Inhibition of hepatic mitochondrial aldehyde dehydrogenase by carbon tetrachloride through JNK-mediated phosphorylation

Abstract

The aim of this study was to investigate the mechanism of inhibition of mitochondrial aldehyde dehydrogenase (ALDH2) by carbon tetrachloride (CCl 4). CCl 4 administration caused marked hepatocyte ballooning and necrosis in the pericentral region. CCl 4 also inhibited hepatic ALDH2 activity in a time-dependent manner without altering the protein level, suggesting ALDH2 inhibition through covalent modifications such as phosphorylation by JNK. To demonstrate phosphorylation, the isoelectric point (p I) of ALDH2 in CCl 4-exposed rats was compared to that of untreated controls. Immunoblot analysis revealed that immunoreactive ALDH2 bands in CCl 4-exposed rats were shifted to acidic p I ranges on two-dimensional electrophoresis (2-DE) gels. Incubation with alkaline phosphatase significantly restored the suppressed ALDH2 activity with a concurrent alkaline p I shift of the ALDH2 spots. Both JNK and activated JNK were translocated to mitochondria after CCl 4 exposure. In addition, incubation with catalytically active JNK led to significant inhibition of ALDH2 activity, with an acidic p I shift on 2-DE gels. Furthermore, immunoprecipitation followed by immunoblot analysis with anti-phospho-Ser–Pro antibody revealed phosphorylation of a Ser residue(s) of ALDH2. These results collectively indicate a novel underlying mechanism by which CCl 4 exposure activates JNK, which translocates to mitochondria and phosphorylates ALDH2, contributing to inhibition of ALDH2 activity accompanied by decreased cellular defense capacity and increased lipid peroxidation.