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변미선,최진,주대명 생화학분자생물학회 2006 Experimental and molecular medicine Vol.38 No.5
IκB kinase β (IKKβ) subunit of IKK complex is essential for the activation of NF-κB in response to various proinflammatory signals. Cys-179 in the activation loop of IKKβ is known to be the target site for IKK inhibitors such as cyclopentenone pros-taglandins, arsenite, and antirheumatic gold com-pounds. Here we show that a mutant IKKβ in which Cys-179 is substituted with alanine had decreased activity when it was exprTNF stimulation did not restore the activity. Phos-phorylation of activation loop serines (Ser-177 and Ser-181) which is required for IKKβ activation was reduced in the IKKβ (C179A) mutant. The activity of IKKβ (C179A) was partially recovered when its phosphorylation was enforced by coexpression with mitogen-activated protein kinase kinase kinases (MAPKKK) such as NF-κB inducing kinase (NIK) and MAPK/extracellular signal-regulated kinase kinase kinase 1(MEKK1) or when the serine residues were replaced with phospho-mimetic glutamate. The IKKβ (C179A) mutant was normal in dimer formation, while its activity abnormally responded to the change in the concentration of substrate Our results suggest that Cys-179 of IKKβ plays a critical role in enzyme activation by promoting phosphorylation of activation-loop serines and interaction with ATP.
Dual effect of oxidative stress on NF-κB activation in HeLa cells
변미선,전계임,최재원,심재용,주대명 생화학분자생물학회 2002 Experimental and molecular medicine Vol.34 No.5
CD9 plays an critical role in the diapedesis of port of MHC molecules. Engagement of CD9 by agonistic monoclonal antibodies has been re-ported to triger multifactorial events including T cell activation as well as cell-cell adhesion during hematopoietic cell diferentiation. In this study, to identify the functional domains partici-pating in the celular events, we mapped the epitopes of CD99, which are recognized by two agonistic CD99 monoclonal antibodies, DN16 and YG32. Using recombinant fusion proteins of GST with whole or parts of CD9, we found that both antibodies interact with CD9 molecules independently of sugar moieties. DN16 mAb de-tected a linear epitope located in the amino terminal region of CD99 while YG32 mAb bound another linear epitope in the center of the extra-celular domain. To confirm that the identified epitopes of CD99 are actualy recognized by the two mAbs, we showed the presence of physical interaction betwen the mAbs and the fusion proteins or synthetic peptides containing the corresponding epitopes using surface plasmon resonance analyses. The dissociation constants of DN16 and YG32 mAbs for the antigen were calculated as 1.2710-7 and 7.0810-9 M, re-spectively. These studies will help understand the functional domains and the subsequent signaling mechanism of CD99.
김경운,조미라,이상헌,민소연,박미경,박성환,주대명,김호연,Kim, Kyoung-Woon,Cho, Mi-La,Lee, Sang-Heon,Min, So-Youn,Park, Mi Kyung,Park, Sung-Hwan,Jue, Dae-Myung,Kim, Ho-Youn 대한면역학회 2003 Immune Network Vol.3 No.4
Inflammatory mediators has been recognized as an important role in the pathogenesis of rheumatoid arthritis (RA). IL-17 is increasingly recognized as an important regulator of immune and inflammatory responses, including induction of proinflammatory cytokines and osteoclastic bone resorption. Evidence of the expression and proinflammatory activity of IL-17 has been demonstrated in RA synovium and in animal models of RA. However, the signaling pathways that regulate IL-17 production remain unknown. In the present study, we investigated the role of the phosphatidylinositol 3 kinase (PI3K)-Akt pathway in the regulation of IL-17 production in RA. PBMC were separated from RA (n=24) patients, and stimulated with various agents (anti CD3, anti CD28, PHA, ConA, IL-15). IL-17 levels were determined by sandwich ELISA and RT-PCR. The production of IL-17 was significantly increased in cells treated with anti-CD3 antibody, PHA, IL-15 or MCP-1 (P<0.05). ConA also strongly induced IL-17 production (P<0.001), whereas TNF-alpha, IL-1beta, IL-18 or TGF-beta did not. IL-17 was detected in the PBMC of patients with osteoarthritis (OA) but their expression levels were much lower than those of RA PBMC. Anti-CD3 antibody activated the PI3K-Akt pathway and activation of the PI3K-Akt pathway resulted in a pronounced augmentation of nuclear factor kappaB ($NF-{\kappa}B$). IL-17 production by activated PBMC in RA is completely or partially blocked in the presence of $NF-{\kappa}B$ inhibitor PDTC and PI3K-Akt inhibitor, wortmannin and LY294002, respectively. Whereas the inhibition of AP-1 and extracellular signal-regulated kinase (ERK)1/2 did not affect IL-17 production. These results provide new insight into that PI3K/Akt and $NF-{\kappa}B$ dependent signal transduction pathway could be involved in the overproduction of key inflammatory cytokine, IL-17 in rheumatoid arthritis.
Yeji Lee,Jin Choi,Kyung-Ho Ha,주대명 생화학분자생물학회 2012 Experimental and molecular medicine Vol.44 No.8
During ischemia-reperfusion injury, brief pre-exposure to oxidative stress renders organs resistant to subsequent severe damage. NF-κB is a transcription factor that is involved in reperfusion-induced inflammatory and immune responses. The activity of NF-κB has been shown to be modulated by oxidative stress in various cell types through different pathways. We studied the effect of pre-exposure to oxidative stress on subsequent NF-κB activation in TNFα-stimulated HEK293 cells. The cells were transiently exposed to 0.5 mM H2O2 for 20 min, prior to stimulation with TNFα, and the subsequent expression of NF-κB-dependent genes and the levels of NF-κB signaling molecules were measured. Pre-exposure to H2O2 significantly delayed the TNFα-induced expression of an NF-κB reporter gene and inflammatory proteins (intercellular adhesion molecule-1 and IL-1β). The degradation of inhibitor of NF-κB α (IκBα) and the nuclear translocation of NF-κB were also delayed by H2O2 treatment,whereas IκBα phosphorylation and IκB kinase activity were not changed. When we examined the ubiquitin/proteosome pathway in H2O2-treated cells,we could not detect significant changes in proteosomal peptidase activities, but we were able to detect a delay of IκBα poly-ubiquitination. Our results suggest that transient exposure to oxidative stress temporally inhibits NF-κB-dependent gene expression by suppressing the poly-ubiquitination of phosphorylated IκBα in HEK293 cells.