Wortmannin-sensitive pathway is required for insulin-stimulated phosphorylation of inhibitor kappaBalpha.
The aim of this study was to examine the signaling pathways by which insulin promotes activation of nuclear factor kappaB (NFkappaB) through the regulation of inhibitor kappaBalpha (IkappaBalpha). We show here that although insulin increased kappaB-dependent reporter gene expression and augmented nuclear translocation of the p65/RelA subunit of NFkappaB and its DNA binding, it was able to induce a time-dependent accumulation of phosphorylated and ubiquitinated IkappaBalpha without its proteolytic degradation. In contrast, cell stimulation with the cytokine TNFalpha allowed activation of NFkappaB through phosphorylation, ubiquitination, and subsequent degradation of IkappaBalpha. Immunofluorescence studies revealed the presence of a large pool of phosphorylated IkappaBalpha in the nucleus of unstimulated and insulin-treated cells. IkappaB kinase alpha and beta, central players in the phosphorylation of IkappaBalpha, were rapidly induced following exposure to TNFalpha but not insulin. Furthermore, insulin-stimulated IkappaBalpha phosphorylation did not depend on activation of the Ras/ERK cascade. Expression of a dominant-negative mutant of Akt1 or class I PI3K inhibited the insulin stimulation of PI3K/Akt1 signaling without affecting phosphorylation of IkappaBalpha. Interestingly, the PI3K inhibitors wortmannin and LY294002 blocked insulin-stimulated class I PI3K-dependent events at much lower doses than that required to inhibit phosphorylation of IkappaBalpha. These data demonstrate that insulin regulates IkappaBalpha function through a distinct low-affinity wortmannin-sensitive pathway.
Pandey
Sanjay K
SK
Diabetes Section, Laboratory of Clinical Investigation, National Institute on Aging, NIH, Baltimore, Maryland 21224-6825, USA.
He
Hua-Jun
HJ
Chesley
Alan
A
Juhaszova
Magdalena
M
Crow
Michael T
MT
Bernier
Michel
M
D000730:Androstadienes / Q000494:pharmacology*; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D020672:I-kappa B Proteins; D007328:Insulin / Q000494:pharmacology*; D008856:Microscopy, Fluorescence; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism; D000077191:Wortmannin
D000730:Androstadienes; D004268:DNA-Binding Proteins; D007004:Hypoglycemic Agents; D020672:I-kappa B Proteins; D007328:Insulin; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B; D019869:Phosphatidylinositol 3-Kinases; D010726:Phosphodiesterase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D019908:Proto-Oncogene Proteins c-raf; D014409:Tumor Necrosis Factor-alpha; D025801:Ubiquitin; D000077191:Wortmannin; C000606204:NFKBIA protein, human; C494918:AKT1 protein, human