As shown in Figure 4, NF-kappaB was identified as a Wip1 target by Chew et al. (6). By using an NF-kappaB reporter and by monitoring the expression levels of NF-kappaB targets, they showed that overexpression of Wip1 or knockdown of Wip1 with siRNA reduced or increased, respectively, NF-kappaB activation resulting from treatment with Interleukin-1 (IL-1) or TNF-alpha. This enhancement of NF-kappaB activity by Wip1 knockdown was not due to alterations in the expression of IkappaBalpha (an inhibitor of NF-kappaB), as determined by immunoblot, or in the DNA binding of the p50:p65 NF-kappaB heterodimeric complex after stimulation, as determined by EMSA. On the other hand, evaluation of the levels of phosphorylation of NF-kappaB p65 on Ser536 (pS536-p65) showed that overexpression of Wip1 or knockdown of Wip1 reduced or enhanced, respectively, the levels of pS536-p65 after stimulation by TNF-alpha. This effect was independent of p38 MAPK since the p38 inhibitor, SB202190, did not rescue the heightened pS536-p65 levels in Wip1-depleted cells. Furthermore, overexpression of Wip1 inhibited the TNF-alpha-induced binding of p65 to its transcriptional cofactor p300. Finally, recombinant Wip1 dephosphorylated immunopurified pS536-p65
