Pasdar M, Li Z, Chan H
Department of Anatomy and Cell Biology, Faculty of Medicine, University of Alberta, Edmonton, Canada.
Cell Motil Cytoskeleton. 1995;30(2):108-21. doi: 10.1002/cm.970300203.
Desmosomes are one component of the intercellular junctional complex in epithelia. In cultures of epithelial cells, desmosome assembly can be regulated by modulating the calcium concentrations of the growth media. At present, very little is known about the intracellular signal transduction mechanisms that regulate desmosome assembly and disassembly in response to changing extracellular calcium concentrations. We have used inhibitors of protein kinases and phosphatases in a combined biochemical and morphological approach to analyze the role of protein phosphorylation in the assembly and disassembly of desmosomes in Madin-Darby canine kidney epithelial cells. Our results suggest that desmosomal proteins (desmoplakins I/II and desmoglein 1) are primarily phosphorylated on serine residues. Electron microscopic analyses of desmosome assembly upon induction of cell-cell contact, in the presence of protein kinase inhibitor, H-7, revealed an apparently normal assembly of desmosomes. However, complete disassembly of desmosomes was inhibited by H-7 upon removal of extracellular calcium. Under these conditions, although desmosomes split, desmosomal plaques and their associated cytokeratin filaments can not be internalized. In contrast, treatment of the cultures with okadaic acid (OA), an inhibitor of protein phosphatases, inhibited desmosome assembly but had no effect on disassembly. In addition, the inhibitory effect of okadaic acid on desmosome assembly was specific to this junction since we observed apparently normal tight junction and adherens junction in okadaic acid-treated cultures. These results suggest that assembly and disassembly of desmosomes may be regulated by extracellular Ca2+ via reversible protein phosphorylation involving both protein kinase and protein phosphatases.
桥粒是上皮细胞间连接复合体的一个组成部分。在上皮细胞培养物中，桥粒组装可通过调节生长培养基中的钙浓度来调控。目前，对于响应细胞外钙浓度变化而调节桥粒组装和拆卸的细胞内信号转导机制知之甚少。我们采用蛋白激酶和磷酸酶抑制剂，结合生化和形态学方法，分析蛋白磷酸化在Madin-Darby犬肾上皮细胞桥粒组装和拆卸中的作用。我们的结果表明，桥粒蛋白（桥粒斑蛋白I/II和桥粒芯糖蛋白1）主要在丝氨酸残基上发生磷酸化。在存在蛋白激酶抑制剂H-7的情况下，对细胞间接触诱导的桥粒组装进行电子显微镜分析，发现桥粒组装明显正常。然而，在去除细胞外钙后，H-7抑制了桥粒的完全拆卸。在这些条件下，尽管桥粒分裂，但桥粒斑及其相关的细胞角蛋白丝不能内化。相反，用蛋白磷酸酶抑制剂冈田酸（OA）处理培养物，抑制了桥粒组装，但对拆卸没有影响。此外，冈田酸对桥粒组装的抑制作用对这种连接是特异性的，因为我们在冈田酸处理的培养物中观察到紧密连接和黏着连接明显正常。这些结果表明，桥粒的组装和拆卸可能通过涉及蛋白激酶和蛋白磷酸酶的可逆蛋白磷酸化，由细胞外Ca2+调控。
