Role of Protein Phosphorylation in the Inhibition of Protein Synthesis Caused by Hypoxia in Rat Hepatocytes

International Journal of Toxycology, 20:21-27, 2001

Sandrine Tinton, Pedro Buc Calderon

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 systhesis 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) are detected by antiphosphotyrosine antibodies. The phosphorylation in the 129- and 83-kDa bands, however, was significantly and progressively decreased under hypoxia. Altough this timedependent decrease was sensitive to changes in oxygen tension, it occured 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 syntheses caused by hypoxia. under aerobic conditions, orthovanadate inhibited the synthesis of proteins, confirming that protein phosphorylation is a major mechanism involved in translation regulation. Once again, this inhibitory effect occured 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.