DIFFERENTIAL MODULATION OF CYP2E1 ACTIVITY BY CAMP-DEPENDENT PROTEIN-KINASE UPON SER(129) REPLACEMENT

Many toxic compounds are activated by cytochrome P450 (CYP) 2E1 to rea ctive metabolites, which represents a potential hazard for cellular ho meostasis. Therefore knowledge about CYP2E1 regulation could be of gre at biological importance. It has been shown that CYP2E1 is controlled transcriptionally and posttranslationally by phosphorylation. In the p resent study we investigated the role of serine-129 (Ser(129)) in the protein kinase A (PKA) recognition sequence motif Arg-Arg-Phe-Ser(129) . To gain further insights into the possible relevance of Ser(129) for CYP2E1 function, Ser(129) was replaced by alanine (Ala) or glycine (G ly) by site-directed mutations of the cDNA coding for CYP2E1, The muta nt cDNAs were transfected into Chinese hamster lung fibroblast V79 cel ls, Despite the mutation in the PKA phosphorylation motif, all strains produced catalytically active CYP2E1. However, there was a marked cha nge in the substrate preference: The Gly(129)-containing strains hydro xylated p-nitrophenol (PNP) to a markedly higher extent than the wild- type cDNA-containing cells, while they demethylated N-nitrosodimethyla mine (NDMA) to a markedly lower extent than the wild-type cells. All t he strains activated NDMA to mutagenic products. Treatment with the me mbrane-permeating cAMP derivative db-cAMP reduced markedly both the PN P hydroxylase and the NDMA demethylase activities as well as the mutat ion frequency induced by NDMA in the Ser(129)-containing strain. This decrease in activity was not accompanied by a decrease in CYP2E1 conte nt. In addition, the catalytic activities of CYP2E1 were decreased in microsomes from rat hepatocytes treated with db-cAMP, Also in this cas e, the decrease in activities was not accompanied by a decrease in enz yme protein. These findings argue that involvement of Ser(129) and its phosphorylation is not in determining CYP2E1 protein level, but rathe r in controlling its catalytic activity. In contrast, in the strains c ontaining Ala(129) or Gly(129), treatment with db-cAMP caused a marked increase in both PNP hydroxylase and NDMA demethylase. In these strai ns a similar db-cAMP-mediated increase was also observed in the mutati on frequency, resulting from the treatment with the promutagen NDMA, w hich is activated by CYP2E1. Our results show that CYP2E1 in V79 cells responds in two separate ways to db-cAMP exposure depending on the am ino acid residue present in the PKA recognition sequence. The enzyme i s committed to a negative regulation by db-cAMP if Ser(129) is the tar get amino acid for PKA, leading to a decrease in the metabolic activat ion to mutagenic and carcinogenic species. On the other hand, Ala(129) or Gly(129) substitution directed CYP2E1 toward a positive regulation by increasing its catalytic activities and metabolic activation to mu tagenic intermediates in the presence of db-cAMP, We also obtained evi dence that cAMP-mediated downregulation of wild-type (Ser(129)) CYP2E1 was not accompanied by its destruction but instead by its stabilizati on, which shows that Ser(129) is not involved in CYP2E1 degradation bu t dictates requirements for its specific activities. (C) 1998 Academic Press.