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<p>IRBIT has been previously shown to interact with the inositol 1,4,5-trisphosphate (IP<sub>3</sub>) receptor (IP<sub>3</sub>R) via a serine-rich segment. Although the interaction between IRBIT and IP<sub>3</sub>R is phosphorylation dependent, it is unclear which particular phosphoisotypes are directly involved in their interaction. In this study, we confirmed that S68 is the predominant site of phosphorylation on IRBIT, but it is not required for IP<sub>3</sub>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<sub>3</sub>-binding core (IBC) on IP<sub>3</sub>R. Computational estimation showed that the IRBIT-pS80pS84pS85 peptide binds IBC in a mode similar to that of IP<sub>3</sub>. Further studies of Ca<sup>2+</sup> imaging in living cells revealed that IRBIT-S68A/S80D/S84D/S85D was sufficient to prohibit IP<sub>3</sub>R-mediated Ca<sup>2+</sup> release. In summary, our results indicate that the phosphorylated S80, S84, and S85 residues on IRBIT may compete with IP<sub>3</sub> for the IP<sub>3</sub>-binding pocket on IP<sub>3</sub>R.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年4月24日

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  • Qing Lin,
  • Hao Yang,
  • Qi Feng,
  • Tomohide Sumi,
  • Kozo Hamada,
  • Yi Qian,
  • Fang Bai,
  • Akihiro Mizutani,
  • Katsuhiko Mikoshiba

摘要

IRBIT has been previously shown to interact with the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) via a serine-rich segment. Although the interaction between IRBIT and IP3R is phosphorylation dependent, it is unclear which particular phosphoisotypes are directly involved in their interaction. In this study, we confirmed that S68 is the predominant site of phosphorylation on IRBIT, but it is not required for IP3R 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 IP3-binding core (IBC) on IP3R. Computational estimation showed that the IRBIT-pS80pS84pS85 peptide binds IBC in a mode similar to that of IP3. Further studies of Ca2+ imaging in living cells revealed that IRBIT-S68A/S80D/S84D/S85D was sufficient to prohibit IP3R-mediated Ca2+ release. In summary, our results indicate that the phosphorylated S80, S84, and S85 residues on IRBIT may compete with IP3 for the IP3-binding pocket on IP3R.