Figure 4. IKKβ phosphorylates RPS3 at serine 209.

(a) Autoradiograph (top) and Coomassie blue staining (bottom) of in vitro kinase assays performed with recombinant GST or GST-RPS3 protein using recombinant human IKKα (rIKKα) or IKKβ (rIKKβ) as kinases. The autophosphorylated IKKs (p-IKKα and p-IKKβ) and phosphorylated RPS3 (p-RPS3) are labeled, respectively. (b) In vitro kinase assays were performed using recombinant RPS3 protein with or without rIKKβ as kinases. After digestion, the phosphorylated peptides were enriched by TiO₂ and fragmented by mass spectrometer. MS/MS of the 1+ fragment ion displays indicative of phosphorylated KKPLPDHVpSIVEPKD based on a Mascot algorithm database search. The y6 ion (in red) shows the incorporation of the site of phosphorylation, which is further confirmed by the loss of H₃PO₄ from several ions (bottom). Schematic diagram of RPS3 with characterized domains (NLS, nuclear localization signal; KH, K homology) and the IKKβ phosphorylation site serine 209 highlighted in red (top). (c) Autoradiograph (top) and Coomassie blue staining (bottom) of in vitro kinase assays performed with recombinant wild-type or S209A mutant GST-RPS3 proteins using recombinant human IKKβ. The phosphorylated (p-RPS3), total GST-RPS3 proteins, and autophosphorylated IKKβ (p-IKKβ) are labeled, respectively. (d) Immunoprecipitation (IP)/immunoblot of phosphoserine for ectopically expressed RPS3 in HEK 293T cells, where wild-type or S209A mutant Flag-RPS3 was transfected with or without an IKKβ plasmid. (e) Immunoblot of phospho-RPS3 (p-RPS3) and RPS3 in whole cell lysates derived from Jurkat cells stimulated with TNF for the indicated periods (bottom). Densitometry of all bands was performed, and the intensity of each p-RPS3 band was normalized to corresponding RPS3 band. The fold change in p-RPS3/RPS3 ratio was further normalized to the unstimulated sample (top). Data are representative of four (a, d), and two (b, c, d) independent experiments, respectively.