EVIDENCE AGAINST A ROLE FOR SERINE-129 IN DETERMINING MURINE CYTOCHROME-P450 CYP2E-1 PROTEIN-LEVELS

The cytochrome P450 CYP2E subfamily plays a central role in drug and c arcinogen metabolism. The cellular content of this protein is regulate d at both the transcriptional and posttranslational levels. CYP2E1 is degraded by both rapid and slow acting proteolytic systems. In the pre sence of a substrate, CYP2E1 becomes stabilized, and the contribution of the rapid actinig proteolytic pathway to its destruction decreases. It has been suggested that phosphorylation at serine 129 acts as a sw itch to initiate the fast acting degradative pathway. Phosphorylation at serine 129 has also been suggested to be the point at which hormone s, such as insulin, exert actions on the stability of this protein. In order to investigate the role of phosphorylation in determining murin e Cyp2e-1 levels, serine 129 was changed by site-directed mutagenesis to amino acids that could not be phosphorylated and the recombinant pr oteins expressed in COS 7 cells. Replacement of serine 129 with alanin e and glycine does not lead to Cyp2e-1 accumulation. In the presence o f insulin, although Cyp2e-1 levels increase slightly, specific stabili zation of the wildtype protein relative to the two mutant forms is not observed. These observations provide evidence that insulin can act by stabilization of Cyp2e-1 protein but suggest that the phosphorylation of serine 129 is not the molecular basis of stabilization observed.