Site-specificity of ethanol-induced dephosphorylation of rat hepatocyte keratins 8 and 18: A 31P NMR study.

Chronic feeding of ethanol to rats results in disorganization of the keratin intermediate filament network within hepatocytes. Previous studies from this laboratory have shown that intermediate filament organization in cultured cells is related to the phosphorylation state of the proteins. Therefore, we have examined the phosphorylation state of hepatocyte keratins from control and ethanol-fed rats. Feeding ethanol to rats results in dephosphorylation of one site on keratin 8 and one site on keratin 18 at all time points beginning with 6 weeks of ethanol treatment. Dephosphorylation was detected by phosphate analysis and by two-dimensional electrophoresis in which a change in isoelectric point of keratins from ethanol-fed rats was observed. These observations indicate that dephosphorylation of keratins in ethanol-fed animals may be an early step in alcoholic hepatitis which has occurred by 6 weeks of ethanol treatment. To further characterize keratin dephosphorylation in ethanol-fed rats, we used 31P NMR spectroscopy to classify the dephosphorylation site(s). Hepatocyte keratins were purified and solubilized in 9.5 M urea, 10 mM Tris-Cl, pH 8.1. 31P NMR spectra were obtained at 109 MHz, in 10 mm tubes at 30 degrees C. Samples of hepatocyte keratins were phosphorylated with A-kinase, protein kinase C, casein kinase II or Ca/CAM kinase and these samples were analyzed by 31P NMR spectroscopy. The resulting spectra were used as standards to compare the 31P chemical shifts of the resonances produced by these kinases with the phosphorus resonances of control and experimental samples. The 31P NMR spectrum of control hepatocyte keratins shows three resonances at 0.7, 4 and 5 ppm. In vitro phosphorylation by A-kinase produces a resonance at 4 ppm which is distinctly different from the resonance produced by each of the other kinases. In hepatocyte keratins from ethanol-fed animals, the resonance at 4 ppm was missing from the spectrum. These observations indicate that the keratin site that is dephosphorylated in ethanol-fed rats is characterized by the same 31P chemical shift as the keratin site that is phosphorylated by A-kinase.