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 Ser(232), Ser(233), and Ser(235) was regulated by IRBIT and determined whether NBCe1 transporters are in active or inactive conformations. The absence of phosphorylation at Ser(232), Ser(233), or Ser(235) produced NBCe1-B in the conformations pSer(233)/pSer(235), pSer(232)/pSer(235), or pSer(232)/pSer(233), respectively. The activity of the pSer(233)/pSer(235) form was similar to that of IRBIT-activated NBCe1-B, but it was insensitive to inhibition by Cl-in(-). The properties of the pSer(232)/pSer(235) form were similar to those of wild-type NBCe1-B, whereas the pSer(232)/pSer(233) 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 Ser(65), which affected Cl-in(-) sensing by the (32)GXXXP(36) motif. IRBIT also recruited the phosphatase calcineurin and the kinase CaMKII to control phosphorylation of Ser(12), which affected Cl-in(-) sensing by the (194)GXXXP(198) motif. Ser(232), Ser(233), and Ser(235) 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.