Cloning and characterization of desmosome and keratin binding proteins.

我们从角蛋白中间丝和桥粒结构结合蛋白的角度研究了角质形成细胞的细胞结构的功能。为了确定这些结构蛋白的新功能，我们使用酵母双杂交系统克隆了角蛋白和桥粒结合蛋白。桥粒与另一种桥粒钙粘蛋白桥粒粘附素构成桥粒复合体的胞外部分。这两种桥粒钙粘附素包括三种不同的亚型，DSC 1-3和DSG 1-3，每个桥粒粘附素亚型都有两种剪接形式，即较长的Dsc1a-3a和较短的Dsc1b-3b。然而，较短的DSC的胞内结构域的功能尚不清楚。再次，我们利用酵母双杂交系统从人皮肤cDNA文库中找到了与DSC 2b的羧基末端结合的蛋白质。Rack1是一种与所有DSC b型的胞内区相互作用的蛋白，是活化的C激酶的受体。清华…此外，S还被认为可能作为蛋白激酶C的锚定位点，通过桥粒蛋白的磷酸化来调节细胞的黏附。接下来，我们试图通过酵母双杂交系统克隆并鉴定一种新的蛋白，该蛋白与人皮肤细胞质区域的KIF相互作用。以I型角蛋白K14、K16的头部结构域为诱饵，对人皮肤基因进行筛选，得到一个候选克隆。该克隆的基因由大约10个氨基酸组成。4.0kbp，5‘端有较大的1.0kbp非翻译区。预测的基因产物为105kD的蛋白质，编码970个氨基酸，与已知蛋白质没有明显的同源性。该蛋白含有多个核定位信号，并在其羧基半部分含有一个可能的跨膜片段。在酵母体系中，该克隆与I型角蛋白、K14和K18有明显的结合，但与II型角蛋白、K6和K8无相互作用。当克隆的基因与绿色荧光蛋白(GFP)融合并用于强制表达培养的角质形成细胞时，GFP信号主要沿核膜观察到。在共聚焦显微镜分析中，这些核信号与细胞核周围的角蛋白定位重叠。这些数据表明，这个新发现的克隆编码了一种交联蛋白，它将KIF结合到核膜上。较少

We have studies the function of the cytoarchitecture of the human keratinocytes in the aspect of the binding proteins to the keratin intermediate filament sand desmosomal apparatus. To identify the novel function of these well characterized structural proteins, we used the yeast two hybrid system to clone the keratin and desmosome binding proteins.Desmocollins constitute the extracellular part of desmosomal complexes with another desmosomal cadherin, desmogleins. These two desmosomal cadherins consist of three distinct isoforms, Dsc 1-3 and Dsg 1-3, and each desmocollin isoform has two splicing forms, the longer Dsc1a-3a and the shorter Dsc1b-3b. However, the function of the intracellular domain of shorter Dsc is yet unknown. Again, we used the yeast two-hybrid system to find out proteins binding to the carboxyl end of Dsc 2b from human skin cDNA library. Rack1, receptor for activated C kinase, was isolated as an interacting protein with the intracellular region of all Dsc b forms. Thu … More s the cytoplasmic tail of desmocollins was suggested to act as an anchoring site of PKC and to have a function modifying the cell adhesion through the phosphorylation of desmosomal proteins.We next attempted to clone and characterize a novel protein, which interact with KIF at the cytoplasmic region of the human skin by yeast two-hybrid system. Employing the head domain of type I keratin K14, K16 as a bait, we screened the human skin cDNA and could obtain a candidate clone. The cloned gene consists of approx. 4.0kbp and has relatively large 1.0kbp 5' untranslating region. The predicted gene product is 105 kD protein encoded by 970 amino acids with no significant homology with known proteins. This protein contains several nuclear localization signals as well as one putative transmembrane segment in its carboxyl half. The isolated clone showed the obvious binding with type I keratin, K14 and K18, but no interaction with type II keratin, K6 and K8 at least in the yeast system. When the cloned gene was fused with green fluorescence protein (GFP) and used for the forced expression into cultured keratinocytes, the GFP signals are observed predominantly along with the nuclear membrane. These nuclear signals were overlapped with keratin localization around the nuclei in the confocal-microscopic analysis. These data suggest that this newly identified clone encodes a cross linking protein, which binds the KIF to the nuclear membrane. Less