Phosphorylation of human keratin 8 in vivo at conserved head domain serine 23 and at epidermal growth factor-stimulated tail domain serine 431

J Biol Chem. 1997 Mar 14;272(11):7556-64. doi: 10.1074/jbc.272.11.7556.

Abstract

Dynamic phosphorylation is one mechanism that regulates the more than 20 keratin type I and II intermediate filament proteins in epithelial cells. The major type II keratin in "simple type" glandular epithelia is keratin 8 (K8). We used biochemical and mutational approaches to localize two major in vivo phosphorylation sites of human K8 to the head (Ser-23) and tail (Ser-431) domains. Since Ser-23 of K8 is highly conserved among all type II keratins, we also examined if the corresponding Ser-59 in stratified epithelial keratin 6e is phosphorylated. Mutation of K6e Ser-59 abolished its phosphorylation in 32PO4-labeled baby hamster kidney cell transfectants. With regard to K8 phosphorylation at Ser-431, it increases dramatically upon stimulation of cells with epidermal growth factor (EGF) or after mitotic arrest and is the major K8 phosphorylated residue after incubating K8 immunoprecipitates with mitogen-activated protein or cdc2 kinases. A monoclonal antibody that specifically recognizes phosphoserine 431-K8 manifests increased reactivity with K8 and recognizes reorganized K8/18 filaments after EGF stimulation. Our results suggest that in vivo serine phosphorylation of K8 and K6e within the conserved head domain motif is likely to reflect a conserved phosphorylation site of most if not all type II keratins. Furthermore, K8 Ser-431 phosphorylation occurs after EGF stimulation and during mitotic arrest and is likely to be mediated by mitogen-activated protein and cdc2 kinases, respectively.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Cell Line
  • Cricetinae
  • Epidermal Growth Factor / metabolism*
  • Humans
  • Keratins / metabolism*
  • Phosphorylation
  • Serine / metabolism

Substances

  • Serine
  • Epidermal Growth Factor
  • Keratins