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

人类 MNB/DYRK1A 基因在 21 号染色体“唐氏综合症关键区”的功能

基本信息

批准号：
14572084
负责人：
ITO Fumiaki
金额：
$ 2.56万
依托单位：
SETSUNAN UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14572084/
关键词：
Down syndrome Cell cycle Human chromosome 21 Centrosome Tubulin MNB DYRK1A Dual-specificity protein kinase Multinucleate cells Mnb

项目摘要

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.

唐氏综合症（21三体）是最常见的出生缺陷，是智力迟钝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位点周围的区域在该综合征的病因学中尤为重要。这个亚染色体区域被称为“唐氏综合症关键区域”。在此之前，我们利用从该染色体区域分离的一系列cosmid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。该基因编码一种双特异性蛋白激酶，催化丝氨酸/苏氨酸和酪氨酸残基的自磷酸化。在本研究中，我们检测了转染了编码绿色荧光蛋白(GFP)-MNB/DYRK1A融合蛋白的cDNA的HeLa细胞，发现了2种表达模式：在一组转染的细胞中，GFP-MNB/DYRK1A在细胞核内以点的形式定位；而在另一组中，它过度表达并在整个细胞核中积累。在过表达GFP-MNB/DYRK1A的细胞中，明显观察到多核；而在那些有核点的细胞中，没有发现这种异常的核。此外，在后者细胞中，有丝分裂和细胞质分裂等基本过程正常发生。多核依赖于MNB/DYRK1A的激酶活性，因为在激酶活性过表达阴性突变体GFP-MNB/DYHK1A（K179R）和GFP-MNB/DYRK1A（Y310F/Y312F）的细胞中没有观察到多核。过表达GFP-MNB/ dyrk1a细胞的免疫染色。α-和γ-微管蛋白特异性抗体显示，这些细胞中产生了多个中心体拷贝和异常多极纺锤体。这些结果表明，在HeLa细胞中，MNB/DYRK1A的过表达通过间期中心体的过度复制、有丝分裂期间异常纺锤体的产生和染色体的错误分离诱导了多核。