Tumor necrosis factor-alpha-induced IKK phosphorylation of NF-kappaB p65 on serine 536 is mediated through the TRAF2, TRAF5, and TAK1 signaling pathway.
The activation of NF-kappaB has been shown to be regulated by multiple phosphorylations of IkappaBs and the NF-kappaB p65 subunit. Here, we characterized the intracellular signaling pathway leading to phosphorylation of p65 on Ser-536 using a novel anti-phospho-p65 (Ser-536) antibody. The Ser-536 of endogenous p65 was rapidly phosphorylated in response to a wide variety of NF-kappaB stimulants including TNF-alpha in the cytoplasm and rapidly dephosphorylated in the nucleus. The TNF-alpha-but not IL-1beta-induced Ser-536 phosphorylation was severely impaired in murine embryonic fibroblasts derived from traf2-/-traf5-/- mice. Bay 11-7082, an inhibitor of IkappaB phosphorylation, inhibited the TNF-alpha-induced phosphorylation in vivo. In addition, overexpression of TGF-beta-activated kinase 1 (TAK1), IKKalpha and IKKbeta stimulated the phosphorylation, and their dominant negative mutants blocked the TNF-alpha-induced phosphorylation. Moreover, small interfering RNAs (siRNAs) against TAK1, IKKalpha and IKKbeta blocked the phosphorylation of endogenous p65. On the other hand, calyculin-A, a protein phosphatase inhibitor, blocked the dephosphorylation in the nucleus in vivo. These results indicate that similar signaling pathways were utilized for the phosphorylations of IkappaBalpha and p65, which further support the idea that both IkappaB and NF-kappaB are substrates for the IKK complex in the activation of NF-kappaB.
Sakurai
Hiroaki
H
Department of Pathogenic Biochemistry, Institute of Natural Medicine, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Toyama 930-0194, Japan. hsakurai@ms.toyama-mpu.ac.jp
Suzuki
Shunsuke
S
Kawasaki
Noritaka
N
Nakano
Hiroyasu
H
Okazaki
Tatsuma
T
Chino
Atsushi
A
Doi
Takahiro
T
Saiki
Ikuo
I
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D005799:Genes, Dominant; D006367:HeLa Cells; D006801:Humans; D051550:I-kappa B Kinase; D015151:Immunoblotting; D051379:Mice; D008954:Models, Biological; D009154:Mutation; D016328:NF-kappa B / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D047992:TNF Receptor-Associated Factor 2; D048015:TNF Receptor-Associated Factor 5; D013997:Time Factors; D051996:Transcription Factor RelA; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D016328:NF-kappa B; D011506:Proteins; D034741:RNA, Small Interfering; D047992:TNF Receptor-Associated Factor 2; D048015:TNF Receptor-Associated Factor 5; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D012694:Serine; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; C496564:Chuk protein, mouse; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human; C496562:Ikbkb protein, mouse; C496566:Ikbke protein, mouse