Investigation of interleukin 1beta-mediated regulation of NF-kappaB activation in colonic cells reveals divergence between PKB and PDK-transduced events.
Recent work has highlighted a role for PDK1 in adaptive immunity, however its contribution to innate immunity has not been addressed. We have investigated the role of PKB and PDK1 in IL-1beta-induced NF-kappaB activation. Over-expression of either in HCT 116 and HEK 293T cells, effected a reproducible NF-kappaB activation. This was validated in a one-hybrid assay utilizing Gal4-RelA and Gal4-luciferase assay. N-tosyl phenylalanyl chloromethyl ketone (TPCK), wortmannin and Ly294002 inhibited IL-1beta-induced NF-kappaB activation in both systems indicating involvement of the PI3K axis in this response. p65 (Rel A) Ser536 phosphorylation was not affected by the PI3K inhibitors but was dose-dependently attenuated by TPCK. Evaluation of IKK-associated activity using GST-p65 substrate phosphorylation in immune complex assays, revealed that whilst TPCK attenuated this, neither of the PI3K inhibitors had any effect. Furthermore whilst TPCK inhibited IL-1beta-induced p65 DNA binding, this was not apparent with either of wortmannin or Ly294002. Similarly, over-expression of PDK1 but not PKB resulted in promotion of p65 DNA binding. Using a p65-S536A reporter construct, we found inhibition of only PDK1 over-expression-induced, but not PKB over-expression-induced NF-kappaB activation. This was supported using biochemical analysis in which immunoprecipitated IKKgamma from IL-1beta-activated cells was unable to phosphorylate a p65-S536A substrate, confirming this as the dominant IKK-dependent site. In further support of a dissociated response, we observed an attenuation of the Ser177/181 IKK phosphorylation by TPCK but not in response to PI3K inhibition. Our data reveals for the first time that PDK1 and PKB may differentially activate NF-kappaB, and that TPCK may subserve a useful anti-inflammatory function by inhibiting IKKbeta.
Parhar
Kuljit
K
Division of Gastroenterology, Faculty of Medicine, The Jack Bell Research Centre, 2660 Oak Street, Vancouver, BC, Canada.
Eivemark
Sharlene
S
Assi
Kiran
K
Gómez-Muñoz
Antonio
A
Yee
Arthur
A
Salh
Baljinder
B
D064413:3-Phosphoinositide-Dependent Protein Kinases; D001665:Binding Sites; D003106:Colon / Q000201:enzymology* / Q000473:pathology*; D004247:DNA / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D015870:Gene Expression / Q000187:drug effects; D045325:HCT116 Cells; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism; D053583:Interleukin-1beta / Q000494:pharmacology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000494:pharmacology; D051996:Transcription Factor RelA / Q000235:genetics*; D015533:Transcriptional Activation / Q000187:drug effects
D064413:3-Phosphoinositide-Dependent Protein Kinases; D004247:DNA; D051550:I-kappa B Kinase; D053583:Interleukin-1beta; D019869:Phosphatidylinositol 3-Kinases; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D012694:Serine; D014108:Tosylphenylalanyl Chloromethyl Ketone; D051996:Transcription Factor RelA; C578562:PDPK1 protein, human