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

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-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.