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 carcinogen metabolism. The cellular content of this protein is regulated at both the transcriptional and posttranslational levels. CYP2E1 is degraded by both rapid and slow acting proteolytic systems. In the presence 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 switch to initiate the fast acting degradative pathway. Phosphorylation at serine 129 has also been suggested to be the point at which hormones, such as insulin, exert actions on the stability of this protein. In order to investigate the role of phosphorylation in determining murine Cyp2e-1 levels, serine 129 was changed by site-directed mutagenesis to amino acids that could not be phosphorylated and the recombinant proteins expressed in COS 7 cells. Replacement of serine 129 with alanine and glycine does not lead to Cyp2e-1 accumulation. In the presence of insulin, although Cyp2e-1 levels increase slightly, specific stabilization of the wild-type 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.
Freeman
J E
JE
Imperial Cancer Research Fund, Ninewells Hospital & Medical School, Dundee, U.K.
Wolf
C R
CR
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D019392:Cytochrome P-450 CYP2E1; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007328:Insulin / Q000494:pharmacology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010089:Oxidoreductases, N-Demethylating / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D012694:Serine / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014162:Transfection
D007328:Insulin; D012694:Serine; D003577:Cytochrome P-450 Enzyme System; D019392:Cytochrome P-450 CYP2E1; D010089:Oxidoreductases, N-Demethylating