Cellular functions of MNB/DYRK1A gene cloned from "Down syndrome critical region"

人类MNB/DYRK基因是果蝇迷你脑（mnb）和大鼠Dyrk（双特异性酪氨酸磷酸化和调节激酶）基因的人类同源物。MNB/DYRK 1A基因首次从21号染色体上的“唐氏综合征关键区”克隆，是与唐氏综合征相关的精神发育迟滞的强有力候选基因。我们以前曾报道过，MNB/DYRK 1A的过度表达诱导HeLa细胞中的多核化，通过过度生产的中心体在间期和生产异常纺锤体在有丝分裂。因此，在这项研究中，我们确定了在细胞周期中的MNB/DYRK 1A的磷酸化状态，因为这种激酶的活性取决于激活环中酪氨酸残基的磷酸化和酪氨酸磷酸化共有基序中Tyr 219的可能磷酸化。用胸苷阻断法将HeLa细胞同步化于G1/S期。然后通过去除胸苷释放细胞以进行细胞周期，并从T ...更多信息 在移除后的不同时间点观察细胞。使用抗MNB/DYRK 1A抗体对这些提取物进行的Western印迹分析显示，所有测试的提取物均在相同位置产生迁移的条带。因此，在细胞周期期间没有发生导致SDS-PAGE上条带移位的MNB/DYRK 1A修饰。接着，在去除胸苷后用诺考达唑处理HeLa细胞并使其在M期同步化。通过蛋白质印迹分析来分析来自同步化细胞的提取物的MNB/DYRK 1A。同步细胞中的MNB/DYRK 1A迁移速度比非同步细胞慢。然而，如果提取物已经用λ-蛋白磷酸酶预处理，则没有观察到条带筛选，这证实了在诺考达唑处理的细胞中MNB/DYRK 1A蛋白被磷酸化。当细胞用诺考达唑处理但不被阻滞在M期时，在这些细胞中未观察到这种带移。因此，这种磷酸化以M期特异性方式诱导。这些结果表明，MNB/DYRK 1A可能与纺锤体附着检查点机制有关，该机制确保所有染色体都正确地附着到纺锤体的微管上。少

The human MNB/DYRK gene is a human homolog of Drosophila minibrain (mnb) and rat Dyrk (Dual-specificity tyrosine-phosphorylated and regulated kinase) genes. MNB/DYRK1A gene was first cloned from "Down syndrome critical region" on chromosome 21 and is a strong candidate for mental retardation associated with Down syndrome. We have previously reported that overexpression of MNB/DYRK1A induces multinucleation in HeLa cells through overproduction of the centrosome during interphase and production of aberrant spindles during mitosis. Thus, in this study, we determined phosphorylation state of MNB/DYRK1A during the cell cycle, because the activity of this kinase depends on the phosphorylation of tyrosine residues in the activation loop and possibly of Tyr219 in a tyrosine phosphorylation consensus motif. HeLa cells were synchronized at the G1/S boundary using thymidine block method. The cells were then released to progress cell cycle by removal of thymidine, and extracts were prepared from t … More hese cells at various time points after the removal. Western blot analysis of these extracts using anti-MNB/DYRK1A antibody revealed that all the extracts tested gave bands migrating at the same position. Thus, no modification of MNB/DYRK1A leading to band shift on SDS-PAGE occurred during the cell cycle. Next, HeLa cells were treated with nocodazole after the removal of thymidine and synchronized at M phase. The extract from the synchronized cells was analyzed for MNB/DYRK1A by western blot analysis. MNB/DYRK1A in the synchronized cells migrated slower than that in asynchronous cells. However, no band sift was observed if the extracts had been pretreated with λ-protein phosphatase, confirming that MNB/DYRK1A protein was phosphorylated in nocodazole-treated cells. When cells were treated with nocodazole but not arrested at M phase, such a band shift was not observed in these cells. Thus, this phosphorylation was induced in an M phase-specific manner. These results indicate that MNB/DYRK1A may be implicated in the spindle-attachment checkpoint mechanism which ensures that all chromosomes are properly attached to the microtubules of the spindle. Less