Differential modulation of CYP2E1 activity by cAMP-dependent protein kinase upon Ser129 replacement.
Many toxic compounds are activated by cytochrome P450 (CYP) 2E1 to reactive metabolites, which represents a potential hazard for cellular homeostasis. Therefore knowledge about CYP2E1 regulation could be of great biological importance. It has been shown that CYP2E1 is controlled transcriptionally and post-translationally by phosphorylation. In the present study we investigated the role of serine-129 (Ser129) in the protein kinase A (PKA) recognition sequence motif Arg-Arg-Phe-Ser129. To gain further insights into the possible relevance of Ser129 for CYP2E1 function, Ser129 was replaced by alanine (Ala) or glycine (Gly) by site-directed mutations of the cDNA coding for CYP2E1. The mutant cDNAs were transfected into Chinese hamster lung fibroblast V79 cells. Despite the mutation in the PKA phosphorylation motif, all strains produced catalytically active CYP2E1. However, there was a marked change in the substrate preference: The Gly129-containing strains hydroxylated p-nitrophenol (PNP) to a markedly higher extent than the wild-type cDNA-containing cells, while they demethylated N-nitrosodimethylamine (NDMA) to a markedly lower extent than the wild-type cells. All the strains activated NDMA to mutagenic products. Treatment with the membrane-permeating cAMP derivative db-cAMP reduced markedly both the PNP hydroxylase and the NDMA demethylase activities as well as the mutation frequency induced by NDMA in the Ser129-containing strain. This decrease in activity was not accompanied by a decrease in CYP2E1 content. In addition, the catalytic activities of CYP2E1 were decreased in microsomes from rat hepatocytes treated with db-cAMP. Also in this case, the decrease in activities was not accompanied by a decrease in enzyme protein. These findings argue that involvement of Ser129 and its phosphorylation is not in determining CYP2E1 protein level, but rather in controlling its catalytic activity. In contrast, in the strains containing Ala129 or Gly129, treatment with db-cAMP caused a marked increase in both PNP hydroxylase and NDMA demethylase. In these strains a similar db-cAMP-mediated increase was also observed in the mutation frequency, resulting from the treatment with the promutagen NDMA, which is activated by CYP2E1. Our results show that CYP2E1 in V79 cells responds in two separate ways to db-cAMP exposure depending on the amino acid residue present in the PKA recognition sequence. The enzyme is committed to a negative regulation by db-cAMP if Ser129 is the target amino acid for PKA, leading to a decrease in the metabolic activation to mutagenic and carcinogenic species. On the other hand, Ala129 or Gly129 substitution directed CYP2E1 toward a positive regulation by increasing its catalytic activities and metabolic activation to mutagenic intermediates in the presence of db-cAMP. We also obtained evidence that cAMP-mediated downregulation of wild-type (Ser129) CYP2E1 was not accompanied by its destruction but instead by its stabilization, which shows that Ser129 is not involved in CYP2E1 degradation but dictates requirements for its specific activities.
Institute of Toxicology, University of Mainz, Germany. Oeschb@mzdmza.zdv.uni-mainz.de
B
B
Oesch-Bartlomowicz
P R
PR
Padma
R
R
Becker
B
B
Richter
J G
JG
Hengstler
J E
JE
Freeman
C R
CR
Wolf
F
F
Oesch
D019943:Amino Acid Substitution; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D019392:Cytochrome P-450 CYP2E1 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D004128:Dimethylnitrosamine / Q000493:pharmacokinetics; D004790:Enzyme Induction; D005347:Fibroblasts; D008658:Inactivation, Metabolic; D007538:Isoniazid / Q000494:pharmacology; D008168:Lung; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism; D009153:Mutagens / Q000493:pharmacokinetics; D009154:Mutation; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D012694:Serine / Q000737:chemistry / Q000502:physiology*; D013379:Substrate Specificity; D014162:Transfection
D009153:Mutagens; D012694:Serine; D003994:Bucladesine; D019392:Cytochrome P-450 CYP2E1; D017868:Cyclic AMP-Dependent Protein Kinases; D004128:Dimethylnitrosamine; D007538:Isoniazid