Identification of novel protein kinases from Beghet patientsser

人类自身抗体已被证明是鉴定新型核抗原的有用试剂。已知有几种自身抗体可以识别在进化过程中高度保守的蛋白质，这些蛋白质参与了必要的细胞内功能，如DNA复制、转录和前mrna的剪接。在本研究中，我们分离了编码转录因子ZF5的cDNA克隆，该转录因子存在于斑点结构中，这是一种新的MAP激酶（MKK6）文库。通过自体免疫患者血清的免疫筛选方法，从HeLa cDNA中分离到一种新的核斑点型POZ蛋白SPOP。此外，我们还克隆出了动力蛋白家族的新成员——人类动力蛋白。全长cDNA序列编码一个由736个氨基酸组成的蛋白，分子量为80 kDa。氨基酸序列与酵母Dnmlp和Vps1p最相似。Dymple缺乏富含脯氨酸的c -末端结构域，dynamin通过该结构域与SH3结构域结合激活。Northern blot分析显示两个转录本大小分别为2.5kb和4.2kb，在脑、骨骼肌和睾丸中选择性多聚腺苷化水平最高。进一步确定了三种选择性剪接模式，以组织特异性的方式在编码序列中产生帧内缺失。当过表达时，野生型dypple表现出点状核周细胞质模式，而n端缺失突变体形成以TGN标记为界的大聚集体。由于动力蛋白通过一种明确的机制参与网格蛋白介导的内吞作用，因此已经预测了在每个特定的囊泡运输途径中存在动力蛋白样分子。我们的研究结果表明，dyniple可能是哺乳动物细胞中除dynamin本身之外的第一个此类亚家族的例子，尽管其确切的作用和膜定位仍有待解决。

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.