Phosphorylation of Human Keratin 8 in Vivo at Conserved Head Domain Serine 23 and at Epidermal Growth Factor-stimulated Tail Domain Serine 431

doi:10.1074/jbc.272.11.7556

Phosphorylation of Human Keratin 8 in Vivo at Conserved Head Domain Serine 23 and at Epidermal Growth Factor-stimulated Tail Domain Serine 431*

Nam-On Ku^‡ and
M. Bishr Omary^§

From the Department of Medicine, Veterans Administration Palo Alto Health Care System, Palo Alto, California 94304, and the Digestive Disease Center, Stanford University School of Medicine, Stanford, California 94305-5487

^§ To whom other correspondence should be addressed:
Palo Alto VA Medical Center, 3801 Miranda Ave., 111-GI, Palo Alto, CA 94304
.

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.

Footnotes

↵^‡ Recipient of an American Heart Association California Affiliate postdoctoral fellowship. To whom reprint requests should be addressed: Palo Alto VA Medical Center, 3801 Miranda Ave., 111-GI, Palo Alto, CA 94304.
↵* This work was supported in part by National Institutes of Health Grant DK 47918, Department of Veterans Affairs merit and career development awards, and Digestive Disease Center Grant DK38707. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

IF

intermediate filament

EGF

epidermal growth factor

K

keratin

MAP

mitogen-activated protein

BHK

baby hamster kidney

mAb

monoclonal antibody

PAGE

polyacrylamide gel electrophoresis.
↵² J. Liao, N.-O. Ku, and M. B. Omary, manuscript in preparation.
- Received August 12, 1996.
- Revision received January 10, 1997.