IKKβ phosphorylates RPS3 at serine 209
Although originally defined as the kinase that phosphorylates IκB19, IKKβ also phosphorylates unrelated substrates including 14-3-3β and Bcl10, which lack the IKK consensus motif (DpSGYXpS/T)28. We therefore hypothesized that IKKβ could directly phosphorylate RPS3. By in vitro kinase assays using recombinant IKK and RPS3 proteins, we observed strong incorporation of 32P in autophosphorylatd IKKα and IKKβ (Fig. 4a, lanes 2-7) as well as phosporylated GST-IκBα (1-54) (Supplementary Fig. 4), but not the GST protein alone (Fig. 4a, lanes 3 and 6), when either IKKα or IKKβ was used. We discovered that GST-RPS3 could be phosphorylated by IKKβ, but not IKKα, in vitro (Fig. 4a, compare lanes 4 and 7).
To identify the RPS3 amino acid residue(s) phosphorylated by IKKβ, we performed liquid chromatography-tandem mass spectrometry analyses using in vitro phosphorylated RPS3. The results indicated that IKKβ phosphorylated S209, located in the RPS3 C-terminus (Fig. 4b). RPS3 amino acid sequence alignment revealed that S209 is conserved in many species throughout phylogeny with the exception of Caenorhabditis elegans and Schizosaccharomyces pombe, two organisms that do not possess the NF-κB signal pathway (Supplementary Fig. 5).
To verify biochemically that S209 is an IKKβ substrate, we performed 32P-labeling in vitro kinase assays with recombinant wild-type or S209A mutant RPS3 proteins. Compared with the wild-type protein, the S209A mutation reduced IKKβ–mediated RPS3 phosphorylation (Fig. 4c). There might be alternative phosphorylation site(s) under these conditions given modest residual phosphorylated RPS3 (Fig. 4c). RPS3 S209 does not fall within a conventional IKK recognition motif, but rather resides in a sequence motif (XXXpS/TXXE), potentially recognized by casein kinase II (CK2). Although IKKβ kinase can display a CK2-like phosphorylation specificity29, no CK2 protein was detectable in our recombinant IKK proteins (Supplementary Fig. 6). Thus, RPS3 S209 phosphorylation was due to the alternate specificity of the IKKβ kinase rather than any trace amount of CK2 bound to IKKs. To determine whether S209 is the critical site at which IKKβ phosphorylates RPS3 in living cells, we transfected the wild-type or S209A mutant Flag-RPS3 alone, or together with IKKβ into cells. Indeed, we observed that overexpressing IKKβ enhanced Flag-RPS3 phosphorylation, but phosphorylation was effectively eliminated by alanine substitution indicating that S209 is the predominant target site for IKKβ phosphorylation (Fig. 4d). We next generated a phospho-S209 RPS3 antibody and confirmed that endogenous RPS3 was phosphorylated at S209 in a time-dependent manner upon TNF stimulation (Fig. 4e). Thus, the RPS3 C-terminal tail potentially contains an important regulatory site.