Phosphorylation of the PEST domain of IκBβ regulates the function of NF-κB/IκBβ complexes

Abstract

Activation of transcription factor NF-κB involves the signal-dependent degradation of basally phosphorylated inhibitors such as IκBα and IκBβ. The gene encoding IκBα is under NF-κB control, which provides a negative feedback loop to terminate the induced NF-κB response. However, recent studies have identified a hypophosphorylated pool of IκBβ that shields nuclear NF-κB from inhibition by newly synthesized IκBα. In the present work, we provide three lines of evidence indicating that this protection mechanism is regulated by the C-terminal PEST domain of IκBβ. First, disruption of two basal phosphoacceptors present in the IκBβ PEST domain (Ser-313 and Sex-315) yields a mutant that forms ternary complexes with NF- κB and its target DNA-binding site. Second, based on in vitro mixing experiments, these ternary complexes are resistant to the inhibitory action of IκBα. Third, mutants of IκBβ that are defective for phosphorylation at Ser-313 and Ser-315 fail to efficiently block NF-κB-directed transcription in vivo, whereas replacement of these two IκBβ residues with a phosphoserine mimetic generates a fully functional repressor. Taken together, our findings suggest that the functional fate of NF-κB when bound to IκBβ is critically dependent on the phosphorylation status of the IκBβ PEST domain.