Pasdar M, Nelson W J
Institute for Cancer Research, Philadelphia, Pennsylvania 19111.
J Cell Biol. 1988 Mar;106(3):677-85. doi: 10.1083/jcb.106.3.677.
The functional interaction of cells in the formation of tissues requires the establishment and maintenance of cell-cell contact by the junctional complex. However, little is known biochemically about the mechanism(s) that regulates junctional complex assembly. To address this problem, we have initiated a study of the regulation of assembly of one component of the junctional complex, the desmosome, during induction of cell-cell contact in cultures of Madin-Darby canine kidney epithelial cells. Here we have analyzed two major protein components of the desmosomal plaque, desmoplakins I (Mr of 250,000) and II (Mr of 215,000). Analysis of protein levels of desmoplakins I and II by immunoprecipitation with an antiserum that reacts specifically with an epitope common to both proteins revealed that desmoplakins I and II are synthesized and accumulate at steady state in a ratio of 3-4:1 (in the absence or presence of cell-cell contact). The kinetics of desmoplakins I and II stabilization and assembly were analyzed after partitioning of newly synthesized proteins into a soluble and insoluble protein fraction by extraction of whole cells in a Triton X-100 high salt buffer. In the absence of cell-cell contact, both the soluble and insoluble pools of desmoplakins I and II are unstable and are degraded rapidly (t1/2 approximately 8 h). Upon induction of cell-cell contact, the capacity of the insoluble pool increases approximately three-fold as a proportion of the soluble pool of newly synthesized desmoplakins I and II is titrated into the insoluble pool. The insoluble pool becomes relatively stable (t1/2 greater than 72 h), whereas proteins remaining in the soluble pool (approximately 25-40% of the total) are degraded rapidly (t1/2 approximately 8 h). Furthermore, we show that desmoplakins I and II can be recruited from this unstable soluble pool of protein to the stable insoluble pool upon induction of cell-cell contact 4 h after synthesis; significantly, the stabilization of this population of newly synthesized desmoplakins I and II is blocked by the addition of cycloheximide at the time of cell-cell contact, indicating that the coordinate synthesis of another protein(s) is required for protein stabilization.
细胞在组织形成过程中的功能相互作用需要通过连接复合体建立和维持细胞间接触。然而，关于调节连接复合体组装机制的生化知识知之甚少。为了解决这个问题，我们启动了一项研究，即在Madin-Darby犬肾上皮细胞培养物中诱导细胞间接触期间，研究连接复合体的一个组成部分——桥粒组装的调节。在此，我们分析了桥粒斑的两个主要蛋白质成分，桥粒斑蛋白I（分子量250,000）和II（分子量215,000）。用一种与这两种蛋白质共有的表位特异性反应的抗血清进行免疫沉淀，分析桥粒斑蛋白I和II的蛋白质水平，结果显示桥粒斑蛋白I和II在稳态下以3 - 4:1的比例合成并积累（无论有无细胞间接触）。在用Triton X - 100高盐缓冲液提取全细胞，将新合成的蛋白质分为可溶性和不溶性蛋白质部分后，分析桥粒斑蛋白I和II的稳定化及组装动力学。在没有细胞间接触的情况下，桥粒斑蛋白I和II的可溶性和不溶性部分均不稳定，且迅速降解（半衰期约8小时）。诱导细胞间接触后，随着新合成的桥粒斑蛋白I和II的可溶性部分被滴定到不溶性部分中，不溶性部分的容量增加约三倍。不溶性部分变得相对稳定（半衰期大于72小时），而留在可溶性部分中的蛋白质（约占总量的25 - 40%）迅速降解（半衰期约8小时）。此外，我们表明，合成后4小时诱导细胞间接触时，桥粒斑蛋白I和II可以从这种不稳定的可溶性蛋白质部分募集到稳定的不溶性部分；值得注意的是，在细胞间接触时添加环己酰亚胺会阻止这群新合成的桥粒斑蛋白I和II的稳定化，这表明蛋白质稳定化需要另一种蛋白质的协同合成。
