Hyperphosphorylation of cytokeratins 8 and 18 by microcystin-LR, a new liver tumor promoter, in primary cultured rat hepatocytes.
Microcystin-LR (MC-LR), an inhibitor of protein phosphatases 1 and 2A, is a potent tumor promoter in rat liver initiated with diethylnitrosamine. To understand its biochemical process in hepatocytes, primary cultured rat hepatocytes were treated with MC-LR. MC-LR (1 microM) induced phosphorylation of various proteins. Two 55 and 49 kDa proteins were phosphorylated at a 3-fold higher rate than other proteins, and these proteins were identified to be cytokeratins 8 and 18 respectively, by immunoprecipitation and Western blot analysis using monoclonal anti-cytokeratin 8 and 18 antibodies. The basic cytokeratins 8 and 18 showed pI 6.4 and 5.4 respectively, in two-dimensional gel electrophoresis. MC-LR dose dependently increased phosphorylation of cytokeratins 8 and 18 in a cell-free system by incubation with a cytosolic fraction of rat liver containing both protein kinases and protein phosphatases 1 and 2A, and with [gamma-32P]ATP. Cytokeratins 8 and 18 were target proteins for phosphorylation induced by inhibition of protein phosphatases 1 and 2A, in vitro and in rat hepatocytes. Thus, the treatment of rat hepatocytes with MC-LR induced hyperphosphorylation of cytokeratins 8 and 18 associated with morphological changes, indicating that intermediate filament networks were rearranged in the cytoplasm. The hyperphosphorylation of cytokeratins is a significant biochemical process associated with liver tumor promotion.
D000818:Animals; D001345:Autoradiography; D015153:Blotting, Western; D002273:Carcinogens / Q000378:metabolism*; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional; D007633:Keratins / Q000378:metabolism*; D008099:Liver / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D008387:Marine Toxins; D052998:Microcystins; D010456:Peptides, Cyclic / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D051381:Rats; D011916:Rats, Inbred F344
D002273:Carcinogens; D008387:Marine Toxins; D052998:Microcystins; D010456:Peptides, Cyclic; D007633:Keratins; D010749:Phosphoprotein Phosphatases; C057862:cyanoginosin LR