RESUMO
Apoptosis plays a crucial role in development and tissue homeostasis. Some key survival pathways, such as DNA damage checkpoints and DNA repair, have been described to be inactivated during apoptosis. Here, we describe the processing of the human checkpoint protein Claspin during apoptosis. We observed cleavage of Claspin into multiple fragments in vivo. In vitro cleavage with caspases 3 and 7 of various fragments of the protein, revealed cut sites near the N- and C-termini of the protein. Using mass spectrometry, we identified a novel caspase cleavage site in Claspin at Asp25. Importantly, in addition to cleavage by caspases, we observed a proteasome-dependent degradation of Claspin under apoptotic conditions, resulting in a reduction of the levels of both full-length Claspin and its cleavage products. This degradation was not dependent upon the DSGxxS phosphodegron motif required for SCF(beta-TrCP)-mediated ubiquitination of Claspin. Finally, downregulation of Claspin protein levels by short interfering RNA resulted in an increase in apoptotic induction both in the presence and absence of DNA damage. We conclude that Claspin has antiapoptotic activity and is degraded by two different pathways during apoptosis. The resulting disappearance of Claspin from the cells further promotes apoptosis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose/fisiologia , Caspases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Motivos de Aminoácidos , Proteínas Mutadas de Ataxia Telangiectasia , Sequência de Bases , Sítios de Ligação , Caspase 3/metabolismo , Caspase 7/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quinase 1 do Ponto de Checagem , Dano ao DNA , Células HL-60 , Células HeLa , Humanos , Técnicas In Vitro , Modelos Biológicos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Polo-like kinases play multiple roles in different phases of mitosis. We have recently shown that the mammalian polo-like kinase, Plk1, is inhibited in response to DNA damage and that this inhibition may lead to cell cycle arrests at multiple points in mitosis. Here we have investigated the role of the checkpoint kinases ATM (ataxia telangiectasia mutated) and ATR (ATM- and Rad3-related) in DNA damage-induced inhibition of Plk1. We show that inhibition of Plk1 kinase activity is efficiently blocked by the radio-sensitizing agent caffeine. Using ATM(-/-) cells we show that under certain circumstances, inhibition of Plk1 by DNA-damaging agents critically depends on ATM. In addition, we show that UV radiation also causes inhibition of Plk1, and we present evidence that this inhibition is mediated by ATR. Taken together, our data demonstrate that ATM and ATR can regulate Plk1 kinase activity in response to a variety of DNA-damaging agents.
Assuntos
Proteínas de Ciclo Celular , Dano ao DNA , Inibidores de Proteínas Quinases , Proteínas Quinases , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ligação a DNA , Humanos , Proteínas Proto-Oncogênicas , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor , Quinase 1 Polo-LikeRESUMO
Tight regulation of cell cycle progression is essential for the maintenance of genomic integrity. The orderly progression from one cell cycle phase to the other is mediated by timed activation of distinct cyclin/cdk complexes. For example, onset of mitosis is regulated by the activation of cyclin B/cdc2 and this event is controlled by several cell cycle checkpoints. Such checkpoints ensure that chromosome segregation does not occur in the case of unreplicated or damaged DNA, or misaligned chromosomes. Recently, new insights into the targets of the DNA damage checkpoint help to unravel more of the complex mechanisms of cell cycle checkpoints. This review focuses on the factors controlling the transition from G(2) phase to mitosis. Also, the pathways contributing to the DNA damage checkpoints in these phases of the cell cycle will be discussed.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiologia , Dano ao DNA , Fase G2 , Mitose , Animais , Ciclina B/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Modelos Químicos , Fosfatases cdc25/metabolismoRESUMO
Polo-like kinases (PLKs) have an important role in several stages of mitosis. They contribute to the activation of cyclin B/Cdc2 and are involved in centrosome maturation and bipolar spindle formation at the onset of mitosis. PLKs also control mitotic exit by regulating the anaphase-promoting complex (APC) and have been implicated in the temporal and spatial coordination of cytokinesis. Experiments in budding yeast have shown that the PLK Cdc5 may be controlled by the DNA damage checkpoint. Here we report the effects of DNA damage on Polo-like kinase-1 (Plk1) in a variety of human cell lines. We show that Plk1 is inhibited by DNA damage in G2 and in mitosis. In line with this, we show that DNA damage blocks mitotic exit. DNA damage does not inhibit the kinase activity of Plk1 mutants in which the conserved threonine residue in the T-loop has been changed to aspartic acid, suggesting that DNA damage interferes with the activation of Plk1. Significantly, expression of these mutants can override the G2 arrest induced by DNA damage. On the basis of these data we propose that Plk1 is an important target of the DNA damage checkpoint, enabling cell-cycle arrests at multiple points in G2 and mitosis.
Assuntos
Dano ao DNA , Proteínas Quinases/metabolismo , Transdução de Sinais , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclina B/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Humanos , Cinetina , Mitose , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas , Purinas/farmacologia , Células Tumorais Cultivadas , Fosfatases cdc25/metabolismo , Quinase 1 Polo-LikeRESUMO
The cyclin-dependent kinase inhibitor p21 is required for a sustained G(2) arrest after activation of the DNA damage checkpoint. Here we have addressed the mechanism by which p21 can contribute to this arrest in G(2). We show that p21 blocks the activating phosphorylation of Cdc2 on Thr(161). p21 does not interfere with the dephosphorylation of two inhibitory phosphorylation sites on Cdc2, Thr(14) and Tyr(15), indicating that p21 targets a different event in Cdc2 activation as the well described DNA damage checkpoint pathway involving Chk1 and Cdc25C. Taken together our data show that a cell is equipped with at least two independent pathways to ensure efficient inhibition of Cdc2 activity in response to DNA damage, influencing both positive and negative regulatory phosphorylation events on Cdc2.
Assuntos
Proteína Quinase CDC2/metabolismo , Ciclina B/metabolismo , Ciclinas/metabolismo , Dano ao DNA , Fase G2 , Compartimento Celular , Núcleo Celular/metabolismo , Inibidor de Quinase Dependente de Ciclina p21 , Quinases Ciclina-Dependentes/antagonistas & inibidores , Modelos Biológicos , Fosforilação , Ligação Proteica , Treonina/metabolismoRESUMO
Basic fibroblast growth factor (bFGF) has been shown to induce growth inhibition of the neuroepithelioma cell line SK-N-MC. Here we show that this growth inhibition occurs in G(2). We show that bFGF is active on these cells during S and early G(2) phase. Therefore, this constitutes a rather unusual mechanism of growth inhibition, because it is generally believed that cells become refractory to extracellular signals after passage through the restriction point. We show that bFGF treatment inhibits Tyr-15 dephosphorylation of cdc2 and prevents activation of Cdc25C, similar to what is seen upon activation of the G(2) DNA damage checkpoint. Interestingly, both DNA damage- and bFGF-induced effects on cdc2 phosphorylation are reverted by caffeine. To confirm the involvement of similar pathways induced by bFGF and DNA damage, we generated tetracycline-regulatable SK-N-MC clones expressing Cdc25C-S216A. Expression of this Cdc25C mutant can revert the bFGF-induced effects on cdc2 phosphorylation and can rescue cells from the block in G(2) imposed by bFGF. Taken together, these data define a growth factor-sensitive point in G(2) that most likely involves regulation of Cdc25C phosphorylation.
Assuntos
Divisão Celular/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fase G2/efeitos dos fármacos , Cafeína/farmacologia , Ciclina B/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Humanos , Fosforilação , Células Tumorais Cultivadas , Tirosina/metabolismoRESUMO
Lithium can interfere with embryonal development in a variety of organisms. We investigated the effect of lithium on the proliferation of early embryonal cells. [3H]Thymidine incorporation of non-committed mouse P19 embryonal carcinoma cells was inhibited by lithium treatment. Similar effects were seen in a variety of other cells. This growth inhibition occurred in the G2 phase, since cells accumulated with a 4N DNA content, but the appearance of mitotic cells was blocked. Lithium could also prevent the activation of cdc2, thereby inhibiting cyclin B/cdc2 kinase activity. These data indicate that lithium might disturb embryonal development through interference in embryonal cell cycle regulation.
Assuntos
Proteína Quinase CDC2/metabolismo , Ativação Enzimática/efeitos dos fármacos , Inibidores do Crescimento/farmacologia , Lítio/farmacologia , Animais , Ciclo Celular/fisiologia , Linhagem Celular , Ciclina B/metabolismo , Relação Dose-Resposta a Droga , Histonas/efeitos dos fármacos , Camundongos , Nocodazol/farmacologia , Fatores de TempoRESUMO
p21waf1 has been shown to mediate the p53-dependent growth arrest induced by DNA-damaging agents. Several functions have been ascribed to p21waf1 that could be involved in this growth arrest. For one, p21waf1 is an efficient inhibitor of cyclin-dependent kinases (CDKs). Also, p21waf1 can interact with proliferating cell nuclear antigen (PCNA), and as such inhibit in vitro DNA-replication. Finally, p21waf1 has been reported to inhibit stress-activated protein kinases (SAPKs). In order to study these multiple functions of p21waf1 we have established U2OS-derived cell lines, in which the expression of p21waf1 can be regulated by the concentration of tetracycline in the culture medium. We observed a virtually complete, but reversible inhibition of cell growth upon induction of p21waf1-expression. Both [3H]thymidine-incorporation and CDK2-activity were strongly inhibited by p21waf1. Upon induction of p21waf1 cells accumulated with a 2N or 4N DNA content suggesting events in G1 and G2 can be inhibited by p21waf1. Indeed, kinase activity associated with cyclin B was reduced dramatically upon induction of p21waf1, although cyclin B continues to be expressed. In contrast, p21waf1 does not seem to inhibit the function of PCNA in ongoing DNA replication, since cells expressing high levels of p21waf1 apparently progressed normally through S-phase. Also, the activity of SAPKs was not substantially affected by the high levels of p21waf1. We conclude that, at least in these U2OS-derived cells, p21waf1 functions as an inhibitor of CDK-activity in G1 and G2, but not as an inhibitor of PCNA or SAPKs.