Heat stress or rotavirus infection of human epithelial cells generates a distinct hyperphosphorylated form of keratin 8.
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.
Liao
J
J
Palo Alto Veterans Affairs Medical Center, California 94304, USA.
Lowthert
L A
LA
Omary
M B
MB
D015153:Blotting, Western; D002460:Cell Line; D003599:Cytoskeleton / Q000648:ultrastructure; D004848:Epithelium / Q000378:metabolism / Q000648:ultrastructure / Q000821:virology; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D006358:Hot Temperature; D006801:Humans; D007633:Keratins / Q000378:metabolism* / Q000648:ultrastructure; D010766:Phosphorylation; D012400:Rotavirus Infections / Q000378:metabolism*; D012995:Solubility
D007633:Keratins