IRBIT interacts with the catalytic core of phosphatidylinositol phosphate kinase type Ia and IIa through conserved catalytic aspartate residues

Abstract

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 Ia (PIPKIa) and type IIa (PIPKIIa) in mouse cerebellum. Site-directed mutagenesis revealed that two conserved catalytic aspartate residues of PIPKIa and PIPKIIa 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 PIPKIa and PIPKIIa. 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 PIPKIa interact with the Na+/HCO3 - cotransporter NBCe1-B. These results suggest that IRBIT forms signaling complexes with PIPKIa and NBCe1-B, whose activity is regulated by PI(4,5)P2.