PHOSPHORYLATION OF HUMAN KERATIN-18 SERINE-33 REGULATES BINDING TO 14-3-3-PROTEINS

Members of the 14-3-3 protein family bind the human intermediate filam ent protein keratin 18 (K18) in vivo, in a cell-cycle-and phosphorylat ion-dependent manner. We identified K18 Ser33 as an interphase phospho rylation site, which increases its phosphorylation during mitosis in c ultured cells and regenerating liver, and as an in vitro cdc2 kinase p hosphorylation site. Comparison of wild-type versus K18 Ser33-->Ala/As p transfected cells showed that K18 Ser33 phosphorylation is essential for the association of K18 with 14-3-3 proteins, and plays a role in keratin organization and distribution. Mutation of another K18 major p hosphorylation site (Ser52) or K18 glycosylation sites had no effect o n the binding of K18 to 14-3-3 proteins. The K18 phospho-Ser33 motif i s different from several 14-3-3-binding phosphomotifs already describe d. Antibodies that are specific to K18 phospho-Ser33 or phospho-Ser52 show that although Ser52 and Ser33 phosphorylated K18 molecules manife st partial colocalization, these phosphorylation events reside predomi nantly on distinct K18 molecules. Our results demonstrate a unique K18 phosphorylation site that is necessary but not sufficient for K18 bin ding to 14-3-3 proteins. This binding is likely to involve one or more mitotic events coupled to K18 Ser33 phosphorylation, and plays a role in keratin subcellular distribution. Physiological Ser52 or Ser33 pho sphorylation on distinct K18 molecules suggests functional compartment alization of these modifications.