An inositol pyrophosphate interaction screen provides insight into the regulation of plant casein kinase II

Inositol pyrophosphates (PP-InsPs) are key nutrient messengers in plants, but their protein receptors remain poorly defined. Using a systems-level affinity screen with biotinylated InsP₆, InsP₇, and InsP₈ in Arabidopsis thaliana , we identify multiple conserved PP-InsP-interacting complexes involved in mRNA metabolism, translation, and cell signaling, including the nuclear α-subunits of casein kinase II (CK2). The CK2 subunit AtCKA1 associates with the PP-InsP kinase AtVIH2, and its 1.9 Å crystal structure with InsP ₆ reveals two conserved PP-InsP binding sites located in the N-and C-terminal lobes. AtCKA1 binds InsP ₆ , InsP ₇ , and InsP ₈ with micromolar affinity. Mutation of both binding sites in the AtCKA ^6xmut mutant abolishes PP-InsP binding in vitro. AtCKA ^6xmut partially rescues the flowering phenotype of ck2a1/2/3 mutants, and equivalent mutations inactivate the yeast orthologs ScCka1 and ScCka2. InsP ₆ competitively inhibits phosphorylation of canonical CK2 substrates by occupying a basic substrate-binding groove. Although incorporating β-subunits strongly enhances the phosphorylation of substrates by the AtCK2 holoenzyme, ck2b1/2/3/4 mutants exhibit only mild growth defects in Arabidopsis. In Marchantia , loss of the single ck2a gene severely impairs growth, whereas deletion of the β subunit has no effect. Together, our findings suggest that InsP ₆ /PP-InsPs modulate the activity of the isolated CK2 α-subunit by regulating access to its substrate-binding site.