Liao J, Lowthert L A, Omary M B
Palo Alto Veterans Affairs Medical Center, California 94304, USA.
Exp Cell Res. 1995 Aug;219(2):348-57. doi: 10.1006/excr.1995.1238.
The two major intermediate filament (IF) phosphoglycoproteins of human simple epithelia are keratins 8 and 18 (K8/18). Previous studies showed that heat stress and, in some cases, viral infection of cultured cells can be associated with alteration in IF organization. Here we show that heat stress of the human colonic cell line HT29 increased K8 and K18 phosphorylation and glycosylation and generated a distinct hyperphosphorylated form of K8 (HK8) that was previously noted upon G2/M arrest of epithelial cells. In contrast, rotavirus infection of HT29 cells resulted in phosphorylation changes similar to those induced by heat stress but did not alter K8/18 glycosylation. The identity of HK8 was determined using phosphatase treatment, tryptic phosphopeptide mapping, and K8-specific antibodies. A time course of heat stress showed that the increased phosphorylation and glycosylation of K8/18 occurred several hours after induction of heat shock protein 70 expression. Rotavirus altered the organization of the K8/18 network in most infected cells, whereas the effect of heat stress on K8/18 organization was less prominent. However, in vitro filament assembly of purified K8/18 was not significantly altered after isolation from heat-stressed or rotavirus-infected cells, despite having increased solubility and hyperphosphorylation. Our results indicate that increased glycosylation and/or phosphorylation of K8 and K18, in association with heat stress or rotavirus infection, does not significantly alter in vitro keratin filament assembly, whereas the association of these modifications with filament reorganization in cells is more dramatic. Generation of HK8 appears to be a late event and to occur in common with several forms of stress including heat, viral infection, and arrest of cells in G2/M.
人类单层上皮细胞的两种主要中间丝（IF）磷酸糖蛋白是角蛋白8和18（K8/18）。先前的研究表明，热应激以及在某些情况下培养细胞的病毒感染可能与中间丝组织的改变有关。在此我们表明，人结肠细胞系HT29的热应激增加了K8和K18的磷酸化和糖基化，并产生了一种独特的K8超磷酸化形式（HK8），这种形式先前在上皮细胞的G2/M期停滞时被注意到。相比之下，轮状病毒感染HT29细胞导致的磷酸化变化与热应激诱导的变化相似，但未改变K8/18的糖基化。使用磷酸酶处理、胰蛋白酶磷酸肽图谱分析和K8特异性抗体确定了HK8的身份。热应激的时间进程表明，K8/18磷酸化和糖基化增加发生在热休克蛋白70表达诱导数小时后。轮状病毒改变了大多数受感染细胞中K8/18网络的组织，而热应激对K8/18组织的影响则不太明显。然而，从热应激或轮状病毒感染的细胞中分离后，纯化的K8/18的体外丝组装没有显著改变，尽管其溶解度增加且发生了超磷酸化。我们的结果表明，与热应激或轮状病毒感染相关的K8和K18糖基化和/或磷酸化增加不会显著改变体外角蛋白丝组装，而这些修饰与细胞中丝重组的关联更为显著。HK8的产生似乎是一个晚期事件，并且与包括热、病毒感染以及细胞在G2/M期停滞在内的多种形式的应激共同发生。
