Evidence for regulation of NF-kappaB by poly(ADP-ribose) polymerase.
The DNA-binding activity of NF-kappaB in nuclear extracts of poly(ADP-ribose) polymerase (PARP)-defective mutant L1210 cell clones was markedly increased and was inversely correlated with the PARP content in these cells. The DNA-binding activity of NF-kappaB in a clone with the lowest PARP content (Cl-3527, contained 6% of PARP of wild type cells) was about 35-fold of that of the wild-type cells, whereas the change in the DNA-binding activity of AP-1 and SP-1 in the mutant was relatively small or not so significant. Transfection of a PARP-expressing plasmid to the mutant cells decreased the abnormally high levels of NF-kappaB complexes, especially p50/p65(Rel A) complex, to near the normal level. Moreover, poly(ADP-ribosyl)ation of nuclear extracts in vitro suppressed the ability of NF-kappaB to form a complex with its specific DNA probe by approx. 80%. Further analysis with purified recombinant NF-kappaB proteins revealed that both rp50 and rMBP-p65 (Rel A) proteins, but not rGST-IkappaB, could be poly(ADP-ribosyl)ated in vitro and that the modification resulted in a marked decrease in the DNA-binding activity of rMBP-p65, whereas a slight activation was observed in rp50. Poly(ADP-ribosyl)ated p65/NF-kappaB was detected in the cytosol of wild type L1210 cells by immunoblotting with anti-poly(ADP-ribose) and anti-p65 antibodies. Taken together, these results strongly suggest that PARP is involved in the regulation of NF-kappaB through the protein modification.
Kameoka
M
M
Department of Biochemistry, Nara Medical University, Kashihara, Nara 634-8521, Japan.
Ota
K
K
Tetsuka
T
T
Tanaka
Y
Y
Itaya
A
A
Okamoto
T
T
Yoshihara
K
K
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D006801:Humans; D007939:Leukemia L1210 / Q000378:metabolism / Q000473:pathology; D016328:NF-kappa B / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D016328:NF-kappa B; D011994:Recombinant Proteins; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases