Phosphorylation of RPS3 and its NF-κB function
We next examined whether S209 phosphorylation plays a role in the nuclear translocation of RPS3 during NF-κB activation. Subcellular fractions from either wild-type or S209A mutant RPS3-transfected cells were prepared and blotted for heat-shock protein 90 (hsp90), a cytoplasmic protein, and poly (ADP-ribose) polymerase (PARP), a nuclear protein, confirming a clean separation (Fig. 5a). As expected, PMA+I stimulation triggered wild-type Flag-RPS3 nuclear translocation (Fig. 5a). However, RPS3 (S209A) nuclear translocation was attenuated (Fig. 5a). We also tested the impact of activating NF-κB by overexpressing IKKβ on RPS3 nuclear translocation. IKKβ overexpression activated NF-κB measured by luciferase assays (Supplementary Fig. 7), and also induced the nuclear translocation of wild-type, but not S209A, RPS3 (Fig. 5b). These data suggest that S209 phosphorylation is critical for the NF-κB activation-induced RPS3 nuclear translocation.
To examine the role of S209 phosphorylation of RPS3 to its NF-κB function6, 7, 30, we silenced endogenous RPS3 expression using an siRNA that targets the 3′ untranslated region (3′ UTR) of RPS3 mRNA, followed by complementation with either wild-type or S209A mutant RPS3 via transfection. As expected, RPS3 siRNA severely reduced endogenous RPS3 abundance compared to NS siRNA, but did not affect the robust expression of Flag-tagged RPS3 from a transfected construct lacking the 3′ UTR (Fig. 5c). We also found that RPS3 knockdown reduced TNF-induced expression of an Ig κB-driven luciferase construct6 (Fig. 5d). The impaired luciferase signal caused by RPS3 deficiency was completely restored by transfecting wild-type, but not by S209A RPS3 (Fig. 5d), despite equivalent expression (Fig. 5c). Moreover, the failure of S209A RPS3 to restore luciferase activity did not result from defective translation because the transient overexpression of green fluorescent protein (GFP) was comparable in cells complemented with wild-type or S029A RPS3 (Supplementary Fig. 8). Taken together, these data suggest that RPS3 S209 phosphorylation is critical for NF-κB activity involving the canonical Ig κB site.
We next used chromatin immunoprecipitation to determine whether S209 phosphorylation affects RPS3 and p65 recruitment to specific κB sites in intact chromatin during NF-κB activation. In RPS3 knockdown cells, PMA+I stimulated the recruitment of ectopically expressed, Flag-tagged wild-type, but not S209A RPS3 to the κB sites of the NFKBIA and IL8 promoters (Fig. 5e). While expressing RPS3 S209A had no impact on p65 nuclear translocation, it substantially attenuated p65 recruitment (Fig. 5e). Additional experiments revealed that p65 attraction to RPS3-independent NF-κB target gene promoters such as CD25 was increased (Supplementary Fig. 9), consistent with our previous observations6. There was no significant Flag-RPS3 or p65 recruitment to ACTB promoter lacking κB sites (Fig. 5e), suggesting the recruitment was κB site-specific. Thus, the recruitment of RPS3 as well as the contingent recruitment of p65 to key promoters depended on S209.
Interleukin 8 (IL-8) secretion induced by either T cell receptor (TCR) agonist stimulation or PMA+I was decreased as a consequence of reduced RPS3/p65 recruitment to the IL8 κB sites in the presence of S209A mutant compared to wild-type RPS3 (Supplementary Fig. 10). However, cell surface CD25 expression was comparable between the wild-type and S209A RPS3 transfected cells (Supplementary Fig. 11). Therefore, RPS3 S209 phosphorylation by IKKβ is apparently required for RPS3 in directing NF-κB to a specific subset of target genes.