Role of protein phosphorylation in the inhibition of protein synthesis caused by hypoxia in rat hepatocytes.
Hypoxia causes a rapid and reversible inhibition of translation in freshly isolated rat hepatocytes. This inhibition is neither due to an ATP loss nor to an increase in cell death. Because protein synthesis is mainly regulated by reversible phosphorylation of initiation and/or elongation factors, we investigated whether translation inhibition by hypoxia may be related to changes in the phosphorylation status of proteins. Whatever the incubation conditions, three phosphoreactive bands (molecular weights 220, 129, and 83 kDa) were detected by antiphosphotyrosine antibodies. The phosphorylation in the 129- and 83-kDa bands, however, was significantly and progressively decreased under hypoxia. Although this time-dependent decrease was sensitive to changes in oxygen tension, it occurred after the early protein synthesis inhibition caused by hypoxia. Moreover, sodium orthovanadate prevented tyrosine dephosphorylation in hypoxic cells, but did not restore the depressed protein synthesis caused by hypoxia. Under aerobic conditions, orthovanadate inhibited the synthesis of proteins, confirming that protein phosphorylation is a major mechanism involved in translational regulation. Once again, this inhibitory effect occurred only after 90 minutes of incubation whereas hypoxia inhibits the protein synthesis at the beginning of the incubation. Labeling cells with [33-32P]-ortho-phosphoric acid allowed detection of several phosphorylated proteins that appeared under hypoxia. Because they were not recognized by the phosphotyrosine antibodies, we suggest that serine/threonine residues of key proteins may be the putative hypoxic targets.
Tinton
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Unité de Pharmacocinétique, Métabolisme, Nutrition et Toxicologie, Université Catholique de Louvain, Brussels, Belgium.
Calderon
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PB
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D015687:Cell Hypoxia / Q000502:physiology*; D002470:Cell Survival; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism*; D008297:Male; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011500:Protein Synthesis Inhibitors / Q000378:metabolism*; D012313:RNA / Q000096:biosynthesis; D051381:Rats; D017208:Rats, Wistar; D014443:Tyrosine / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology
D004791:Enzyme Inhibitors; D011500:Protein Synthesis Inhibitors; D014638:Vanadates; D014443:Tyrosine; D012313:RNA; D000255:Adenosine Triphosphate