Molecular and Cellular Biochemistry

, Volume 300, Issue 1–2, pp 113–127 | Cite as

Investigation of interleukin 1β-mediated regulation of NF-κB activation in colonic cells reveals divergence between PKB and PDK-transduced events

  • Kuljit Parhar
  • Sharlene Eivemark
  • Kiran Assi
  • Antonio Gómez-Muñoz
  • Arthur Yee
  • Baljinder Salh
Original Paper


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-1β-induced NF-κB activation. Over-expression of either in HCT 116 and HEK 293T cells, effected a reproducible NF-κB 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-1β-induced NF-κB 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-1β-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-κB activation. This was supported using biochemical analysis in which immunoprecipitated IKKγ from IL-1β-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-κB, and that TPCK may subserve a useful anti-inflammatory function by inhibiting IKKβ.





interleukin 1 beta


interleukin 1 receptor


nuclear factor kappa B


myeloid differentiation primary response gene 88


IL-1 receptor associated kinase 1

Traf 6

TNF-receptor associated factor 6


I kappa kinase


protein kinase B


phosphoinositide-dependent kinase 1


phosphatidylinositol 3 kinase


integrin-linked kinase


intestinal epithelial cells


CREB binding protein


TGFβ-activated kinase


transactivation domain


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Copyright information

© Springer Science+Business Media, LLC 2006

Authors and Affiliations

  • Kuljit Parhar
    • 1
  • Sharlene Eivemark
    • 1
  • Kiran Assi
    • 1
  • Antonio Gómez-Muñoz
    • 1
  • Arthur Yee
    • 1
  • Baljinder Salh
    • 1
    • 2
  1. 1.The Division of Gastroenterology, The Faculty of MedicineThe Jack Bell Research CentreVancouverCanada
  2. 2.Faculty of MedicineUniversity of British ColumbiaVancouverCanada

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