Identification of the major physiologic phosphorylation site of human keratin 18: potential kinases and a role in filament reorganization.
There is ample in vitro evidence that phosphorylation of intermediate filaments, including keratins, plays an important role in filament reorganization. In order to gain a better understanding of the function of intermediate filament phosphorylation, we sought to identify the major phosphorylation site of human keratin polypeptide 18 (K18) and study its role in filament assembly or reorganization. We generated a series of K18 ser-->ala mutations at potential phosphorylation sites, followed by expression in insect cells and comparison of the tryptic 32PO4-labeled patterns of the generated constructs. Using this approach, coupled with Edman degradation of the 32PO4-labeled tryptic peptides, and comparison with tryptic peptides analyzed after labeling normal human colonic tissues, we identified ser-52 as the major K18 physiologic phosphorylation site. Ser-52 in K18 is not glycosylated and matches consensus sequences for phosphorylation by CAM kinase, S6 kinase and protein kinase C, and all these kinases can phosphorylate K18 in vitro predominantly at that site. Expression of K18 ser-52-->ala mutant in mammalian cells showed minimal phosphorylation but no distinguishable difference in filament assembly when compared with wild-type K18. In contrast, the ser-52 mutation played a clear but nonexclusive role in filament reorganization, based on analysis of filament alterations in cells treated with okadaic acid or arrested at the G2/M stage of the cell cycle. Our results show that ser-52 is the major physiologic phosphorylation site of human K18 in interphase cells, and that its phosphorylation may play an in vivo role in filament reorganization.
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Palo Alto Veterans Administration Medical Center, CA 94304.
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D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D004988:Ethers, Cyclic / Q000494:pharmacology; D006801:Humans; D007382:Intermediate Filaments / Q000378:metabolism*; D007633:Keratins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation / Q000502:physiology; D019319:Okadaic Acid; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000032:analysis; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D018411:Spodoptera
D004988:Ethers, Cyclic; D010748:Phosphopeptides; D019319:Okadaic Acid; D012694:Serine; D007633:Keratins; D017346:Protein Serine-Threonine Kinases; D010749:Phosphoprotein Phosphatases