibits centriole duplication.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Centriolos/enzimología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Línea Celular , Centriolos/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/genética , Transporte de Proteínas , Transducción de SeñalRESUMEN
Centrioles are highly structured organelles whose size is remarkably consistent within any given cell type. New centrioles are born when Polo-like kinase 4 (Plk4) recruits Ana2/STIL and Sas-6 to the side of an existing "mother" centriole. These two proteins then assemble into a cartwheel, which grows outwards to form the structural core of a new daughter. Here, we show that in early Drosophila melanogaster embryos, daughter centrioles grow at a linear rate during early S-phase and abruptly stop growing when they reach their correct size in mid- to late S-phase. Unexpectedly, the cartwheel grows from its proximal end, and Plk4 determines both the rate and period of centriole growth: the more active the centriolar Plk4, the faster centrioles grow, but the faster centriolar Plk4 is inactivated and growth ceases. Thus, Plk4 functions as a homeostatic clock, establishing an inverse relationship between growth rate and period to ensure that daughter centrioles grow to the correct size.
Asunto(s)
Centriolos/enzimología , Péptidos y Proteínas de Señalización del Ritmo Circadiano/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Fase S , Animales , Conducta Animal , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Centriolos/genética , Péptidos y Proteínas de Señalización del Ritmo Circadiano/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Embrión no Mamífero/enzimología , Homeostasis , Locomoción , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Transporte de Proteínas , Transducción de Señal , Factores de TiempoRESUMEN
Establishing the bipolar spindle in mammalian oocytes after their prolonged arrest is crucial for meiotic fidelity and subsequent development. In contrast to somatic cells, the first meiotic spindle assembles in the absence of centriole-containing centrosomes. Ran-GTP can promote microtubule nucleation near chromatin, but additional unidentified factors are postulated for the activity of multiple acentriolar microtubule organizing centers in the oocyte. We now demonstrate that partially overlapping, nonredundant functions of Aurora A and Plk4 kinases contribute to initiate acentriolar meiosis I spindle formation. Loss of microtubule nucleation after simultaneous chemical inhibition of both kinases can be significantly rescued by drug-resistant Aurora A alone. Drug-resistant Plk4 can enhance Aurora A-mediated rescue, and, accordingly, Plk4 can phosphorylate and potentiate the activity of Aurora A in vitro. Both kinases function distinctly from Ran, which amplifies microtubule growth. We conclude that Aurora A and Plk4 are rate-limiting factors contributing to microtubule growth as the acentriolar oocyte resumes meiosis.
Asunto(s)
Aurora Quinasa A/metabolismo , Centriolos/enzimología , Meiosis , Microtúbulos/enzimología , Oocitos/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Aurora Quinasa A/antagonistas & inhibidores , Aurora Quinasa A/genética , Células Cultivadas , Centriolos/efectos de los fármacos , Técnicas de Cultivo de Embriones , Femenino , Cinética , Meiosis/efectos de los fármacos , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Microtúbulos/efectos de los fármacos , Oocitos/efectos de los fármacos , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal , Proteína de Unión al GTP ran/metabolismoRESUMEN
Primary cilia protrude from the surface of quiescent cells and disassemble at cell cycle reentry. We previously showed that ciliary reassembly is suppressed by trichoplein-mediated Aurora A activation pathway in growing cells. Here, we report that Ndel1, a well-known modulator of dynein activity, localizes at the subdistal appendage of the mother centriole, which nucleates a primary cilium. In the presence of serum, Ndel1 depletion reduces trichoplein at the mother centriole and induces unscheduled primary cilia formation, which is reverted by forced trichoplein expression or coknockdown of KCTD17 (an E3 ligase component protein for trichoplein). Serum starvation induced transient Ndel1 degradation, subsequent to the disappearance of trichoplein at the mother centriole. Forced expression of Ndel1 suppressed trichoplein degradation and axonemal microtubule extension during ciliogenesis, similar to trichoplein induction or KCTD17 knockdown. Most importantly, the proportion of ciliated and quiescent cells was increased in the kidney tubular epithelia of newborn Ndel1-hypomorphic mice. Thus, Ndel1 acts as a novel upstream regulator of the trichoplein-Aurora A pathway to inhibit primary cilia assembly.
Asunto(s)
Aurora Quinasa A/metabolismo , Proteínas Portadoras/metabolismo , Proliferación Celular , Células Epiteliales/enzimología , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Animales Recién Nacidos , Aurora Quinasa A/genética , Proteínas Portadoras/genética , Puntos de Control del Ciclo Celular , Centriolos/enzimología , Cilios/enzimología , Genotipo , Células HeLa , Humanos , Túbulos Renales/citología , Túbulos Renales/enzimología , Ratones , Ratones Noqueados , Microtúbulos/enzimología , Fenotipo , Estabilidad Proteica , Proteolisis , Interferencia de ARN , Células 3T3 Swiss , Factores de Tiempo , TransfecciónRESUMEN
Centriolar satellites (CS) are small granular structures that cluster in the vicinity of centrosomes. CS are highly susceptible to stress stimuli, triggering abrupt displacement of key CS factors. Here we discover a linear p38-MK2-14-3-3 signalling pathway that specifically targets CEP131 to trigger CS remodelling after cell stress. We identify CEP131 as a substrate of the p38 effector kinase MK2 and pinpoint S47 and S78 as critical MK2 phosphorylation sites in CEP131. Ultraviolet-induced phosphorylation of these residues generates direct binding sites for 14-3-3 proteins, which sequester CEP131 in the cytoplasm to block formation of new CS, thereby leading to rapid depletion of these structures. Mutating S47 and S78 in CEP131 is sufficient to abolish stress-induced CS reorganization, demonstrating that CEP131 is the key regulatory target of MK2 and 14-3-3 in these structures. Our findings reveal the molecular mechanism underlying dynamic CS remodelling to modulate centrosome functions on cell stress.
Asunto(s)
Proteínas 14-3-3/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centriolos/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas 14-3-3/genética , Proteínas de Ciclo Celular/genética , Centriolos/enzimología , Centriolos/genética , Proteínas del Citoesqueleto , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de Microtúbulos/genética , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
Centrioles are duplicated and segregated in close link to the cell cycle. During mitosis, daughter centrioles are disengaged and eventually separated from mother centrioles. New daughter centrioles may be generated only after centriole separation. Therefore, centriole separation is considered a licensing step for centriole duplication. It was previously known that separase specifically cleaves pericentrin (PCNT) during mitotic exit. Here we report that PCNT has to be phosphorylated by PLK1 to be a suitable substrate of separase. Phospho-resistant mutants of PCNT are not cleaved by separase and eventually inhibit centriole separation. Furthermore, phospho-mimetic PCNT mutants rescue centriole separation even in the presence of a PLK1 inhibitor. On the basis on these results, we propose that PLK1 phosphorylation is a priming step for separase-mediated cleavage of PCNT and eventually for centriole separation. PLK1 phosphorylation of PCNT provides an additional layer of regulatory mechanism to ensure the fidelity of centriole separation during mitotic exit.
Asunto(s)
Antígenos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centriolos/metabolismo , Mitosis , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Antígenos/genética , Proteínas de Ciclo Celular/genética , Centriolos/enzimología , Centriolos/genética , Células HeLa , Humanos , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Separasa/metabolismo , Quinasa Tipo Polo 1RESUMEN
Primary cilia are microtubule-based sensory organelles that organize numerous key signals during developments and tissue homeostasis. Ciliary microtubule doublet, named axoneme, is grown directly from the distal end of mother centrioles through a multistep process upon cell cycle exit; however, the instructive signals that initiate these events are poorly understood. Here we show that ubiquitin-proteasome machinery removes trichoplein, a negative regulator of ciliogenesis, from mother centrioles and thereby causes Aurora-A inactivation, leading to ciliogenesis. Ciliogenesis is blocked if centriolar trichoplein is stabilized by treatment with proteasome inhibitors or by expression of non-ubiquitylatable trichoplein mutant (K50/57R). Started from two-stepped global E3 screening, we have identified KCTD17 as a substrate-adaptor for Cul3-RING E3 ligases (CRL3s) that polyubiquitylates trichoplein. Depletion of KCTD17 specifically arrests ciliogenesis at the initial step of axoneme extension through aberrant trichoplein-Aurora-A activity. Thus, CRL3-KCTD17 targets trichoplein to proteolysis to initiate the axoneme extension during ciliogenesis.
Asunto(s)
Axonema/enzimología , Axonema/metabolismo , Centriolos/metabolismo , Cilios/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Centriolos/enzimología , Cilios/enzimología , Cilios/genética , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Humanos , Complejo de la Endopetidasa Proteasomal/genéticaRESUMEN
Newly formed centrioles in cycling cells undergo a maturation process that is almost two cell cycles long before they become competent to function as microtubule-organizing centers and basal bodies. As a result, each cell contains three generations of centrioles, only one of which is able to form cilia. It is not known how this long and complex process is regulated. We show that controlled Plk1 activity is required for gradual biochemical and structural maturation of the centrioles and timely appendage assembly. Inhibition of Plk1 impeded accumulation of appendage proteins and appendage formation. Unscheduled Plk1 activity, either in cycling or interphase-arrested cells, accelerated centriole maturation and appendage and cilia formation on the nascent centrioles, erasing the age difference between centrioles in one cell. These findings provide a new understanding of how the centriole cycle is regulated and how proper cilia and centrosome numbers are maintained in the cells.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Centriolos/enzimología , Mitosis , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Ciclo Celular , Centriolos/fisiología , Centriolos/ultraestructura , Células HeLa , Humanos , Proteínas de Microtúbulos/metabolismo , Transporte de Proteínas , Quinasa Tipo Polo 1RESUMEN
Centriole biogenesis depends on the polo-like kinase (PLK4) and a small group of structural proteins. The spatiotemporal regulation of these proteins at pre-existing centrioles is essential to ensure that centriole duplication occurs once per cell cycle. Here, we report that phosphatidylinositol 4-phosphate 5-kinase type-1 gamma (PIP5K1C, hereafter referred to as PIPKIγ) plays an important role in centriole fidelity. PIPKIγ localized in a ring-like pattern in the intermediate pericentriolar materials around the proximal end of the centriole in G1, S and G2 phases, but not in M phase. This localization was dependent upon an association with centrosomal protein of 152 KDa (CEP152). Without detaining cells in S or M phase, the depletion of PIPKIγ led to centriole amplification in a manner that was dependent upon PLK4 and spindle assembly abnormal protein 6 homolog (SAS6). The expression of exogenous PIPKIγ reduced centriole amplification that occurred as a result of endogenous PIPKIγ depletion, hydroxyurea treatment or PLK4 overexpression, suggesting that PIPKIγ is likely to function at the PLK4 level to restrain centriole duplication. Importantly, we found that PIPKIγ bound to the cryptic polo-box domain of PLK4 and that this binding reduced the kinase activity of PLK4. Together, our findings suggest that PIPKIγ is a novel negative regulator of centriole duplication, which acts by modulating the homeostasis of PLK4 activity.
Asunto(s)
Centriolos/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Ubiquitinación , Animales , Proteínas de Unión al Calcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células HEK293 , Células HeLa , Humanos , Ratones , Células 3T3 NIH , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Dominios y Motivos de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas , ProteolisisRESUMEN
The centrosomes in dividing cells follow a series of cyclical events of duplication and separation, which are tightly linked to the cell cycle. Serine/threonine-protein kinase NEK7 (NEK7) is a centrosomal kinase that is required for proper spindle formation during mitosis. In this study, we observed that centriole duplication was inhibited in NEK7-depleted cells. Ectopic expression of centrosome-directed NEK7 led to the formation of extra centrioles in a kinase-activity-dependent manner. We also observed extra centriole formation in centrosome-directed NEK6-expressing cells, suggesting that NEK6 and NEK7 might share biological activities that induce centriole duplication. The centrosomal pericentriolar material (PCM) proteins were significantly reduced in NEK7-depleted cells. The PCM proteins in NEK7-depleted cells did not accumulate at the centrosomes, even if the cells exited mitosis and progressed to the G2 phase. These results revealed that NEK7 is essential for PCM accumulation in a cell cycle stage-specific manner. Furthermore, HeLa cells depleted of NEK7 during S phase retained a higher quantity of PCM proteins and exhibited a less severe mitotic phenotype. On the basis of these results, we propose that NEK7 is involved in the recruitment of PCM proteins, which are necessary for both centriole duplication and spindle pole formation. Our study revealed that NEK7 activity is required for centrosome cycle progression not only at M phase, but also at G1 phase.
Asunto(s)
Autoantígenos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centriolos/enzimología , Centrosoma/enzimología , Interfase , Proteínas Serina-Treonina Quinasas/metabolismo , Autoantígenos/genética , Proteínas de Ciclo Celular/genética , Línea Celular , Centriolos/genética , Centriolos/metabolismo , Centrosoma/metabolismo , Humanos , Mitosis , Quinasas Relacionadas con NIMA , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
In many organisms, homolog pairing and synapsis at meiotic prophase depend on interactions between chromosomes and the nuclear membrane. Male Drosophila lack synapsis, but nonetheless, their chromosomes closely associate with the nuclear periphery at prophase I. To explore the functional significance of this association, we characterize mutations in nuclear blebber (nbl), a gene required for both spermatocyte nuclear shape and meiotic chromosome transmission. We demonstrate that nbl corresponds to dtopors, the Drosophila homolog of the mammalian dual ubiquitin/small ubiquitin-related modifier (SUMO) ligase Topors. We show that mutations in dtopors cause abnormalities in lamin localizations, centriole separation, and prophase I chromatin condensation and also cause anaphase I bridges that likely result from unresolved homolog connections. Bridge formation does not require mod(mdg4) in meiosis, suggesting that bridges do not result from misregulation of the male homolog conjunction complex. At the ultrastructural level, we observe disruption of nuclear shape, an uneven perinuclear space, and excess membranous structures. We show that dTopors localizes to the nuclear lamina at prophase, and also transiently to intranuclear foci. As a role of dtopors at gypsy insulator has been reported, we also asked whether these new alleles affected expression of the gypsy-induced mutation ct(6) and found that it was unaltered in dtopors homozygotes. Our results indicate that dTopors is required for germline nuclear structure and meiotic chromosome segregation, but in contrast, is not necessary for gypsy insulator function. We suggest that dtopors plays a structural role in spermatocyte lamina that is critical for multiple aspects of meiotic chromosome transmission.
Asunto(s)
Núcleo Celular/metabolismo , Segregación Cromosómica , Cromosomas de Insectos/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/enzimología , Meiosis/genética , Ubiquitina-Proteína Ligasas/metabolismo , Alelos , Secuencia de Aminoácidos , Anafase/genética , Animales , Núcleo Celular/enzimología , Núcleo Celular/genética , Centriolos/enzimología , Centriolos/genética , Centriolos/metabolismo , Cromatina/genética , Cromatina/metabolismo , Segregación Cromosómica/genética , Cromosomas de Insectos/enzimología , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Epistasis Genética/genética , Femenino , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Mutación , Proteínas de Unión al ARN/genética , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Centrioles are highly conserved structures that fulfil important cellular functions, such as nucleation of cilia and flagella (basal-body function) and organisation of pericentriolar material to form the centrosome. The evolution of these functions can be inferred from the distribution of the molecular components of extant centrioles and centrosomes. Here, we undertake an evolutionary analysis of 53 proteins known either for centriolar association or for involvement in cilia-associated pathologies. By linking protein distribution in 45 diverse eukaryotes with organism biology, we provide molecular evidence to show that basal-body function is ancestral, whereas the presence of the centrosome is specific to the Holozoa. We define an ancestral centriolar inventory of 14 core proteins, Polo-like-kinase, and proteins associated with Bardet-Biedl syndrome (BBS) and Meckel-Gruber syndrome. We show that the BBSome is absent from organisms that produce cilia only for motility, predicting a dominant and ancient role for this complex in sensory function. We also show that the unusual centriole of Caenorhabditis elegans is highly divergent in both protein composition and sequence. Finally, we demonstrate a correlation between the presence of specific centriolar proteins and eye evolution. This correlation is used to predict proteins with functions in the development of ciliary, but not rhabdomeric, eyes.
Asunto(s)
Centriolos/química , Centriolos/metabolismo , Evolución Molecular , Proteínas/metabolismo , Animales , Caenorhabditis elegans/metabolismo , Centriolos/enzimología , Centriolos/genética , Cilios/metabolismo , Células Eucariotas/metabolismo , Humanos , Fosfotransferasas/metabolismo , Filogenia , Tubulina (Proteína)/genéticaRESUMEN
Previously, we have identified a novel centrosomal protein centrobin that asymmetrically localizes to the daughter centriole. We found that depletion of centrobin expression inhibited the centriole duplication and impaired cytokinesis. However, the biological significance of centrobin in the cell cycle remains unknown. In the current study, we observed that silencing centrobin significantly inhibited the proliferation of lung cancer cell A549 and prevented the cells from G1 to S transition, whereas the growth rate of lung cancer cell line H1299, a p53-null cell line, was not affected. Furthermore, we demonstrated that the G1-S-phase arrest induced by centrobin knockdown in A549 cells is mediated by the upregulation of cell-cycle regulator p53, which is associated with the activation of cellular stress induced p38 pathway instead of DNA damage induced ATM pathway. Inhibition of p38 activity or downregulation of p38 expression could overcome the cell-cycle arrest caused by centrobin depletion. Taken together, our current findings demonstrated that centrobin plays an important role in the progression of cell cycle, and a tight association between the cell-cycle progression and defective centrosomes caused by depletion of centrobin.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Centriolos/enzimología , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Bromodesoxiuridina/metabolismo , Recuento de Células , Línea Celular Tumoral , Proliferación Celular , Fase G1 , Técnicas de Silenciamiento del Gen , Humanos , Antígeno Ki-67/metabolismo , Fase S , Transducción de SeñalRESUMEN
Proper progression of mitosis requires spatio-temporal regulation of protein phosphorylation by orchestrated activities of kinases and phosphatases. Although many kinases, such as Aurora kinases, polo-like kinases (Plks), and cyclin B-Cdk1 are relatively well characterized in the context of their physiological functions at mitosis and regulation of their enzymatic activities during mitotic progression, phosphatases involved are largely unknown. Here we identified a novel protein tyrosine phosphatase containing domain 1 (Ptpcd 1) as a mitotic phosphatase, which shares sequence homology to Cdc14. Immunofluorescence studies revealed that Ptpcd1 partially colocalized with gamma-tubulin, an archetypical centrosomal marker. Overexpression of this phosphatase prevented unscheduled centrosomal amplification in hydroxyurea arrested U2OS cells. Intriguingly, Ptpcd 1-associated and colocalized with polo-like kinase 1(Plk1). Hence, overexpression of Ptpcd1 rescued prometaphase arrest of Plk-1 depleted cells, but resulted in aberrant cytokinesis as did as Plk1 overexpression. These results suggested that Ptpcd1 is involved in centrosomal duplication and cytokinesis.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Centriolos/enzimología , Citocinesis , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Animales , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Clonación Molecular , Citocinesis/genética , Prueba de Complementación Genética , Células HeLa , Humanos , Ratones , Mitosis/genética , Monoéster Fosfórico Hidrolasas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Fosfatasas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Quinasa Tipo Polo 1RESUMEN
Centrosomal coiled-coil proteins paired with kinases play critical roles in centrosomal functions within somatic cells, however knowledge regarding gamete centriolar proteins is limited. In this study, the substrate of TSSK1 and 2, TSKS, was localized during spermiogenesis to the centrioles of post-meiotic spermatids, where it reached its greatest concentration during the period of flagellogenesis. This centriolar localization persisted in ejaculated human spermatozoa, while centriolar TSKS diminished in mouse sperm, where centrioles are known to undergo complete degeneration. In addition to the centriolar localization during flagellogenesis, mouse TSKS and the TSSK2 kinase localized in the tail and acrosomal regions of mouse epididymal sperm, while TSSK2 was found in the equatorial segment, neck and the midpiece of human spermatozoa. TSSK2/TSKS is the first kinase/substrate pair localized to the centrioles of spermatids and spermatozoa. Coupled with the infertility due to haploinsufficiency noted in chimeric mice with deletion of Tssk1 and 2 (companion paper) this centriolar kinase/substrate pair is predicted to play an indispensable role during spermiogenesis.
Asunto(s)
Centriolos/enzimología , Flagelos/fisiología , Proteínas Serina-Treonina Quinasas/genética , Espermátides/fisiología , Reacción Acrosómica , Animales , Centriolos/ultraestructura , Proteínas del Citoesqueleto , Flagelos/enzimología , Flagelos/ultraestructura , Masculino , Ratones , Ratones Noqueados , Fosfoproteínas , Proteínas Serina-Treonina Quinasas/deficiencia , ARN Mensajero/genética , Espermátides/citología , Espermátides/enzimología , Espermatozoides/enzimologíaRESUMEN
The mechanisms that co-ordinate centrosome maturation and the migration of human cells remain elusive. Protein phosphatase 4 (Ppp4) is a ubiquitous protein serine/threonine phosphatase in eukaryotes that is enriched at centrosomes. HEK293 cells cultures depleted to 30% Ppp4c levels by lentivirus-delivered stable gene silencing were delayed in mitosis at the prometaphase/metaphase boundary and displayed cells with aberrant chromosome organisation and microtubules unconnected to the centrosomes. The levels of alpha- and gamma-tubulin and aurora A were decreased; in mitotic cells, the cytological localisations of polo-like kinase 1, alpha- and gamma-tubulin and aurora A were aberrant and the phosphorylation of Aurora A-Thr 288 was decreased. The novel localisation of endogenous Ppp4 regulatory subunit, R3A, to centrosomes in human mitotic cells suggests that a Ppp4c-R2-R3 trimeric complex mediates centrosome maturation. We demonstrate for the first time that human cells depleted to 30% Ppp4c showed severely decreased migration and exhibit decreased levels of both total beta-actin and filamentous actin in cell extensions, filopodia and lamellopodia-like structures. Our studies show that Ppp4c is required for the organisation of the actin cytoskeleton at the leading edge of human cells during migration. We also demonstrate that the active forms of the RhoGTPases, Rac1 and Cdc42, are substantially decreased in the presence and absence of growth factor in Ppp4c depleted cells, implicating Ppp4c in the regulation of these GTPases. The results suggest that Ppp4c-R2-R3 complexes may co-ordinate centrosome maturation and cell migration via regulation of RhoGTPases and that Ppp4 may be a useful anticancer target.
Asunto(s)
Movimiento Celular , Centrosoma/enzimología , Fosfoproteínas Fosfatasas/deficiencia , Proteínas de Unión al GTP rho/metabolismo , Aurora Quinasas , Caspasas/metabolismo , Recuento de Células , Muerte Celular , Línea Celular , Forma de la Célula , Centriolos/enzimología , Humanos , Lentivirus/metabolismo , Mitosis , Modelos Biológicos , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Cuaternaria de Proteína , Transporte de Proteínas , Tubulina (Proteína)/metabolismoRESUMEN
The basic helix-loop-helix transcription factor, Id1, was shown to induce tetraploidy in telomerase-immortalized nasopharyngeal epithelial cells in this study. Using both transient and stable Id1-expressing cell models, multiple mitotic aberrations were detected, including centrosome amplification, binucleation, spindle defects, and microtubule perturbation. Many of these abnormal phenotypes have previously been reported in cells overexpressing Aurora A. Further experiments showed that Id1 could stabilize Aurora A, whereas knocking down Aurora A expression in Id1-expressing cells could rescue some of the mitotic defects. The mechanisms by which Aurora A could be modulated by Id1 were explored. DNA amplification of the Aurora A locus was not involved. Id1 could only weakly activate the transcriptional activity of the Aurora A promoter. We found that Id1 overexpression could affect Aurora A degradation, leading to its stabilization. Aurora A is normally degraded from mitosis exit by the APC/C(Cdh1)-mediated proteasomal proteolysis pathway. Our results revealed that Id1 and Cdh1 are binding partners. The association of Id1 and Cdh1 was found to be dependent on the canonical destruction box motif of Id1, the increased binding of which may compete with the interaction between Cdh1 and Aurora A, leading to stabilization of Aurora A in Id1-overexpressing cells.