Identification of novel protein kinases from Beghet patientsser

人类自身抗体已被证明是识别新的核抗原的有用试剂。已知一些自身抗体识别在进化中高度保守的蛋白质，这些蛋白质参与细胞内的基本功能，如DNA复制、转录和剪接前mRNA。在本研究中，我们分离了存在于斑点结构中的转录因子ZF5的cDNA克隆，一个新的MAP激酶(MKK6)文库，以及一个新的核斑点型POZ蛋白SPOP，通过这种免疫筛选方法从HeLa患者血清中分离到一个新的核斑点型POZ蛋白。此外，我们还克隆了一个新的动力蛋白家族成员--人类Dymple。该基因全长编码一个由736个氨基酸组成的蛋白质，分子质量为80 kDa。氨基酸序列与酵母Dnmlp和Vps1p最为相似。Dymple缺乏一个富含Pro的C-末端结构域，Dynamin通过该结构域与SH3结构域结合以被激活。Northern印迹分析显示，在脑、骨骼肌和睾丸中有两个大小分别为2.5kb和4.2kb的转录本，其中多聚腺苷酸化水平最高。进一步证实，在DIMPLE的编码序列中，有三种选择性剪接模式以组织特异性的方式产生框内缺失。当过表达时，野生型Dymple表现为点状的核周细胞质模式，而N端缺失突变体形成由TGN标记结合的大聚集体。由于Dynamin通过一种已被很好描述的机制参与了胞内蛋白介导的内吞作用，因此在每个特定的囊泡运输途径中都可以预测到Dynamin样分子的存在。我们的发现表明，Dyniple可能是除了Dynamin本身之外，哺乳动物细胞中第一个这样一个亚家族的例子，尽管它的确切作用和膜的定位仍然有待解决。

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.