Deciphering the multi-site phos-code of IRBIT underlying its binding to IP3R

IRBIT has been previously shown to interact with the inositol 1,4,5-trisphosphate (IP ₃ ) receptor (IP ₃ R) via a serine-rich segment containing numerous phosphorylation sites. Although the interaction between IRBIT and IP ₃ R is phosphorylation dependent, it is unclear which particular phosphoisotypes are directly involved in their interaction. In this study, we investigated the mechanism of how phosphorylation states of IRBIT regulate its interaction with IP ₃ R. Site-directed mutagenesis confirmed that S68 is the predominant site of phosphorylation on IRBIT, but it is not required for IP ₃ R binding. In vitro kinase assay showed that protein kinase A and casein kinase 2 phosphorylated residues S62/S64/S66 and S80/T82/S84/S85, respectively. Subsequent pulldown assays demonstrated that S71/S74/S77 and S80/S84/S85 provided two binding sites for IP ₃ -binding core (IBC) on IP ₃ R. Computational estimation showed that the IRBIT-pS80pS84pS85 peptide binds IBC with the lower binding free energy in a mode similar to that of IP ₃ . Further studies of Ca ²⁺ imaging in living cells revealed that IRBIT-S80GS84GS85G mutant was not able to inhibit the activation of IP ₃ R, while IRBIT-S68AS80DS84DS85D was sufficient to prohibit IP ₃ R-mediated Ca ²⁺ release. In summary, our results indicate that the phosphorylated S80, S84, and S85 residues on IRBIT may compete with IP ₃ for the IP ₃ -binding pocket on IP ₃ R.