RESUMO
The spindle checkpoint coordinates the cell biology of mitosis with cell-cycle progression. It ensures that sister-chromatid separation only takes place when all kinetochores have formed stable bipolar microtubule attachments. Here, we discuss recent advances in our understanding of what activates this checkpoint pathway, the molecular nature of the checkpoint signal and its mode of transmission, and how the checkpoint might be inactivated.
Assuntos
Microtúbulos/metabolismo , Fuso Acromático , Anáfase , Animais , Humanos , Cinética , Metáfase , Mitose , Modelos Biológicos , Transdução de Sinais , XenopusRESUMO
The transcriptional repressor CBFA2T3 is a putative breast tumor suppressor. To define the role of CBFA2T3, we used a segment of this protein as bait in a yeast two-hybrid screen and identified a novel uncharacterized protein, ZNF652. In general, primary tumors and cancer cell lines showed lower expression of ZNF652 than normal tissues. Together with the location of this gene on the long arm of chromosome 17q, a region of frequent loss of heterozygosity in cancer, these results suggest a possible role of ZNF652 in tumorigenesis. In silico analysis of this protein revealed that it contains multiple classic zinc finger domains that are predicted to bind DNA. Coimmunoprecipitation assays showed that ZNF652 strongly interacts with CBFA2T3 and this interaction occurs through the COOH-terminal 109 amino acids of ZNF652. In contrast, there was a weak interaction of ZNF652 with CBFA2T1 and CBFA2T2, the other two members of this ETO family. Transcriptional reporter assays further confirmed the strength and selectivity of the ZNF652-CBFA2T3 interaction. The transcriptional repression of growth factor independent-1 (GFI-1), a previously characterized ETO effector zinc finger protein, was shown to be enhanced by CBFA2T1, but to a lesser extent by CBFA2T2 and CBFA2T3. We therefore suggest that each of the various gene effector zinc finger proteins may specifically interact with one or more of the ETO proteins to generate a defined range of transcriptional repressor complexes.
Assuntos
Neoplasias da Mama/genética , Proteínas de Ligação a DNA/genética , Fosfoproteínas/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Dedos de Zinco/fisiologia , Sequência de Aminoácidos , Animais , Neoplasias da Mama/metabolismo , Proteínas de Ligação a DNA/metabolismo , Genes Supressores de Tumor , Humanos , Camundongos , Dados de Sequência Molecular , Fosfoproteínas/biossíntese , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteína 1 Parceira de Translocação de RUNX1 , Coelhos , Ratos , Proteínas Repressoras/biossíntese , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteínas Supressoras de Tumor/biossíntese , Proteínas Supressoras de Tumor/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Dedos de Zinco/genéticaRESUMO
The spindle checkpoint delays the metaphase-to-anaphase transition in response to spindle and kinetochore defects. Genetic screens in budding yeast identified the Mad and Bub proteins as key components of this conserved regulatory pathway. Here we present the fission yeast homologue of Mad3p. Cells devoid of mad3(+) are unable to arrest their cell cycle in the presence of microtubule defects. Mad3p coimmunoprecipitates Bub3p, Mad2p, and the spindle checkpoint effector Slp1/Cdc20p. We demonstrate that Mad3p function is required for the overexpression of Mad2p to result in a metaphase arrest. Mad1p, Bub1p, and Bub3p are not required for this arrest. Thus, Mad3p appears to have a crucial role in transducing the inhibitory "wait anaphase" signal to the anaphase-promoting complex (APC). Mad3-green fluorescent protein (GFP) is recruited to unattached kinetochores early in mitosis and accumulates there upon prolonged checkpoint activation. For the first time, we have systematically studied the dependency of Mad3/BubR1 protein recruitment to kinetochores. We find Mad3-GFP kinetochore localization to be dependent upon Bub1p, Bub3p, and the Mph1p kinase, but not upon Mad1p or Mad2p. We discuss the implications of these findings in the context of our current understanding of spindle checkpoint function.