IKKβ is required for RPS3 nuclear translocation
To examine whether the RPS3-IKKβ interaction is required for RPS3 nuclear translocation, we knocked down IKKα or IKKβ expression with siRNAs (Supplementary Fig. 1) and then observed stimulation-induced RPS3 nuclear migration by confocal microscopy. Both TNF and PMA+I triggered RPS3 nuclear translocation in Jurkat cells transfected with a scrambled nonspecific (NS) siRNA (Fig. 2a)6. RPS3 nuclear translocation was only slightly, if at all, impaired by IKKα-silencing. Conversely, knockdown of IKKβ attenuated 60-70% of RPS3 nuclear accumulation following stimulation (Fig. 2a). Immunoblotting of nuclear fractions confirmed that full expression of IKKβ, but not IKKα, was necessary for activation-induced RPS3 nuclear translocation (Fig. 2b). Control immunoblots revealed that p65 nuclear translocation was blocked under the same conditions (Fig. 2b).
We next examined the nuclear translocation of RPS3 in cells ectopically expressing either kinase-dead (SSAA) or constitutively-active (SSEE) mutant IKKβ proteins. As expected, the SSEE, but not SSAA, mutant of IKKβ induced NF-κB-dependent luciferase reporter activity (Fig. 2c, left). Whereas RPS3 remained cytosolic in IKKβ (SSAA)-expressing cells (Fig. 2c, right), a substantial proportion of RPS3 translocated to the nucleus in cells expressing IKKβ (SSEE) (Fig. 2c, right). The percentage of cells containing detectable nuclear RPS3 increased 5-fold in IKKβ (SSEE)-expressing cells, but not in IKKβ (SSAA)-expressing ones (Fig. 2d and Supplementary Fig. 2). Thus, IKKβ activity is necessary and sufficient for RPS3 nuclear translocation in response to NF-κB activating stimuli.