Phosphorylation of human keratin 8 in vivo at conserved head domain serine 23 and at epidermal growth factor-stimulated tail domain serine 431.
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.
Ku
N O
NO
Department of Medicine, Veterans Administration Palo Alto Health Care System, Palo Alto, California 94304, USA.
Omary
M B
MB
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D004815:Epidermal Growth Factor / Q000378:metabolism*; D006801:Humans; D007633:Keratins / Q000378:metabolism*; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism
D012694:Serine; D004815:Epidermal Growth Factor; D007633:Keratins