Inositol 1,4,5-Triphosphate Receptor-binding Protein Released with Inositol 1,4,5-Triphosphate (IRBIT) Associates with Components of the mRNA 3′ Processing Machinery in a Phosphorylation-dependent Manner and Inhibits Polyadenylation

Abstract

IRBIT is a recently identified protein that modulates the activities of both inositol 1,4,5-triphosphate receptor and pancreas-type Na ؉ /HCO 3 ؊ cotransporter 1, and the multisite phosphorylation of IRBIT is required for achieving this modulatory action. Here, we report the identification of the cleavage and polyadenylation specificity factor (CPSF), which is a multi-protein complex involved in 3 processing of mRNA precursors, as an additional binding partner for IRBIT. We found that IRBIT interacted with CPSF and was recruited to an exogenous polyadenylation signal-containing RNA. The main target for IRBIT in CPSF was Fip1 subunit, and the phosphorylation of the serine-rich region of IRBIT was required both for direct association with Fip1 in vitro and for redistribution of Fip1 into the cytoplasm of intact cells. Furthermore, tert-butylhydroquinone (tBHQ), an agent that induces oxidative stress, increased the phosphorylation level of IRBIT in vivo and in parallel enhanced the interaction between IRBIT and CPSF and promoted the cytoplasmic distribution of endogenous Fip1. In addition to CPSF, IRBIT interacted in vitro with poly(A) polymerase (PAP), which is the enzyme recruited by CPSF to elongate the poly(A) tail, and inhibited PAP activity in a phosphorylation-dependent manner. These findings raise the possibility that IRBIT modulates the polyadenylation state of specific mRNAs, both by controlling the cytoplasmic/nuclear partitioning of Fip1 and by inhibiting PAP activity, in response to a stimulus that alters its phosphorylation state.The inositol 1,4,5-triphosphate (IP 3 ) 3 receptors (IP 3 Rs) are IP 3 -gated Ca 2ϩ channels located on intracellular Ca 2ϩ stores. When the cell is exposed to a stimulus, IP 3 is produced as a second messenger and mediates the release of Ca 2ϩ by interacting with IP 3 Rs. We previously identified an IP 3 R-binding protein termed IRBIT (IP 3 R-binding protein released with inositol 1,4,5-triphosphate) that interacts with the IP 3 -binding core domain of IP 3 R and is dissociated from IP 3 R by physiological concentrations of IP 3 (1). IRBIT binds to IP 3 R through its N-terminal phosphorylated region and suppresses IP 3 R activity at the resting state by blocking IP 3 access to IP 3 R. When cells are exposed to a stimulus resulting in high concentration of IP 3 , IP 3 displaces IRBIT and activates IP 3 R. From these findings, it was speculated that IRBIT regulates Ca 2ϩ release by setting the threshold of IP 3 concentration required for activation of IP 3 R (2). Besides its function in the regulation of IP 3 R activity, IRBIT also binds to and activates pancreas-type Na ϩ /HCO 3 Ϫ cotransporter 1 (pNBC1), indicating a role in the regulation of intracellular and extracellular pH (3). Interestingly, although interactions with IP 3 R and pNBC1 each show unique properties, they also share common features, such the requirement of the N-terminal phosphorylated region (2, 3). The finding that IRBIT binds to and regulates both IP 3 R and pNBC1 suggests that IRBIT is a multifunctional...

Splicing variation of Long-IRBIT determines the target selectivity of IRBIT family proteins

“…Therefore, it is possible that the number of glutamine residues in the Long-IRBIT splicing site affects the binding affinity for PP1 and the subsequent phosphorylation states of Long-IRBIT splice variants. It was reported that the phosphorylation state of IRBIT contributes to the binding to target molecules (3,4,7,10,13,15). Therefore, the number of glutamine residues in the Long-IRBIT splicing site may determine the binding affinity of Long-IRBIT to target molecules.…”

“…It also contributes to intracellular pH regulation and fluid secretion (6)(7)(8). In addition, IRBIT binds to cleavage and polyadenylation-specific factor subunit (Fip1) and modulates the polyadenylation state of specific mRNA (13). It was also reported that IRBIT regulates ribonucleotide reductase and contributes to cell cycle progression (14).…”

Activation of Na+/H+ Exchanger NHE3 by Angiotensin II Is Mediated by Inositol 1,4,5-Triphosphate (IP3) Receptor-binding Protein Released with IP3 (IRBIT) and Ca2+/Calmodulin-dependent Protein Kinase II

“…The importance of phosphorylation of the serine residues within the PEST domain of IRBIT has been shown for interaction of IRBIT with the IP 3 receptor, pNBC1, or the cleavage and polyadenylation specificity factor (14,42,43). Protein kinases phosphorylating IRBIT in vivo remain unknown, but several candidate protein kinases, including CaMKII, casein kinase, and protein kinase A, have been suggested (44).…”

Calcium Channels in the Development, Maturation, and Function of Spermatozoa

“…After its release, IRBIT was found to interact and activate the pancreatic but not the kidney Na ϩ /HCO 3 Ϫ cotransporter 1 (NBC1), suggesting a role in HCO 3 Ϫ secretion and pH regulation in epithelial tissues (467). IRBIT has a multisite strict phosphorylation requirement for its dual modulation activities (289). Regulation of HCO 3 Ϫ secretion and Cl Ϫ absorption is an important but incompletely resolved issue for different epithelia.…”