Cysteine-179 of I kappa B kinase beta plays a critical role in enzyme activation by promoting phosphorylation of activation loop serines

I kappa B kinase beta (IKK beta) subunit of IKK complex is essential for the activation of NF-kappa B in response to various proinflammatory signals. Cys-179 in the activation loop of IKK beta is known to be the target site for IKK inhibitors such as cyclopentenone prostaglandins, arsenite, and antirheumatic gold compounds. Here we show that a mutant IKK beta in which Cys-179 is substituted with alanine had decreased activity when it was expressed in HEK-293 cells, and TNF stimulation did not restore the activity. Phosphorylation of activation loop serines (Ser-177 and Ser-181) which is required for IKK beta activation was reduced in the IKK beta (C179A) mutant. The activity of IKK beta (C179A) was partially recovered when its phosphorylation was enforced by coexpression with mitogen-activated protein kinase kinase kinases (MAPKKK) such as NF-kappa B 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 IKK beta (C179A) mutant was normal in dimer formation, while its activity abnormally responded to the change in the concentration of substrate ATP in reaction mixture. Our results suggest that Cys-179 of IKK beta plays a critical role in enzyme activation by promoting phosphorylation of activation-loop serines and interaction with ATP.

Experimental Study on the Preventive Mechanism of Anisodamine on Multiple Organ Dysfunction Syndrome / 中国医师杂志

Objective To investigate the preventive mechanism of Anisodamine (654-2) on multiple organ dysfunction syndrome (MODS) of rabbits. Methods Rabbit model of MODS induced by hemorrhagic shock and endotoxin was used in this study. Twenty-four rabbits were randomly divided into the control group (C group) , hemorrhagic shock plus endotoxin group (M group) and 654-2 treatment group (T group). The expression of IKK-? of pulmonary alveolar macrophage (PAM) and kuffer cell(KC), the NF-?B activity of nuclear protein extracted from PAM and KC and the concentration of tumor necrosis factor-? (TNF-?) in the culture supernatant were measured by in situ hybridization (ISH), electrophoretic mobility shift assay (EMSA) and enzyme linked immune absorbent analysis(ELISA), respectively. Then the blood air, biochemical and pathological changes in visceral organs were examined in each groups. Results In PAM and KC of M group, The expression of IKK-? mRNA [(0 15?0 03);(0 17?0 04)], the activity of NF-?B [(1 49?0 30);(1 72?0 36)] and the secretion level of TNF-? [( 279 74?25 91);(300 05?30 86)ng/L] were significantly higher than those of control group (P

Expression and Significance of I?B kinase-? mRNA in the Lung Tissues of Rabbits Following Hemorrhagic Shock / 中国医师杂志

Objective To investigate the changes and significance of I?B kinase-?(I?K-?) in the lung tissues of rabbits with hemorrhagic shock.Methods The expressions of I?K-? and NF-?B in the lung tissues and the concentration of tumor necrosis factor-?(TNF-?) in the plasma were measured by in situ hybridization(ISH),immunohitochemistry and enzyme linked immune adsorbent analysis(ELISA), respectively. And the pathological changes were examined with light microscope in lung tissues.Results In hemorrhagic shock group,the expressions of I?K-?(0 1685?0 0164)and NF-?B( 0 1469?0 0083)in lung tissues , the level of TNF-?(636 72?100 23) in the plasma were obviously higher than those of normal group [I?K-?(0 0427?0 0241),NF-?B(0 0358?0 0048),TNF-?(199 51?35 69)ng/L](all P

THE CHANGES IN NF-?B SIGNAL PATHWAYS IN PULMONARY ALVEOLAR MACROPHAGES (PAM) STIMULATED BY LPS / 解放军医学杂志

Objective To investigate changes in NF ?B signal pathway in PAM stimulated by lipopolysaccharide(LPS) in vitro , and to explore the molecular pathological mechanism of acute lung injury(ALI). Methods After PAM were stimulated by LPS, the changes in expression of IKK mRNA, activation of NF ?B, degradation of I?B, and secretion of TNF ? in PAM were measured at 0, 15min, 30min, 1h, 2h, and 4h by in situ hybridization, electrophoretic mobility shift assay (EMSA), and enzyme linked immune absorbing analysis (ELISA), respectively. Results The expression of IKK ? mRNA was increased 15min after LPS stimulation and reached the peak at 30 min, then returned to the base line after 1 hour. The changes in I?B ? mRNA were opposite. The activity of NF ?B was increased 15min after LPS stimulation, peaking at 1 hours, and returned to the pre stimulation level after 2 hours. The content of TNF ? was increased initially at 30min, reached the peak at 1 hour, and gradually returned to the pre stimulation level in 2～4 hour. Conclusion The transduction pathway of activation of IKK ? degradation of I?B/activation of NF ?B/synthesization of TNF ? might play a critical role in the molecular pathological mechanism of LPS induced ALI.

Function of I?B kinase links inflammation and cancer / 中国病理生理杂志

It has long been acknowledged that there is a link between inflammation and cancer, but its molecular mechanism remains unclear. A key player in inflammation is nuclear transcription factor NF-?B, that activity is triggered in response to infectious agents and proinflammatory cytokines via the I?B kinase. In parenchyma cells, inflammation through I?B kinase/NF-?B pathway suppresses apoptosis, accelerates cell cycle, then promote tumorigenesis. In mesenchyma cells inflammation through I?B kinase/NF-?B pathway produces cytokines and chemokines that may serve as tumor growth factors. To sum up, I?B kinase/NF -?B pathway represents a critical molecular link between inflammation and cancer.