Role of phosphorylation in ethanol-induced aggregation of keratin intermediate filaments

Keratins are members of a diverse group of tissue-specific cytoskeletal components known as intermediate filaments. Regulation of the structure and intracellular distribution of intermediate filaments is known to be related to the phosphorylation state of their structural subunits. It also is known that disruption of the keratin filaments of hepatocytes in response to chronic ethanol ingestion is characteristic of alcoholic liver disease.

To characterize the mechanism of ethanol-induced keratin filament reorganization and dephosphorylation, cells were grown in culture with and without ethanol, and then were treated at the end of the incubation period for 1 hr with either 8-bromo-adenosine 3':5'-cyclic monophosphate (8Br), water-soluble forskolin (ws-forskolin), H-89 diHCL, or okadaic acid. Morphology of the cells was examined by immunofluorescence microscopy, and keratin phosphorylation levels were determined by analysis of 32p labeling.

We found that treatment of hepatoma cells with 300 mM ethanol results in disruption and aggregation of the keratin network in the vicinity of the nucleus as well as a hypophosphorylation of keratin subunits from ethanol-treated cells compared with non-ethanol-treated controls. 8Br and ws-forskolin treatment of ethanol groups restored keratin phosphorylation to control levels and reversed the ethanol-induced aggregation of keratin filaments. When H-89, an inhibitor of A-kinase, was added to control cells, keratin filament disorganization and dephosphorylation was observed. H-89 produced only a slight additional decrease in keratin phosphorylation in ethanol-treated cells, with no change in keratin distribution. Okadaic acid treatment of control cells produced hyperphosphorylation and filament network disruption, whereas in ethanol groups a reversal of the ethanol-mediated hypophosphorylation was observed but without reversal of the keratin filament aggregation.

These results suggest that site-specific phosphorylation of keratin filaments is important in maintaining their integrity and that activation of the A-kinase system can antagonize the effects of ethanol, whereas its inhibition results in filament dephosphorylation and reorganization, mimicking effects of ethanol treatment.