RESUMO
The tumor suppressor Liver Kinase B1 (LKB1) is a multifunctional serine/threonine protein kinase that regulates cell metabolism, polarity, and growth and is associated with Peutz-Jeghers Syndrome and cancer predisposition. The LKB1 gene comprises 10 exons and 9 introns. Three spliced LKB1 variants have been documented, and they reside mainly in the cytoplasm, although two possess a nuclear-localization sequence (NLS) and are able to shuttle into the nucleus. Here, we report the identification of a fourth and novel LKB1 isoform that is, interestingly, targeted to the mitochondria. We show that this mitochondria-localized LKB1 (mLKB1) is generated from alternative splicing in the 5' region of the transcript and translated from an alternative initiation codon encoded by a previously unknown exon 1b (131 bp) hidden within the long intron 1 of LKB1 gene. We found by replacing the N-terminal NLS of the canonical LKB1 isoform, the N-terminus of the alternatively spliced mLKB1 variant encodes a mitochondrial transit peptide that allows it to localize to the mitochondria. We further demonstrate that mLKB1 colocalizes histologically with mitochondria-resident ATP Synthase and NAD-dependent deacetylase sirtuin-3, mitochondrial (SIRT3) and that its expression is rapidly and transiently upregulated by oxidative stress. We conclude that this novel LKB1 isoform, mLKB1, plays a critical role in regulating mitochondrial metabolic activity and oxidative stress response.
Assuntos
Quinases Proteína-Quinases Ativadas por AMP , Mitocôndrias , Mutação , Estresse Oxidativo , Proteínas Serina-Treonina Quinases , Quinases Proteína-Quinases Ativadas por AMP/genética , Quinases Proteína-Quinases Ativadas por AMP/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Estresse Oxidativo/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Sirtuína 3/metabolismo , Sinais Direcionadores de Proteínas , Transporte Proteico , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Processamento Alternativo , Códon de IniciaçãoRESUMO
The DNA damage checkpoint halts cell cycle progression in G2 in response to genotoxic insults. Central to the execution of cell cycle arrest is the checkpoint-induced stabilization of securin-separase complex (yeast Pds1-Esp1). The checkpoint kinases Chk1 and Chk2 (yeast Chk1 and Rad53) are thought to critically contribute to the stability of securin-separase complex by phosphorylation of securin, rendering it resistant to proteolytic destruction by the anaphase promoting complex (APC). Dun1, a Rad53 paralog related to Chk2, is also essential for checkpoint-imposed arrest. Dun1 is required for the DNA damage-induced transcription of DNA repair genes; however, its role in the execution of cell cycle arrest remains unknown. Here, we show that Dun1's role in checkpoint arrest is independent of its involvement in the transcription of repair genes. Instead, Dun1 is necessary to prevent Pds1 destruction during DNA damage in that the Dun1-deficient cells degrade Pds1, escape G2 arrest and undergo mitosis despite the presence of checkpoint-active Chk1 and Rad53. Interestingly, proteolytic degradation of Pds1 in the absence of Dun1 is mediated not by APC but by the HECT domain-containing E3 ligase Rsp5. Our results suggest a regulatory scheme in which Dun1 prevents chromosome segregation during DNA damage by inhibiting Rsp5-mediated proteolytic degradation of securin Pds1.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , Dano ao DNA , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Securina/metabolismo , Separase/metabolismo , Transdução de Sinais , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular/deficiência , Segregação de Cromossomos , Reparo do DNA/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Fase G2 , Deleção de Genes , Mitose , Proteínas Serina-Treonina Quinases/deficiência , Proteólise , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Transcrição Gênica , Complexos Ubiquitina-Proteína Ligase/metabolismoRESUMO
Activating mutations within the epidermal growth factor receptor (EGFR) kinase domain give rise to several cancers including Non-Small Cell Lung Cancer (NSCLC). Small molecule inhibitors targeted at these mutants have proven to be clinically successful drugs. These molecules are ATP competitive and rapidly result in the emergence of resistance. Recently Jia et al. [Nature, 2016, 534, 129-132] reported a small molecule inhibitor (called EAI045) that binds at an allosteric pocket, does not compete with ATP and displays high potency and selectivity towards certain activating mutants (L858R, T790M, L858R/T790M) of EGFR, with IC50 values ranging from 3 nM to 49 nM. We present here a study combining extensive molecular dynamics simulations with binding assays to provide a structural basis underlying the mechanism of binding of this molecule. It appears that in mutants, conformational destabilization of the short helix (that carries Leu858 in the wildtype), is key to the exposure of the allosteric pocket which otherwise is occluded by a set of sidechains including L858. We extend this hypothesis to show that a similar mechanism would enable the molecule to inhibit EGFRL861Q which is another oncogenic mutant and validate this with binding experiments. The screening of the human structural kinome revealed at least 12 other oncogenic kinases which carry at least one activating mutant in this disorder-prone region and hence would be amenable to allosteric inhibition by molecules such as EAI045. Our study characterizes a druggable allosteric pocket which appears to be specific to certain oncogenic mutants of the EGFR and holds therapeutic potential.
RESUMO
Targeting the mitotic pathways of rapidly proliferating tumor cells has been an effective strategy in traditional cancer therapy. Chemotherapeutics such as taxanes and vinca alkaloids, which disrupt microtubule function, have enjoyed clinical success; however, the accompanying side effects, toxicity and multi drug resistance remain as serious concerns. The emerging classes of inhibitors targeting mitotic kinases and proteasome face their own set of challenges. It is hoped that elucidation of the regulatory interface between mitotic checkpoints, mitochondria and mitotic death will aid the development of more efficacious anti-mitotic agents and improved treatment protocols. The links between the spindle assembly checkpoint (SAC) and mitochondrial dynamics that control the progression of anti-mitotic agent-induced apoptosis have been under investigation for several years and the functional integration of these various signaling networks is now beginning to emerge. In this review, we highlight current research on the regulation of SAC, the death pathway and mitochondria with particular focus on their regulatory interconnections.
RESUMO
Maintaining genome integrity requires the accurate and complete replication of chromosomal DNA. This is of the utmost importance for embryonic stem cells (ESCs), which differentiate into cells of all lineages, including germ cells. However, endogenous and exogenous factors frequently induce stalling of replication forks in every cell cycle, which can trigger mutations and chromosomal instabilities. We show here that the oncofetal, nonhistone chromatin factor HMGA2 equips cells with a highly effective first-line defense mechanism against endonucleolytic collapse of stalled forks. This fork-stabilizing function most likely employs scaffold formation at branched DNA via multiple DNA-binding domains. Moreover, HMGA2 works independently of other human factors in two heterologous cell systems to prevent DNA strand breaks. This fork chaperone function seemingly evolved to preserve ESC genome integrity. It is hijacked by tumor (stem) cells to also guard their genomes against DNA-damaging agents widely used to treat cancer patients.
Assuntos
Replicação do DNA , Células-Tronco Embrionárias/metabolismo , Proteína HMGA2/metabolismo , Neoplasias/metabolismo , Animais , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Instabilidade Genômica , Proteína HMGA2/genética , Humanos , Camundongos , Neoplasias/genéticaRESUMO
DNA damage checkpoint prevents segregation of damaged chromosomes by imposing cell-cycle arrest. In budding yeast, Mec1, Chk1, and Rad53 (homologous to human ATM/ATR, Chk1, and Chk2 kinases, respectively) are among the main effectors of this pathway. The DNA damage checkpoint is thought to inhibit chromosome segregation by preventing separase-mediated cleavage of cohesins. Here, we describe a regulatory network that prevents segregation of damaged chromosomes by restricting spindle elongation and acts in parallel with inhibition of cohesin cleavage. This control circuit involves Rad53, polo kinase, the anaphase-promoting complex activator Cdh1, and the bimC kinesin family proteins Cin8 and Kip1. The inhibition of polo kinase by Rad53-dependent phosphorylation prevents it from inactivating Cdh1. As a result, Cdh1 remains in a partially active state and limits Cin8 and Kip1 accumulation, thereby restraining spindle elongation. Hence, the DNA damage checkpoint suppresses both cohesin cleavage and spindle elongation to preserve chromosome stability.
Assuntos
Anáfase/fisiologia , Dano ao DNA , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fuso Acromático , Western Blotting , Proteínas Cdh1 , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2 , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos , Imunoprecipitação , Cinesinas , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Motores Moleculares/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , CoesinasRESUMO
Separation of duplicated centrosomes (spindle-pole bodies or SPBs in yeast) is a crucial step in the biogenesis of the mitotic spindle. In vertebrates, centrosome separation requires the BimC family kinesin Eg5 and the activities of Cdk1 and polo kinase; however, the roles of these kinases are not fully understood. In Saccharomyces cerevisiae, SPB separation also requires activated Cdk1 and the plus-end kinesins Cin8 (homologous to vertebrate Eg5) and Kip1. Here we report that polo kinase has a role in the separation of SPBs. We show that adequate accumulation of Cin8 and Kip1 requires inactivation of the anaphase-promoting complex-activator Cdh1 through sequential phosphorylation by Cdk1 and polo kinase. In this process, Cdk1 functions as a priming kinase in that Cdk1-mediated phosphorylation creates a binding site for polo kinase,which further phosphorylates Cdh1. Thus, Cdh1 inactivation through the synergistic action of Cdk1 and polo kinase provides a new model for inactivation of cell-cycle effectors.
Assuntos
Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fuso Acromático , Ciclossomo-Complexo Promotor de Anáfase , Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Proteínas Cdh1 , Ciclo Celular , Quinase 5 Dependente de Ciclina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Biológicos , Modelos Genéticos , Proteínas Motores Moleculares , Fosforilação , Proteínas Repressoras/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Quinase 1 Polo-LikeRESUMO
DNA replication checkpoint is activated in response to replication stresses. It maintains the integrity of stalled replication forks and prevents premature segregation of largely unreplicated chromosomes. In budding yeast, Mec1 and Rad53 kinases (homologous to mammalian ATM/ATR and Chk2 kinases, respectively) are the main effectors of this checkpoint control. Using a yeast based screen, we have identified a compound (named here ENA) which inhibits DNA replication and activates Mec1/Rad53 checkpoint. A brief exposure to this compound stops fork progression at or near replication origin and renders the forks incompetent to resume replication despite the presence of a functional checkpoint. ENA also inhibits DNA synthesis in mammalian cells leading to the activation of ATM/ATR pathway and the induction of apoptosis in a p53 independent manner. Interestingly, ENA acts as an effective anti-proliferative agent against a subset of cancer cell lines and as an anti-tumor agent against human xenografts in mice. Thus, ENA is a potent cell cycle inhibitor with conceivable therapeutic potential.
Assuntos
Antineoplásicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Compostos de Epóxi/farmacologia , Naftalimidas/farmacologia , Fase S/efeitos dos fármacos , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Avaliação Pré-Clínica de Medicamentos , Genes cdc/efeitos dos fármacos , Células HCT116 , Células HeLa , Humanos , Camundongos , Camundongos Nus , Modelos Biológicos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The cohesin complex prevents premature segregation of duplicated chromosomes by providing resistance to the pole-ward pull by spindle microtubules. The centromeric region (or sister kinetochores) bears the majority of this force and undergoes transient separation prior to anaphase, indicative of its elastic nature. A cysteine protease, separase, cleaves the cohesin subunit Scc1 and dissolves cohesion between sister chromatids, initiating their separation. Separase also cleaves the kinetochore protein Slk19 during anaphase. Slk19 has been implicated in stabilization of the mitotic spindle and regulation of mitotic exit, but it is not known what role it plays at the kinetochores. We show that during pre-anaphase arrest, the spindle in slk19Delta cells is excessively dynamic and the nuclei move into mother-daughter junction prematurely. As a result, the chromatin mass undergoes partial division that requires neither anaphase promoting complex (APC) activity nor Scc1 cleavage. Partial division of the chromatin mass is accompanied by the loss of the centromeric region's ability to resist pole-ward pull by the spindle. Slk19 physically associates with Scc1 and this association appears necessary for efficient cleavage of Slk19 by separase. Our results suggest that Slk19 participates in regulating nuclear migration and, in conjunction with cohesin complex, may be involved in the maintenance of centromeric tensile strength to resist the pole-ward pull.