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

人类MNB/Dyrk基因是果蝇小脑(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/DYRK1A基因首次从21号染色体上的“唐氏综合征临界区”克隆，是唐氏综合征相关精神发育迟滞的有力候选基因。我们之前已经报道过MNB/DYRK1A的过表达通过在间期过度生产中心体和在有丝分裂时产生异常纺锤体来诱导HeLa细胞的多核。因此，在本研究中，我们确定了MNB/DYRK1A在细胞周期中的磷酸化状态，因为该激酶的活性依赖于激活环中酪氨酸残基的磷酸化，并可能依赖于酪氨酸磷酸化共识基序中的Tyr219的磷酸化。用胸腺嘧啶核苷阻断法在G1/S交界处同步HeLa细胞。然后通过去除胸腺嘧啶核苷来释放细胞以进行细胞周期，并从t…中制备提取物。摘除后不同时间点的HSE细胞较多。用抗MNB/DYRK1A抗体对这些提取物进行Western印迹分析表明，所有被测提取物都有相同位置的条带迁移。因此，在细胞周期中，MNB/DYRK1A的修饰不会导致其在SDS-PAGE上的条带漂移。接下来，去除胸腺嘧啶核苷后用诺可达唑处理HeLa细胞，并使其同步于M期。用免疫印迹法检测同步化细胞提取液中MNB/DYRK1A的表达。MNB/DYRK1A在同步化细胞中的迁移速度慢于异步化细胞。然而，经λ蛋白磷酸酶处理后，未观察到条带筛选，证实在诺可达唑处理的细胞中，MNBDYRK1a蛋白被磷酸化。当诺考达唑处理细胞但未使其停滞于M期时，这些细胞没有观察到这种带状移位。因此，这种磷酸化是以M期特异的方式诱导的。这些结果表明，MNB/DYRK1A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在纺锤体的微管上。较少

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