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 filament protein keratin 18 (K18) in vivo, in a cell-cycle- and phosphorylation-dependent manner. We identified K18 Ser33 as an interphase phosphorylation site, which increases its phosphorylation during mitosis in cultured cells and regenerating liver, and as an in vitro cdc2 kinase phosphorylation site. Comparison of wild-type versus K18 Ser33-->Ala/Asp 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 phosphorylation site (Ser52) or K18 glycosylation sites had no effect on the binding of K18 to 14-3-3 proteins. The K18 phospho-Ser33 motif is different from several 14-3-3-binding phosphomotifs already described. Antibodies that are specific to K18 phospho-Ser33 or phospho-Ser52 show that although Ser52 and Ser33 phosphorylated K18 molecules manifest partial colocalization, these phosphorylation events reside predominantly on distinct K18 molecules. Our results demonstrate a unique K18 phosphorylation site that is necessary but not sufficient for K18 binding 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 phosphorylation on distinct K18 molecules suggests functional compartmentalization of these modifications.
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VA Palo Alto Health Care System, 3801 Miranda Avenue, 154J, Palo Alto, CA 94304, USA.
Liao
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D048948:14-3-3 Proteins; D016475:3T3 Cells; D000818:Animals; D000918:Antibody Specificity; D002460:Cell Line; D006224:Cricetinae; D019073:HT29 Cells; D006801:Humans; D007382:Intermediate Filaments / Q000737:chemistry; D007633:Keratins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D008115:Liver Regeneration / Q000502:physiology; D051379:Mice; D008938:Mitosis / Q000502:physiology; D009154:Mutation; D010449:Peptide Mapping; D010766:Phosphorylation; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*; D014446:Tyrosine 3-Monooxygenase
D048948:14-3-3 Proteins; D011506:Proteins; D012694:Serine; D007633:Keratins; D014446:Tyrosine 3-Monooxygenase