Kouklis P D, Hutton E, Fuchs E
Howard Hughes Medical Institute, Department of Molecular Genetics and Cell Biology, University of Chicago, Illinois 60637.
J Cell Biol. 1994 Nov;127(4):1049-60. doi: 10.1083/jcb.127.4.1049.
In epidermal cells, keratin intermediate filaments connect with desmosomes to form extensive cadherin-mediated cytoskeletal architectures. Desmoplakin (DPI), a desmosomal component lacking a transmembrane domain, has been implicated in this interaction, although most studies have been conducted with cells that contain few or no desmosomes, and efforts to demonstrate direct interactions between desmoplakin and intermediate filaments have not been successful. In this report, we explore the biochemical nature of the connections between keratin filaments and desmosomes in epidermal keratinocytes. We show that the carboxy terminal "tail" of DPI associates directly with the amino terminal "head" of type II epidermal keratins, including K1, K2, K5, and K6. We have engineered and purified recombinant K5 head and DPI tail, and we demonstrate direct interaction in vitro by solution-binding assays and by ligand blot assays. This marked association is not seen with simple epithelial type II keratins, vimentin, or with type I keratins, providing a possible explanation for the greater stability of the epidermal keratin filament architecture over that of other cell types. We have identified an 18-amino acid residue stretch in the K5 head that is conserved only among type II epidermal keratins and that appears to play some role in DPI tail binding. This finding might have important implications for understanding a recent point mutation found within this binding site in a family with a blistering skin disorder.
在表皮细胞中，角蛋白中间丝与桥粒相连，形成广泛的钙黏蛋白介导的细胞骨架结构。桥粒斑蛋白（DPI）是一种缺乏跨膜结构域的桥粒成分，尽管大多数研究是在几乎没有或没有桥粒的细胞中进行的，但它被认为参与了这种相互作用，而且证明桥粒斑蛋白与中间丝之间直接相互作用的努力尚未成功。在本报告中，我们探讨了表皮角质形成细胞中角蛋白丝与桥粒之间连接的生化性质。我们发现DPI的羧基末端“尾巴”直接与II型表皮角蛋白（包括K1、K2、K5和K6）的氨基末端“头部”结合。我们构建并纯化了重组K5头部和DPI尾巴，并通过溶液结合试验和配体印迹试验在体外证明了它们的直接相互作用。简单上皮II型角蛋白、波形蛋白或I型角蛋白之间没有这种明显的结合，这为表皮角蛋白丝结构比其他细胞类型具有更高稳定性提供了一种可能的解释。我们在K5头部鉴定出一个18个氨基酸残基的片段，该片段仅在II型表皮角蛋白中保守，并且似乎在DPI尾巴结合中起一定作用。这一发现可能对理解最近在一个患有皮肤水疱病的家族中该结合位点发现的点突变具有重要意义。
