Protein phosphatase-1 is a novel regulator of the interaction between IRBIT and the inositol 1,4,5-trisphosphate receptor

IRBIT is an IP₃R [IP₃ (inositol 1,4,5-trisphosphate) receptor]-binding protein that competes with IP₃ for binding to the IP₃R. Phosphorylation of IRBIT is essential for the interaction with the IP₃R. The unique N-terminal region of IRBIT, residues 1–104 for mouse IRBIT, contains a PEST (Pro-Glu-Ser-Thr) domain with many putative phosphorylation sites. In the present study, we have identified a well-conserved PP1 (protein phosphatase-1)-binding site preceeding this PEST domain which enabled the binding of PP1 to IRBIT both in vitro and in vivo. IRBIT emerged as a mediator of its own dephosphorylation by associated PP1 and, hence, as a novel substrate specifier for PP1. Moreover, IRBIT-associated PP1 specifically dephosphorylated Ser⁶⁸ of IRBIT. Phosphorylation of Ser⁶⁸ was required for subsequent phosphorylation of Ser⁷¹ and Ser⁷⁴, but the latter two sites were not targeted by PP1. We found that phosphorylation of Ser⁷¹ and Ser⁷⁴ were sufficient to enable inhibition of IP₃ binding to the IP₃R by IRBIT. Finally, we have shown that mutational inactivation of the docking site for PP1 on IRBIT increased the affinity of IRBIT for the IP₃R. This pinpoints PP1 as a key player in the regulation of IP₃R-controlled Ca²⁺ signals.