IRBIT Interacts with the Catalytic Core of Phosphatidylinositol Phosphate Kinase Type Iα and IIα through Conserved Catalytic Aspartate Residues

PLoS One. 2015 Oct 28;10(10):e0141569. doi: 10.1371/journal.pone.0141569. eCollection 2015.


Phosphatidylinositol phosphate kinases (PIPKs) are lipid kinases that generate phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a critical lipid signaling molecule that regulates diverse cellular functions, including the activities of membrane channels and transporters. IRBIT (IP3R-binding protein released with inositol 1,4,5-trisphosphate) is a multifunctional protein that regulates diverse target proteins. Here, we report that IRBIT forms signaling complexes with members of the PIPK family. IRBIT bound to all PIPK isoforms in heterologous expression systems and specifically interacted with PIPK type Iα (PIPKIα) and type IIα (PIPKIIα) in mouse cerebellum. Site-directed mutagenesis revealed that two conserved catalytic aspartate residues of PIPKIα and PIPKIIα are involved in the interaction with IRBIT. Furthermore, phosphatidylinositol 4-phosphate, Mg2+, and/or ATP interfered with the interaction, suggesting that IRBIT interacts with catalytic cores of PIPKs. Mutations of phosphorylation sites in the serine-rich region of IRBIT affected the selectivity of its interaction with PIPKIα and PIPKIIα. The structural flexibility of the serine-rich region, located in the intrinsically disordered protein region, is assumed to underlie the mechanism of this interaction. Furthermore, in vitro binding experiments and immunocytochemistry suggest that IRBIT and PIPKIα interact with the Na+/HCO3- cotransporter NBCe1-B. These results suggest that IRBIT forms signaling complexes with PIPKIα and NBCe1-B, whose activity is regulated by PI(4,5)P2.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Amino Acid Sequence
  • Animals
  • Aspartic Acid*
  • Catalytic Domain*
  • Cell Line
  • Cerebellum / metabolism
  • Conserved Sequence
  • Enzyme Activation
  • Humans
  • Inositol 1,4,5-Trisphosphate / metabolism
  • Lectins, C-Type / genetics
  • Lectins, C-Type / metabolism*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Mice
  • Mice, Knockout
  • Molecular Sequence Data
  • Phosphorylation
  • Phosphotransferases (Alcohol Group Acceptor) / chemistry*
  • Phosphotransferases (Alcohol Group Acceptor) / metabolism*
  • Protein Binding
  • Protein Interaction Domains and Motifs*
  • Protein Transport
  • Rats
  • Sequence Deletion


  • CLECL1 protein, human
  • Lectins, C-Type
  • Membrane Proteins
  • Aspartic Acid
  • Inositol 1,4,5-Trisphosphate
  • Adenosine Triphosphate
  • Phosphotransferases (Alcohol Group Acceptor)

Grant support

This work was supported by the Japan Society for the Promotion of Science Grants-in-Aid for Scientific research (Grant numbers 17770120, 21870049, and 23500458 to HA, and 25221002 to KM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.