A distinct role of neutrophil lactoferrin in RelA/p65 phosphorylation on Ser536 by recruiting TNF receptor-associated factors to IkappaB kinase signaling complex.
The activation of NF-kappaB by neutrophil lactoferrin (Lf) is regulated via the IkappaB kinase (IKK) signaling cascade, resulting in the sequential phosphorylation and degradation of IkappaB. In this study, we observed that Lf protein augmented p65 phosphorylation at the Ser(536), but not the Ser(276) residue, and stimulated the translocation of p65 into the nucleus. Lf was also shown to enhance the association between p65 and CREB-binding protein/p300 in vivo. To elucidate the mechanism by which Lf triggers these signaling pathways, we attempted to delineate the roles of the upstream components of the IKK complex, using their dominant-negative mutants and IKKalpha(-/-) and IKKbeta(-/-) mouse embryonic cells. We demonstrated that both IKKalpha and IKKbeta as well as NF-kappaB-inducing kinase are indispensable for Lf-induced p65 phosphorylation. However, MAPK kinase kinase 1 is not essentially required for this activation. We also observed that Lf-induced p65 phosphorylation was either partially or completely abrogated as the result of treatment with the mutant forms of TNFR-associated factor (TRAF) 2, TRAF5, or TRAF6. Moreover, we demonstrated that Lf directly interacted with TRAF5. Expression of the dominant-negative mutant of TRAF5 or its small interfering RNA almost completely abrogated the Lf-induced p65 phosphorylation. These results suggest that signaling pathways, including TRAFs/NF-kappaB-inducing kinase/IKKs, may be involved in the regulation of Lf-induced p65 activation, thereby resulting in the activation of members of the NF-kappaB family.
Oh
Sang-Muk
SM
School of Life Sciences and Biotechnology, Korea University, Seoul, Korea.
Lee
Shin-Hee
SH
Lee
Bum-Jin
BJ
Pyo
Chul-Woong
CW
Yoo
Na-Kyung
NK
Lee
Soo Young
SY
Kim
Jiyoung
J
Choi
Sang-Yun
SY
D050882:CREB-Binding Protein / Q000378:metabolism; D002460:Cell Line; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism*; D007781:Lactoferrin / Q000494:pharmacology*; D009504:Neutrophils / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D051996:Transcription Factor RelA / Q000378:metabolism*; D015533:Transcriptional Activation / Q000235:genetics; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins / Q000378:metabolism*
D021122:Protein Subunits; D051996:Transcription Factor RelA; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins; D012694:Serine; D050882:CREB-Binding Protein; D051550:I-kappa B Kinase; D007781:Lactoferrin