INVOLVEMENT OF SYNTHESIS AND PHOSPHORYLATION OF NUCLEAR-PROTEIN FACTORS THAT BIND TO THE POSITIVE CIS-ACTING ELEMENT IN THE TRANSCRIPTIONALACTIVATION OF THE CYP2B1 B2 GENE BY PHENOBARBITONE IN-VIVO/
Cs. Nirodi et al., INVOLVEMENT OF SYNTHESIS AND PHOSPHORYLATION OF NUCLEAR-PROTEIN FACTORS THAT BIND TO THE POSITIVE CIS-ACTING ELEMENT IN THE TRANSCRIPTIONALACTIVATION OF THE CYP2B1 B2 GENE BY PHENOBARBITONE IN-VIVO/, Archives of biochemistry and biophysics, 331(1), 1996, pp. 79-86
The synthesis and phosphorylation of protein factor(s) that bind to th
e positive cis-acting element (-69 to -98 nt) of the CYP2B1/B2 gene ha
ve been examined in vivo in the rat. Treatment of rats with cyclohexim
ide, a protein synthetic inhibitor, suppresses basal as well as phenob
arbitone-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 by gel shift assays
. Treatment of rats with a-aminopurine, a general protein kinase inhib
itor, blocks the phenobarbitone-mediated increase in CYP2B1/B2 mRNA ce
ll-free transcription of a minigene construct containing the positive
element, pP450e179DNA, and binding of nuclear proteins to the positive
element. Treatment of rats with okadaic acid, a protein phosphatase i
nhibitor, mimics the effects of phenobarbitone, but only partially. Th
us, both phenobarbitone and okadaic acid individually enhance binding
of the nuclear protein(s) to the positive element, cell-free transcrip
tion of the minigene construct, and phosphorylation of the similar to
28- and 94-kDa proteins binding to the positive element. But unlike ph
enobarbitone, okadaic acid is not an inducer of CYP2B1/B2 mRNA or its
run-on transcription. Thus, phenobarbitone-responsive positive element
interactions constitute only a minimal requirement, and okadaic acid
is perhaps not able to bring about the total requirement for activatio
n of CYP2B1/B2 gene transcription that should include interaction betw
een the minimal promoter and further upstream elements. An intriguing
feature is the antagonistic effect of okadaic acid on phenobarbitone-m
ediated effects on CYP2B1/B2 mRNA levels, cell-free and run-on transcr
iption, and nuclear protein binding to the positive element. The reaso
n for this antagonism is not clear. It is concluded that phenobarbiton
e treatment enhances in vivo the synthesis and phosphorylation of prot
ein factors binding to the positive element and these constitute a min
imal requirement for the transcriptional activation of the CYP2B1/B2 g
ene. (C) 1996 Academic Press, Inc.