RESUMO
Cyclin A2 is predominantly, but not exclusively, localized in the nucleus from G1/S transition onwards. It is degraded when cells enter mitosis after nuclear envelope breakdown. We previously showed that a fusion protein (S2A) between the hepatitis B virus (HBV) surface antigen protein and a non-degradable fragment of human cyclin A2 (Delta152) resides in the endoplasmic reticulum membranes, escapes degradation and transforms normal rat fibroblasts. The present study investigates whether cytoplasmic cyclin A2 may play a role in oncogenesis. We show that the sequestration of non-degradable cyclin A2-Delta152 by a cellular ER targeting domain (PRL-A2) leads to cell transformation when coexpressed with activated Ha-ras. REF52 cells constitutively expressing PRL-A2 are found to have a high incidence of multinucleate giant cells, polyploidy and abnormal centrosome numbers, giving rise to the nucleation of multipolar spindles. Injection of these cells into athymic nude mice causes tumors, even in the absence of a cooperating Ha-ras oncogene. These results demonstrate that, independently of any viral context, an intracellular redistribution of non-degradable cyclin A2 is capable of deregulating the normal cell cycle to the point where it promotes aneuploidy and cancer.
Assuntos
Transformação Celular Neoplásica , Centrossomo/ultraestrutura , Ciclina A/fisiologia , Retículo Endoplasmático/química , Poliploidia , Animais , Ciclo Celular , Células Cultivadas , Ciclina A/genética , Ciclina A/imunologia , Ciclina A2 , Genes ras , Imuno-Histoquímica , Camundongos , Camundongos Nus , Microscopia de Fluorescência , Neoplasias Experimentais/etiologia , Proteínas Oncogênicas/fisiologia , Ratos , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The protein kinase MELK is implicated in the control of cell proliferation, cell cycle and mRNA splicing. We previously showed that MELK activity is correlated with its phosphorylation level, is cell cycle dependent, and maximal during mitosis. Here we report on the identification of T414, T449, T451, T481 and S498 as residues phosphorylated in Xenopus MELK (xMELK) in M-phase egg extract. Phosphorylations of T449, T451, T481 are specifically detected during mitosis. Results obtained in vivo showed that MPF and MAPK pathways are involved in xMELK phosphorylation. In vitro, MPF and MAPK directly phosphorylate xMELK and MPF phosphorylates xMELK on T481. In addition, phosphorylation by MPF and MAPK enhances MELK activity in vitro. Taken together our results indicate that MELK phosphorylation by MPF and MAPK enhance its activity during M-phase.
Assuntos
Divisão Celular , Sistema de Sinalização das MAP Quinases , Fator Promotor de Maturação/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/fisiologia , Processamento Alternativo , Sequência de Aminoácidos , Animais , Linhagem Celular , Mitose , Dados de Sequência Molecular , Oócitos/metabolismo , Fosforilação , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Xenopus laevisRESUMO
The stability of DNA ends generated by the HO endonuclease in yeast is surprisingly high with a half-life of more than an hour. This transient stability is unaffected by mutations that abolish nonhomologous end joining (NHEJ). The unprocessed ends interact with Yku70p and Yku80p, two proteins required for NHEJ, but not significantly with Rad52p, a protein involved in homologous recombination (HR). Repair of a double-strand break by NHEJ is unaffected by the possibility of HR, although the use of HR is increased in NHEJ-defective cells. Partial in vitro 5' strand processing suppresses NHEJ but not HR. These results show that NHEJ precedes HR temporally, and that the availability of substrate dictates the particular pathway used. We propose that transient stability of DNA ends is a foundation for the permanent stability of telomeres.