RESUMO
The spindle assembly checkpoint (SAC) is essential for ensuring the proper attachment of kinetochores to the spindle and, thus, the precise separation of paired sister chromatids during mitosis. The SAC proteins are recruited to the unattached kinetochores for activation of the SAC in prometaphase. However, it has been less studied whether activation of the SAC also requires the proteins that do not localize to the kinetochores. Here, we show that the nuclear protein RED, also called IK, a down-regulator of human leukocyte antigen (HLA) II, interacts with the human SAC protein MAD1. Two RED-interacting regions identified in MAD1 are from amino acid residues 301-340 and 439-480, designated as MAD1(301-340) and MAD1(439-480), respectively. Our observations reveal that RED is a spindle pole-associated protein that colocalizes with MAD1 at the spindle poles in metaphase and anaphase. Depletion of RED can cause a shorter mitotic timing, a failure in the kinetochore localization of MAD1 in prometaphase, and a defect in the SAC. Furthermore, the RED-interacting peptides MAD1(301-340) and MAD1(439-480), fused to enhanced green fluorescence protein, can colocalize with RED at the spindle poles in prometaphase, and their expression can abrogate the SAC. Taken together, we conclude that RED is required for kinetochore localization of MAD1, mitotic progression, and activation of the SAC.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Citocinas/fisiologia , Cinetocoros/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular , Proteínas Nucleares/metabolismo , Pontos de Checagem do Ciclo Celular , Citocinas/genética , Citocinas/metabolismo , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Mitose , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Interferência de RNA , Proteínas Recombinantes de Fusão/metabolismo , Análise de Célula Única , Imagem com Lapso de Tempo , Técnicas do Sistema de Duplo-HíbridoRESUMO
PARK2, an ubiquitin ligase closely correlated with Parkinson's disease and cancer, has been shown to accumulate at centrosomes to ubiquitinate misfolded proteins accumulated during interphase. In the present study, we demonstrated that PARK2 can also localize to centrosomes in mitosis and that the protein does not fluctuate through the S- to M-phase. A C-terminal truncation of PARK2 resulted in a spindle assembly checkpoint defect, characterized by HeLa cells able to bypass mitotic arrest induced by nocodazole and form multinucleated cells when overexpressing the C-terminal truncated PARK2 protein. The spindle assembly checkpoint defect may be due to a change in a biochemical or structural property of PARK2 caused by the C-terminal truncation, resulting in a loss of self-interaction between PARK2 proteins.
Assuntos
Fuso Acromático , Ubiquitina-Proteína Ligases/fisiologia , Western Blotting , Dicroísmo Circular , Células HeLa , Humanos , Microscopia de Fluorescência , Solubilidade , Ubiquitina-Proteína Ligases/químicaRESUMO
Drug abuse patterns are different due to cultural, social and geographical differences. Methamphetamine (MA) is the most important drug of abuse in Taiwan followed by opiates. Recently, there has been an increase of ketamine and MDMA abuse in disco dancing clubs. Here, we report the patterns of drug abuse by the participants in a metropolitan city disco-dancing club and the general public in Taiwan. The positive rates of common drugs of abuse detected in samples collected from participants in a dancing club were as follows: MDMA, 75.7%; ketamine, 47.0%; MA, 41.6%; opiates, 0%. Marijuana and cocaine were detected at much lower rates (3.4 and 4.7%, respectively). Ketamine and one of the amphetamines were detected together in 42.9% of the samples. The positive rates in samples collected from police detainees suspected of drug abuse in the general public were as follows: MA, 76.0%; OPA, 37.0%; MDMA, 6.0%; ketamine, 2.0%.
Assuntos
Ketamina/urina , N-Metil-3,4-Metilenodioxianfetamina/urina , Transtornos Relacionados ao Uso de Substâncias/epidemiologia , Anfetaminas/sangue , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Prevalência , Transtornos Relacionados ao Uso de Substâncias/urina , Taiwan/epidemiologia , População UrbanaRESUMO
Aneuploidy is a common characteristic of human solid tumors. It has been proposed that a defect of the spindle assembly checkpoint (SAC) generates aneuploidy and might facilitate tumorigenesis. However, a direct link between the SAC proteins and tumorigenesis has not yet been elucidated. Here, we demonstrate the association of the SAC protein MAD1 with the RNA polymerase II complex and its role in gene expression. Furthermore, MAD1 binds to the E-cadherin promoter region. Knockdown of endogenous MAD1 by siRNA reduces E-cadherin expression and enhances the migration ability of non-metastatic breast cancer cells, indicating that reduced MAD1 expression is a new potential diagnostic symptom of tumor metastasis.