FUNCTION OF THE HUMAN MNB/DYRK1A GENE ON THE "DOWN SYNDROME CRITICAL REGION" OF CHROMOSOME 21

唐氏综合征(21三体)是最常见的出生缺陷，也是智力低下和先天性心脏病的主要原因。对21号染色体部分三体病例的研究表明，D21S55基因座周围的区域在该综合征的病因学中尤为重要。这一亚染色体区域被称为“唐氏综合征临界区”。早些时候，我们利用从该染色体区域分离的一系列粘粒克隆进行了外显子捕获实验，并鉴定了该区域人类MNB/DYRK1A基因的基因组结构和cDNA序列。该基因编码双特异性蛋白激酶，催化其在丝氨酸/苏氨酸和酪氨酸残基上的自动磷酸化。在本研究中，我们检测了转染绿色荧光蛋白(GFP)-MNB/DYRK1A融合蛋白的HeLa细胞，发现有两种表达模式：在一组细胞中，GFP-MNB/DYRK1A以点状分布在细胞核内；在另一组细胞中，GFP-MNB/DYRK1A过表达并聚集在整个细胞核中。在高表达GFP-MNB/DYRK1a的细胞中，可观察到明显的多核现象，而在有核点的细胞中，未发现此类异常核。此外，在后一种细胞中，有丝分裂和胞质分裂等基本过程发生正常。多核作用依赖于MNB/DYRK1A的活性，因为在高表达K179R的GFP-MNB/DYHK1A(K179R)和GFP-MNB/DYRK1A(Y310F/Y312F)细胞中没有观察到多核现象。GFP-MNB/DYRK1A-高表达细胞的免疫组织化学染色用针对α和γ的特异性抗体，微管蛋白显示在这些细胞中产生了中心体的多个拷贝和异常的多极纺锤体。这些结果表明，MNB/DYRK1A的过表达通过在间期中心体的过度复制和有丝分裂期间异常纺锤体的产生和染色体的错误分离而诱导HeLa细胞的多核。

Down syndrome(trisomy 21) is the most frequent birth defect and is a major cause of mental retardation and congenital heart disease. Studies of cases with partial trisomy of chromosome 21 have suggested that the region around locus D21S55 is particularly important in the etiology of the syndrome. This subchromosomal region is called the "Down syndrome critical region". Earlier we performed exon trapping experiments using a series of cosmid clones isolated from this chromosomal region, and identified the genomic structure and cDNA sequence of the human MNB/DYRK1A gene in this region. This gene encodes a dual specificity protein kinase that catalyzes its autophosphorylation on serine/threonine and tyrosine residues. In this research, we examined HeLa cells transfected with cDNA encoding a green fluorescent protein(GFP)-MNB/DYRK1A fusion protein and found 2 patterns of expression : In one group of transfected cells, GFP-MNB/DYRK1A was localized as dots within the nucleus ; and in the other group, it was overexpressed and had accumulated all over the nucleus. In the cells overexpressing GFP-MNB/DYRK1A, multinucleation was clearly observed ; whereas in those with the nuclear dots, such aberrant nuclei were not found. Furthermore, in the latter cells essential processes such as mitosis and cytokinesis occurred normally. Multinucleation was dependent on the kinase activity of MNB/DYRK1A, because it was not observed in cells overexpressing kinase activity-negative mutants, GFP-MNB/DYHK1A(K179R) and GFP-MNB/DYRK1A(Y310F/Y312F). Immunostainihg of GFP-MNB/DYRK1A-overexpressing cells. with specific antibodies against α-and γ-tubulin revealed that multiple copies of centrosomes and aberrant multipolar spindles were generated in these cells. These results indicate that overexpression of MNB/DYRK1A induces multinucleation in HeLa cells through overduplication of the centrosome during interphase and production of aberrant spindles and missegregation of chromosomes during mitosis.