Borar, P. | Search bioRxiv

bioRxiv2026

IKK2/β mediated phosphorylation of transcription factor Ets2 at site(s) distal to DNA binding domain negatively modulates its DNA binding activity

Transcription factor Ets2 coalesce with the NF-{kappa}B pathway to regulate gene expression in specific signaling contexts. IKK2/{beta}-mediated phosphorylation events critically regulate the NF-{kappa}B pathway. However, any link between Ets2 and IKK2 remains elusive. Here we report Ets2 as a direct substrate of IKK2. In-vitro kinase assays using deletion constructs, high resolution MS-MS and site directed mutagenesis identified S295 as a prominent phosphorylation site distal to the DNA binding domain, substitution of which to phosphor-mimetic Glutamate triggers further phosphorylation of Ets2. MD simulations clearly indicate conformational constriction of the otherwise disordered N-terminal region and inhibition of DNA binding activity upon phosphorylation, which was further confirmed by Electrophoretic mobility shift assays. Our results uncover a phosphoregulatory connection between Ets2 and IKK2.

biochemistry↗

bioRxiv2023

Dual-specific autophosphorylation of kinase IKK2 enables phosphorylation of substrate IkappaBalpha without requiring ATP

Rapid and high-fidelity phosphorylation of serine residues at positions 32 and 36 of I{kappa}B by IKK2, a prototypical Ser/Thr kinase, is critical for canonical NF-{kappa}B activation. Here, we report that IKK2 not only phosphorylates substrate serine residues and autophosphorylates its own activation loop, but also autophosphorylates at a tyrosine residue proximal to the active site and is, therefore, a dual-specificity kinase. We observed that mutation of Y169, an autophosphorylatable tyrosine located at the DFG+1 (DLG in IKK1) position, to phenylalanine renders IKK2 incapable of catalyzing phosphorylation at S32 within its I{kappa}B substrate. We also observed that mutation of the phylogenetically conserved ATP-contacting residue K44 in IKK2 to methionine converts IKK2 to an enzyme that no longer catalyzes specific phosphorylation of I{kappa}B at S32 or S36, but rather directs phosphorylation of I{kappa}B at other residues. Lastly, we report evidence of a phospho-relay from autophosphorylated IKK2 to I{kappa}B in the presence of ADP. These observations suggest an unusual evolution of IKK2, in which autophosphorylation of tyrosine(s) in the activation loop, and the conserved ATP-contacting K44 residue provide its signal-responsive substrate specificity and ensure fidelity during NF-{kappa}B activation.

biochemistry↗