ALBERT

Description: 〈p〉IRBIT is a multifunctional protein that controls the activity of various epithelial ion transporters including NBCe1-B. Interaction with IRBIT increases NBCe1-B activity and exposes two cryptic Cl〈sup〉–〈/sup〉-sensing GXXXP sites that enable regulation of NBCe1-B by intracellular Cl〈sup〉–〈/sup〉 (Cl〈sup〉–〈/sup〉〈sub〉in〈/sub〉). Here, phosphoproteomic analysis revealed that IRBIT controlled five phosphorylation sites in NBCe1-B that determined both the active conformation of the transporter and its regulation by Cl〈sup〉–〈/sup〉〈sub〉in〈/sub〉. Mutational analysis suggested that the phosphorylation status of Ser〈sup〉232〈/sup〉, Ser〈sup〉233〈/sup〉, and Ser〈sup〉235〈/sup〉 was regulated by IRBIT and determined whether NBCe1 transporters are in active or inactive conformations. The absence of phosphorylation at Ser〈sup〉232〈/sup〉, Ser〈sup〉233〈/sup〉, or Ser〈sup〉235〈/sup〉 produced NBCe1-B in the conformations pSer〈sup〉233〈/sup〉/pSer〈sup〉235〈/sup〉, pSer〈sup〉232〈/sup〉/pSer〈sup〉235〈/sup〉, or pSer〈sup〉232〈/sup〉/pSer〈sup〉233〈/sup〉, respectively. The activity of the pSer〈sup〉233〈/sup〉/pSer〈sup〉235〈/sup〉 form was similar to that of IRBIT-activated NBCe1-B, but it was insensitive to inhibition by Cl〈sup〉–〈/sup〉〈sub〉in〈/sub〉. The properties of the pSer〈sup〉232〈/sup〉/pSer〈sup〉235〈/sup〉 form were similar to those of wild-type NBCe1-B, whereas the pSer〈sup〉232〈/sup〉/pSer〈sup〉233〈/sup〉 form was partially active, further activated by IRBIT, but retained inhibition by Cl〈sup〉–〈/sup〉〈sub〉in〈/sub〉. Furthermore, IRBIT recruited the phosphatase PP1 and the kinase SPAK to control phosphorylation of Ser〈sup〉65〈/sup〉, which affected Cl〈sup〉–〈/sup〉〈sub〉in〈/sub〉 sensing by the 〈sup〉32〈/sup〉GXXXP〈sup〉36〈/sup〉 motif. IRBIT also recruited the phosphatase calcineurin and the kinase CaMKII to control phosphorylation of Ser〈sup〉12〈/sup〉, which affected Cl〈sup〉–〈/sup〉〈sub〉in〈/sub〉 sensing by the 〈sup〉194〈/sup〉GXXXP〈sup〉198〈/sup〉 motif. Ser〈sup〉232〈/sup〉, Ser〈sup〉233〈/sup〉, and Ser〈sup〉235〈/sup〉 are conserved in all NBCe1 variants and affect their activity. These findings reveal how multiple kinase and phosphatase pathways use phosphorylation sites to fine-tune a transporter, which have important implications for epithelial fluid and HCO〈sub〉3〈/sub〉〈sup〉–〈/sup〉 secretion.〈/p〉