Phosphatase inhibition leads to activation of IkappaB kinase in murine macrophages.
We have been interested in elucidating the role of intracellular phosphatase activity in the regulation of immune cell activation. To this end, we treated RAW 264.7 murine macrophages with the phosphatase inhibitor, calyculin-A. Treatment with calyculin-A led to activation of IkappaB kinase, degradation of IkappaBalpha, and induced nuclear translocation and DNA binding of NF-kappaB. Each of these effects occurred in both a time- and dose-dependent manner. In addition, each of these effects was negatively modulated by prior induction of the heat-shock response. Despite clear activation of the IkappaB kinase/IkappaBalpha/NF-kappaB pathway, however, phosphatase inhibition did not lead to increased expression of NF-kappaB-dependent genes. Thus, intracellular phosphatase activity is a central regulator of the NF-kappaB signal transduction pathway and is negatively modulated by heat shock. Inhibition of intracellular phosphatase activity with calyculin-A is not sufficient to induce NF-kappaB-dependent gene expression, demonstrating the complexity of NF-kappaB regulation in immune cells.
Grossman
Bruce J
BJ
Division of Critical Care Medicine, Cincinnati Children's Hospital Medical Center and The Children's Hospital Research Foundation, Division of Critical Care Medicine-OSB-5, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA.
Shanley
Thomas P
TP
Denenberg
Alvin G
AG
Zhao
Bin
B
Wong
Hector R
HR
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation; D051550:I-kappa B Kinase; D008156:Luciferases / Q000378:metabolism; D008264:Macrophages / Q000201:enzymology* / Q000378:metabolism; D008387:Marine Toxins; D051379:Mice; D010080:Oxazoles / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D021381:Protein Transport; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D004791:Enzyme Inhibitors; D008387:Marine Toxins; D010080:Oxazoles; D014409:Tumor Necrosis Factor-alpha; C059041:calyculin A; D004247:DNA; D008156:Luciferases; D017346:Protein Serine-Threonine Kinases; C496564:Chuk protein, mouse; D051550:I-kappa B Kinase; C496562:Ikbkb protein, mouse; C496566:Ikbke protein, mouse; D010744:Phosphoric Monoester Hydrolases