Modulation of Cl - signaling and ion transport by recruitment of kinases and phosphatases mediated by the regulatory protein IRBIT.

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- -sensing GXXXP sites that enable regulation of NBCe1-B by intracellular Cl- (Cl- in ). 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- in Mutational analysis suggested that the phosphorylation status of Ser232 , Ser233 , and Ser235 was regulated by IRBIT and determined whether NBCe1 transporters are in active or inactive conformations. The absence of phosphorylation at Ser232 , Ser233 , or Ser235 produced NBCe1-B in the conformations pSer233 /pSer235 , pSer232 /pSer235 , or pSer232 /pSer233 , respectively. The activity of the pSer233 /pSer235 form was similar to that of IRBIT-activated NBCe1-B, but it was insensitive to inhibition by Cl- in The properties of the pSer232 /pSer235 form were similar to those of wild-type NBCe1-B, whereas the pSer232 /pSer233 form was partially active, further activated by IRBIT, but retained inhibition by Cl- in Furthermore, IRBIT recruited the phosphatase PP1 and the kinase SPAK to control phosphorylation of Ser65 , which affected Cl- in sensing by the 32 GXXXP36 motif. IRBIT also recruited the phosphatase calcineurin and the kinase CaMKII to control phosphorylation of Ser12 , which affected Cl- in sensing by the 194 GXXXP198 motif. Ser232 , Ser233 , and Ser235 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 HCO3 - secretion.