Identification of novel protein kinases from Beghet patientsser

Beghet 患者中新型蛋白激酶的鉴定

基本信息

批准号：
08457046
负责人：
HAGIWARA Masatoshi
金额：
$ 4.74万
依托单位：
Tokyo Medical and Dental University (1997)Nagoya University (1996)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08457046/
关键词：
auto antigen immunoscreening MKK6 SPOP ZF5 Dymple Ki67 Mitosin 自己抗体核内蛋白 Behcet病 TAK1 P38 HOG

项目摘要

Human autoantibodies have proven to be useful reagents for identifying novel nuclear antigens. Several autoantibodies are known to recognize proteins which are highly conserved in evolution and which are involved in essential intracellular functions such as DNA replication, transcription, and splicing of pre-mRNA, In this study, we isolated cDNA clones encoding transcripton factor ZF5, which is present in speckle structures, a novel MAP kinase kinase (MKK6) library, and a novel nuclear speckle-type POZ protein designated SPOP were isolated from a HeLa cDNA by this immunoscreening approach with autoimmune patient sera. Moreover, we have cloned human dymple, a novel dynamin-family member. The full-length cDNA sequence encodes a protein composed of 736 amino acids with a molecular mass of 80 kDa. Amino acid sequence most resembles yeast Dnmlp and Vps1p. Dymple lacks a proline-rich C-terminal domain, through which dynamin binds to SH3 domains to be activated. Northern blot analysis revealed two transcript sizes of 2.5kb and 4.2kb with alternative polyadenylation at highest levels in brain, skeletal muscle and testis. It was further established that there are three patterns of alternative splicing producing in-frame deletions in the coding sequence of dymple in a tissue-specific manner. When overexpressed, wild-type dymple exhibited a punctate perinuclear cytoplasmic pattern, while an N-terminal deletion mutant formed large aggregates bounded by a TGN marker. Since dynamin participates in clathrin-mediated endocytosis through a well-characterized mechanism, the existence of a dynamin-like molecules in each specific vesicle transport pathway has been predicted. Our findings suggest that dyniple may be the first example of such a subfamily in mammalian cells other than dynamin-itself, though its precise role and membrane localization remain to be resolved.

人类自身抗体已被证明是鉴定新型核抗原的有用试剂。 Several autoantibodies are known to recognize proteins which are highly conserved in evolution and which are involved in essential intracellular functions such as DNA replication, transcription, and splicing of pre-mRNA, In this study, we isolated cDNA clones encoding transcripton factor ZF5, which is present in speckle structures, a novel MAP kinase kinase (MKK6) library, and a novel nuclear speckle-type POZ通过自身免疫性患者血清从HELA cDNA中分离出蛋白质指定的汤匙。此外，我们已经克隆了一个新型的Dynamin家庭成员人类Dymple。全长cDNA序列编码由736个氨基酸组成的蛋白质，其分子质量为80 kDa。氨基酸序列最类似于酵母DNMLP和VPS1P。 Dymple缺少富含脯氨酸的C末端结构域，通过该结构蛋白与SH3结构域结合以被激活。 Northern印迹分析显示，在大脑，骨骼肌和睾丸中，最高水平的替代聚腺苷酸化的2.5kb和4.2kb的转录物大小。进一步确定，在组织特异性方式中，在Dymple的编码序列中产生框架内缺失的替代剪接模式。当过表达时，野生型dymple表现出点状细胞质模式，而N端缺失突变体形成了由TGN标记界定的大骨料。由于动力蛋白通过特征良好的机制参与网格蛋白介导的内吞作用，因此已经预测了每个特定囊泡转运途径中动力蛋白样分子的存在。我们的发现表明，驱动器可能是在哺乳动物细胞中以外的哺乳动物细胞中这种亚科的第一个例子，尽管其精确的作用和膜定位仍有待解决。