scholarly journals Phosphoinositide 3 Kinase Mediates Toll-Like Receptor 4-Induced Activation of NF-κB in Endothelial Cells

2003 ◽  
Vol 71 (8) ◽  
pp. 4414-4420 ◽  
Author(s):  
Xianwu Li ◽  
Joan C. Tupper ◽  
Douglas D. Bannerman ◽  
Robert K. Winn ◽  
Christopher J. Rhodes ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Kinase Activity ◽  
Luciferase Activity ◽  
Pi3 Kinase ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Toll Like Receptor 4 ◽  
Kinase Activation ◽  
Phosphoinositide 3 Kinase ◽  
Negative Mutant

ABSTRACT Many of the proinflammatory effects of gram-negative bacteria are elicited by the interaction of bacterial lipopolysaccharide (LPS) with Toll-like receptor 4 (TLR4) expressed on host cells. TLR4 signaling leads to activation of NF-κB and transcription of many genes involved in the inflammatory response. In this study, we examined the signaling pathways involved in NF-κB activation by TLR4 signaling in human microvascular endothelial cells. Akt is a major downstream target of phosphoinositide 3 kinase (PI3-kinase), and PI3-kinase activation is necessary and sufficient for Akt phosphorylation. Consequently, Akt kinase activation was used as a measure of PI3-kinase activity. In a stable transfection system, dominant-negative mutants of myeloid differentiation factor 88 (MyD88) and interleukin-1 (IL-1) receptor-associated kinase 1 (IRAK-1) (MyD88-TIR and IRAK-DD, respectively) blocked Akt kinase activity in response to LPS and IL-1β. A dominant-negative mutant (Mal-P/H) of MyD88 adapter-like protein (Mal), a protein with homology to MyD88, failed to inhibit LPS- or IL-1β-induced Akt activity. Moreover, a dominant-negative mutant of p85 (p85-DN) inhibited the NF-κB luciferase activity, IL-6 production, and IκBα degradation elicited by LPS and IL-1β but not that stimulated by tumor necrosis factor alpha. The dominant-negative mutant of Akt partially inhibited the NF-κB luciferase activity evoked by LPS and IL-1β. However, expression of a constitutively activated Akt failed to induce NF-κB luciferase activity. These findings indicate that TLR4- and IL-1R-induced PI3-kinase activity is mediated by the adapter proteins MyD88 and IRAK-1 but not Mal. Further, these studies suggest that PI3-kinase is an important mediator of LPS and IL-1β signaling leading to NF-κB activation in endothelial cells and that Akt is necessary but not sufficient for NF-κB activation by TLR4.

scholarly journals The 14-3-3 Protein Translates the NA+,K+-ATPase α1-Subunit Phosphorylation Signal into Binding and Activation of Phosphoinositide 3-Kinase during Endocytosis

2005 ◽  
Vol 280 (16) ◽  
pp. 16272-16277 ◽  
Author(s):  
Riad Efendiev ◽  
Zongpei Chen ◽  
Rafael T. Krmar ◽  
Sabine Uhles ◽  
Adrian I. Katz ◽  
...  
Keyword(s):  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Α Subunit ◽  
Opossum Kidney ◽  
Rich Domain ◽  
Opossum Kidney Cells ◽  
Basolateral Plasma Membrane ◽  
Functional Relevance ◽  
Phosphoinositide 3 Kinase ◽  
Negative Mutant

Clathrin-dependent endocytosis of Na+,K+-ATPase molecules in response to G protein-coupled receptor signals is triggered by phosphorylation of the α-subunit and the binding of phosphoinositide 3-kinase. In this study, we describe a molecular mechanism linking phosphorylation of Na+,K+-ATPase α-subunit to binding and activation of phosphoinositide 3-kinase. Co-immunoprecipitation studies, as well as experiments using confocal microscopy, revealed that dopamine favored the association of 14-3-3 protein with the basolateral plasma membrane and its co-localization with the Na+,K+-ATPase α-subunit. The functional relevance of this interaction was established in opossum kidney cells expressing a 14-3-3 dominant negative mutant, where dopamine failed to decrease Na+,K+-ATPase activity and to promote its endocytosis. The phosphorylated Ser-18 residue within the α-subunit N terminus is critical for 14-3-3 binding. Activation of phosphoinositide 3-kinase by dopamine during Na+,K+-ATPase endocytosis requires the binding of the kinase to a proline-rich domain within the α-subunit, and this effect was blocked by the presence of a 14-3-3 dominant negative mutant. Thus, the 14-3-3 protein represents a critical linking mechanism for recruiting phosphoinositide 3-kinase to the site of Na+,K+-ATPase endocytosis.

TNF-α induces protein synthesis through PI3-kinase-Akt/PKB pathway in cardiac myocytes

2001 ◽  
Vol 280 (4) ◽  
pp. H1861-H1868 ◽  
Author(s):  
Eiji Hiraoka ◽  
Seinosuke Kawashima ◽  
Tomosaburo Takahashi ◽  
Yoshiyuki Rikitake ◽  
Tadahiro Kitamura ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Cardiac Myocytes ◽  
Specific Inhibitor ◽  
Pi3 Kinase ◽  
Dominant Negative ◽  
Neonatal Rat ◽  
Dominant Negative Mutant ◽  
Tnf Α ◽  
Negative Mutant ◽  
Stimulation Of

The activation of phosphatidylinositol (PI) 3-kinase and Akt/protein kinase B (PKB) by tumor necrosis factor (TNF)-α and their roles on stimulation of protein synthesis were investigated in cultured neonatal rat cardiac myocytes. Treatment of cells with TNF-α resulted in enlargement of cell surface area and stimulation of protein synthesis without affecting myocyte viability. TNF-α induced marked activation of PI3-kinase and Akt/PKB, and the activation of PI3-kinase and Akt/PKB was rapid (maximal at 10 and 15 min, respectively) and concentration dependent. Akt/PKB activation by TNF-α was inhibited by a PI3-kinase-specific inhibitor LY-294002 and adenovirus-mediated expression of a dominant negative mutant of PI3-kinase, indicating that TNF-α activates Akt/PKB through PI3-kinase activation. Furthermore, TNF-α-induced protein synthesis was inhibited by pretreatment with LY-294002 and expression of a dominant negative mutant of PI3-kinase or Akt/PKB. These results indicate that activation of the PI3-kinase-Akt/PKB pathway plays an essential role in protein synthesis induced by TNF-α in cardiac myocytes.

scholarly journals Exploring the Functional Interactions between Aurora B, INCENP, and Survivin in Mitosis

2003 ◽  
Vol 14 (8) ◽  
pp. 3325-3341 ◽  
Author(s):  
Reiko Honda ◽  
Roman Körner ◽  
Erich A. Nigg
Keyword(s):  
Chromosome Segregation ◽  
Kinase Activity ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Aurora B ◽  
Mitotic Progression ◽  
Aurora B Kinase ◽  
Kinase Activation ◽  
Central Spindle ◽  
Assay Conditions

The function of the Aurora B kinase at centromeres and the central spindle is crucial for chromosome segregation and cytokinesis, respectively. Herein, we have investigated the regulation of human Aurora B by its complex partners inner centromere protein (INCENP) and survivin. We found that overexpression of a catalytically inactive, dominant-negative mutant of Aurora B impaired the localization of the entire Aurora B/INCENP/survivin complex to centromeres and the central spindle and severely disturbed mitotic progression. Similar results were also observed after depletion, by RNA interference, of either Aurora B, INCENP, or survivin. These data suggest that Aurora B kinase activity and the formation of the Aurora B/INCENP/survivin complex both contribute to its proper localization. Using recombinant proteins, we found that Aurora B kinase activity was stimulated by INCENP and that the C-terminal region of INCENP was sufficient for activation. Under identical assay conditions, survivin did not detectably influence kinase activity. Human INCENP was a substrate of Aurora B and mass spectrometry identified three consecutive residues (threonine 893, serine 894, and serine 895) containing at least two phosphorylation sites. A nonphosphorylatable mutant (TSS893–895AAA) was a poor activator of Aurora B, demonstrating that INCENP phosphorylation is important for kinase activation.

scholarly journals Dynamin 2 Is Required for Phagocytosis in Macrophages

1999 ◽  
Vol 190 (12) ◽  
pp. 1849-1856 ◽  
Author(s):  
Elizabeth S. Gold ◽  
David M. Underhill ◽  
Naomi S. Morrissette ◽  
Jian Guo ◽  
Mark A. McNiven ◽  
...  
Keyword(s):  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Membrane Extension ◽  
Large Particles ◽  
Particle Binding ◽  
Phosphoinositide 3 Kinase ◽  
Endocytic Vesicles ◽  
Particle Internalization ◽  
Dynamin 2 ◽  
Negative Mutant

Cells internalize soluble ligands through endocytosis and large particles through actin-based phagocytosis. The dynamin family of GTPases mediates the scission of endocytic vesicles from the plasma membrane. We report here that dynamin 2, a ubiquitously expressed dynamin isoform, has a role in phagocytosis in macrophages. Dynamin 2 is enriched on early phagosomes, and expression of a dominant-negative mutant of dynamin 2 significantly inhibits particle internalization at the stage of membrane extension around the particle. This arrest in phagocytosis resembles that seen with inhibitors of phosphoinositide 3-kinase (PI3K), and inhibition of PI3K prevents the recruitment of dynamin to the site of particle binding. Although expression of mutant dynamin in macrophages inhibited particle internalization, it had no effect on the production of inflammatory mediators elicited by particle binding.

scholarly journals A Dominant-negative Mutant of Androgen Receptor Coregulator ARA54 Inhibits Androgen Receptor-mediated Prostate Cancer Growth

2001 ◽  
Vol 277 (7) ◽  
pp. 4609-4617 ◽  
Author(s):  
Hiroshi Miyamoto ◽  
Mujib Rahman ◽  
Hiroshi Takatera ◽  
Hong-Yo Kang ◽  
Shuyuan Yeh ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Androgen Receptor ◽  
Dominant Negative ◽  
Cancer Growth ◽  
Dominant Negative Mutant ◽  
Negative Mutant

scholarly journals NF-κB-Inducing Kinase (NIK) Mediates Skeletal Muscle Insulin Resistance: Blockade by Adiponectin

Endocrinology ◽  
2011 ◽  
Vol 152 (10) ◽  
pp. 3622-3627 ◽  
Author(s):  
Sanjeev Choudhary ◽  
Sandeep Sinha ◽  
Yanhua Zhao ◽  
Srijita Banerjee ◽  
Padma Sathyanarayana ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Skeletal Muscle ◽  
Glucose Uptake ◽  
Kinase Activity ◽  
Pi3 Kinase ◽  
Dominant Negative ◽  
Muscle Insulin Resistance ◽  
Akt Phosphorylation ◽  
Skeletal Muscle Insulin Resistance ◽  
Obese Subjects

Enhanced levels of nuclear factor (NF)-κB-inducing kinase (NIK), an upstream kinase in the NF-κB pathway, have been implicated in the pathogenesis of chronic inflammation in diabetes. We investigated whether increased levels of NIK could induce skeletal muscle insulin resistance. Six obese subjects with metabolic syndrome underwent skeletal muscle biopsies before and six months after gastric bypass surgery to quantitate NIK protein levels. L6 skeletal myotubes, transfected with NIK wild-type or NIK kinase-dead dominant negative plasmids, were treated with insulin alone or with adiponectin and insulin. Effects of NIK overexpression on insulin-stimulated glucose uptake were estimated using tritiated 2-deoxyglucose uptake. NF-κB activation (EMSA), phosphatidylinositol 3 (PI3) kinase activity, and phosphorylation of inhibitor κB kinase β and serine-threonine kinase (Akt) were measured. After weight loss, skeletal muscle NIK protein was significantly reduced in association with increased plasma adiponectin and enhanced AMP kinase phosphorylation and insulin sensitivity in obese subjects. Enhanced NIK expression in cultured L6 myotubes induced a dose-dependent decrease in insulin-stimulated glucose uptake. The decrease in insulin-stimulated glucose uptake was associated with a significant decrease in PI3 kinase activity and protein kinase B/Akt phosphorylation. Overexpression of NIK kinase-dead dominant negative did not affect insulin-stimulated glucose uptake. Adiponectin treatment inhibited NIK-induced NF-κB activation and restored insulin sensitivity by restoring PI3 kinase activation and subsequent Akt phosphorylation. These results indicate that NIK induces insulin resistance and further indicate that adiponectin exerts its insulin-sensitizing effect by suppressing NIK-induced skeletal muscle inflammation. These observations suggest that NIK could be an important therapeutic target for the treatment of insulin resistance associated with inflammation in obesity and type 2 diabetes.

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