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

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Abstract

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.

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Abbreviations

IL-1β:

interleukin 1 beta

IL-1R:

interleukin 1 receptor

NF-κB:

nuclear factor kappa B

MyD88:

myeloid differentiation primary response gene 88

IRAK 1:

IL-1 receptor associated kinase 1

Traf 6:

TNF-receptor associated factor 6

IKK:

I kappa kinase

PKB:

protein kinase B

PDK:

phosphoinositide-dependent kinase 1

PI3K:

phosphatidylinositol 3 kinase

ILK:

integrin-linked kinase

IECs:

intestinal epithelial cells

CBP:

CREB binding protein

TAK1:

TGFβ-activated kinase

TAD:

transactivation domain

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Authors and Affiliations

  1. The Division of Gastroenterology, The Faculty of Medicine, The Jack Bell Research Centre, 2660 Oak Street, Vancouver, BC, Canada, V6H 3Z6

    Kuljit Parhar, Sharlene Eivemark, Kiran Assi, Antonio Gómez-Muñoz, Arthur Yee & Baljinder Salh

  2. Faculty of Medicine, University of British Columbia, 2775 Laurel Street, Vancouver, BC, Canada, V5Z 1M9

    Baljinder Salh

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  1. Kuljit Parhar
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  2. Sharlene Eivemark
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  4. Antonio Gómez-Muñoz
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Correspondence to Baljinder Salh.

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This study was supported in part by grants from the Crohn’s and Colitis Foundation of Canada (BS) and the Canadian Society for Intestinal Research (BS) and funds from the Geraldine Dow Foundation to B. S. K.P. was supported by a Michael Smith Graduate Studentship. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked, “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Parhar, K., Eivemark, S., Assi, K. et al. Investigation of interleukin 1β-mediated regulation of NF-κB activation in colonic cells reveals divergence between PKB and PDK-transduced events. Mol Cell Biochem 300, 113–127 (2007). https://doi.org/10.1007/s11010-006-9375-4

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