Kameoka M, Ota K, Tetsuka T, Tanaka Y, Itaya A, Okamoto T, Yoshihara K
Department of Biochemistry, Nara Medical University, Kashihara, Nara 634-8521, Japan.
Biochem J. 2000 Mar 15;346 Pt 3(Pt 3):641-9.
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.
聚（ADP - 核糖）聚合酶（PARP）缺陷型突变L1210细胞克隆的核提取物中NF-κB的DNA结合活性显著增加，且与这些细胞中的PARP含量呈负相关。在PARP含量最低的克隆（Cl-3527，仅含野生型细胞PARP的6%）中，NF-κB的DNA结合活性约为野生型细胞的35倍，而突变体中AP-1和SP-1的DNA结合活性变化相对较小或不显著。将PARP表达质粒转染至突变细胞可使异常高水平的NF-κB复合物，尤其是p50/p65（Rel A）复合物降至接近正常水平。此外，体外对核提取物进行聚（ADP - 核糖）基化可使NF-κB与其特异性DNA探针形成复合物的能力降低约80%。对纯化的重组NF-κB蛋白进行进一步分析发现，rp50和rMBP-p65（Rel A）蛋白在体外均可被聚（ADP - 核糖）基化，但rGST-IκB不能，这种修饰导致rMBP-p65的DNA结合活性显著降低，而rp50则出现轻微激活。通过用抗聚（ADP - 核糖）抗体和抗p65抗体进行免疫印迹，在野生型L1210细胞的细胞质中检测到了聚（ADP - 核糖）基化的p65/NF-κB。综上所述，这些结果有力地表明PARP通过蛋白质修饰参与了NF-κB的调节。
