Keratins turn over by ubiquitination in a phosphorylation-modulated fashion.
Keratin polypeptides 8 and 18 (K8/18) are intermediate filament (IF) proteins that are expressed in glandular epithelia. Although the mechanism of keratin turnover is poorly understood, caspase-mediated degradation of type I keratins occurs during apoptosis and the proteasome pathway has been indirectly implicated in keratin turnover based on colocalization of keratin-ubiquitin antibody staining. Here we show that K8 and K18 are ubiquitinated based on cotransfection of His-tagged ubiquitin and human K8 and/or K18 cDNAs, followed by purification of ubiquitinated proteins and immunoblotting with keratin antibodies. Transfection of K8 or K18 alone yields higher levels of keratin ubiquitination as compared with cotransfection of K8/18, likely due to stabilization of the keratin heteropolymer. Most of the ubiquitinated species partition with the noncytosolic keratin fraction. Proteasome inhibition stabilizes K8 and K18 turnover, and is associated with accumulation of phosphorylated keratins, which indicates that although keratins are stable they still turnover. Analysis of K8 and K18 ubiquitination and degradation showed that K8 phosphorylation contributes to its stabilization. Our results provide direct evidence for K8 and K18 ubiquitination, in a phosphorylation modulated fashion, as a mechanism for regulating their turnover and suggest that other IF proteins could undergo similar regulation. These and other data offer a model that links keratin ubiquitination and hyperphosphorylation that, in turn, are associated with Mallory body deposits in a variety of liver diseases.
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Palo Alto VA Medical Center and Stanford University School of Medicine, Stanford University, Palo Alto, California 94304, USA.
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D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007381:Intermediate Filament Proteins / Q000378:metabolism*; D007633:Keratins / Q000235:genetics / Q000378:metabolism*; D007976:Leupeptins / Q000494:pharmacology; D009097:Multienzyme Complexes / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex; D014162:Transfection; D014452:Ubiquitins / Q000378:metabolism*
D004791:Enzyme Inhibitors; D007381:Intermediate Filament Proteins; D007976:Leupeptins; D009097:Multienzyme Complexes; D014452:Ubiquitins; C063261:acetylleucyl-leucyl-norleucinal; D007633:Keratins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex