The long term objective of our studies is to understand the function, disease association, and regulation of the intermediate filament proteins, keratin polypeptide 8 and 18 (K8 and K18), that are expressed in glandular-type epithelia as found in intestine, livers, and exocrine pancreases. Although the functions of these cytoskeletal proteins remain poorly understood, several blistering/scaling skin diseases are caused by mutations in the epidermal (non-glandular) keratins, and a mutation in K18 was identified in a patient with crypotegenic cirrhosis. Recently, a group of proteins termed 14-3-3 were shown to associate with K18 during the S and G2/M phases of the cell cycle in a stoichiometrically significant, phosphorylation- dependent and reversible manner, and act to sequester K8/18 from the insoluble cytoskeletal compartment in to the cytosol. The 14-3-3 proteins constitute a large family that plays an important role in the timing of mitosis in yeast, and binds to several signaling molecules including protein kinase C, Raf-1 and PI-3 kinases, and cdc25 phosphatases. Although several putative functions have been suggested for 14-3-3 proteins, their role in mammalian cells remains unknown. Our hypothesis is that manipulating the interaction between K8/18 and 14-3-3 proteins will provide important functional information regarding 14-3-3, K8/18, the significance of keratin-14-3-3 interaction, and a potential human disease model. We propose to test our hypothesis by: (1) mutating the phosphylation site that dictates 14-3-3 binding to K8/18, and testing the effect in cultured cells, (2) reconstituting into K18-null mice wild type or a phosphorylation mutant K18 that does not bind 14-3-3, followed by testing the organ-specific functional and potential disease-related consequences of altering the keratin-14-3-3 binding, (3) determining how keratin-14-3-3 binding effects the interaction of 14-3-3 with other proteins such as Raf-1 kinase and protein kinase C, (4) introducing K18 mutations that increase 14-3-3 binding and testing their functional consequences, and (5) identifying the binding domain on 14-3-3 that regulates its interaction with keratin, and testing the role of this domain in binding to other 14-3-3 target proteins. Our proposed studies are likely to generate important new biologic information regarding two major protein families in the digestive system, and help clarify their function and potential role in human disease.
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