ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 331, No. 1, July 1, pp. 79–86, 1996 Article No. 0285 Involvement of Synthesis and Phosphorylation of Nuclear Protein Factors That Bind to the Positive cis-Acting Element in the Transcriptional Activation of the CYP2B1/B2 Gene by Phenobarbitone in Vivo C. S. Nirodi, S. Sultana, N. Ram, L. Prabhu, and G. Padmanaban 1 Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India Received January 29, 1996, and in revised form April 10, 1996 binding to the positive element. The reason for this antagonism is not clear. It is concluded that phenobar- The synthesis and phosphorylation of protein fac- bitone treatment enhances in vivo the synthesis and tor(s) that bind to the positive cis-acting element (069 phosphorylation of protein factors binding to the posi- to 098 nt) of the CYP2B1/B2 gene have been examined tive element and these constitute a minimal require- in vivo in the rat. Treatment of rats with cyclohexi- ment for the transcriptional activation of the CYP2B1/ mide, a protein synthetic inhibitor, suppresses basal B2 gene.  1996 Academic Press, Inc. as well as phenobarbitone-induced levels of CYP2B1/ B2 mRNA and its run-on transcription. Under these conditions, complex formation of the nuclear extract with the positive element is also inhibited, as judged The cytochrome P450 group of proteins constitute a by gel shift assays. Treatment of rats with 2-aminopu- multigene superfamily involved in the metabolism of a rine, a general protein kinase inhibitor, blocks the phenobarbitone-mediated increase in CYP2B1/B2 wide variety of compounds of exogenous and endoge- mRNA, cell-free transcription of a minigene construct nous origin (1). These have been classified into 36 dis- containing the positive element, pP450e179DNA, and tinct families, 10 of which exist in mammals (2). Many binding of nuclear proteins to the positive element. members of the family are inducible. The mechanism Treatment of rats with okadaic acid, a protein phos- of induction of the CYP1A1 gene in the rat by polycyclic phatase inhibitor, mimics the effects of phenobarbi- aromatic hydrocarbons is reasonably well understood. tone, but only partially. Thus, both phenobarbitone It involves the binding of the ligand to a cytosolic recep- and okadaic acid individually enhance binding of the tor, translocation to the nucleus in an active protein nuclear protein(s) to the positive element, cell-free assembly, and interaction with 5-upstream DNA ele- transcription of the minigene construct, and phos- ments to activate the gene (3, 4). The details regarding phorylation of the Ç26- and 94-kDa proteins binding the transcriptional activation of CYP2B1/B2 gene by to the positive element. But unlike phenobarbitone, the prototype inducer, phenobarbitone (PB), 2 are just okadaic acid is not an inducer of CYP2B1/B2 mRNA or beginning to emerge. its run-on transcription. Thus, phenobarbitone-re- Studies in this laboratory have led to the identifica- sponsive positive element interactions constitute only tion of PB-responsive, positive (069 to 098 nt), and a minimal requirement, and okadaic acid is perhaps negative cis-elements of the CYP2B2 gene (5–7). The not able to bring about the total requirement for acti- positive element includes the 17-nt barbiturate-respon- vation of CYP2B1/B2 gene transcription that should sive consensus element (barbie box), first identified by include interaction between the minimal promoter Fulco and co-workers in B. megaterium and the rat and further upstream elements. An intriguing feature (8, 9). The barbie box has also been identified in the is the antagonistic effect of okadaic acid on phenobar- barbiturate-responsive cytochrome P450 gene in S. bitone-mediated effects on CYP2B1/B2 mRNA levels, cell-free and run-on transcription, and nuclear protein 2 Abbreviations used: PB, phenobarbitone; OK, okadaic acid; AP, 1 To whom correspondence should be addressed. Fax: (080) 2-aminopurine; nt, nucleotide; PEPCK, phosphoenolpyruvate car- boxykinase. 3341683. 79 0003-9861/96 $18.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved.