IκBα degradation and RPS3 nuclear translocation
Importin-α regulates the nuclear import of NF-κB Rel subunits23, 24. RPS3 harbors a nuclear localization signal (NLS) sequence and its nuclear translocation occurs in parallel to, but independently of, p65 translocation6. We envisioned that RPS3 could also utilize the importin-α/β pathway. Consistent with this notion, RPS3 association with importin-α, but not importin-β, was enhanced in TNF–stimulated cells (Fig. 3a). Therefore, we examined whether RPS3 binding to importin-α is essential for nuclear translocation during NF-κB activation.
Since IκBα degradation is a prerequisite to unmask the NLS of p65, and both RPS3 and IκBα bind to p65 in the cytoplasmic inhibitory complex, we tested whether IκBα degradation is required for the liberation of RPS3. We measured the association of RPS3 with importin-α in 293T cells overexpressing wild-type IκBα or an IκBα mutant (SSAA) resistant to IKKβ-induced phosphorylation and degradation. In cells transfected with wild-type IκBα, TNF stimulation augmented the interaction of RPS3 and importin-α to a similar degree as in non-transfected cells. By contrast, we observed that the RPS3-importin-α association was abolished by the presence of non-degradable IκBα (Fig. 3b).
To examine whether IκBα is the only cytoplasmic barrier precluding RPS3 nuclear translocation, we measured both RPS3-importin-α association and nuclear RPS3 after reducing IκBα expression. Compared with nonspecific siRNA, siRNA targeting of IκBα completely depleted IκBα in Jurkat cells (Fig. 3c, input). Nevertheless, the RPS3-importin-α association was not augmented (Fig. 3c), nor was significant nuclear RPS3 detected (Fig. 3d). Moreover, cells treated with sodium pervanadate (Pv) to induce IκBα degradation through an IKK-independent mechanism25-27 did not show increased association between RPS3 and importin-α (Fig. 3e and Supplementary Fig. 3b) or nuclear accumulation of RPS3, despite complete IκBα degradation (Supplementary Fig. 3c). We further examined whether a subsequent NF-κB activation signal independently promotes the importin-α association and nuclear transport of RPS3 after IκBα degradation. We found that TNF stimulation following Pv treatment was required for the RPS3-importin-α association, comparable to TNF stimulation alone (Fig. 3e). Thus, IκBα phosphorylation and degradation itself is required but not sufficient to cause RPS3 association with importin-α followed by nuclear translocation. Rather, an additional signal, potentially IKKβ phosphorylation of RPS3, is required.