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 to control phosphorylation of Ser⁶⁵, which affected Cl^- _in sensing by the ³²GXXXP³⁶ motif. IRBIT also recruited the phosphatase calcineurin and the kinase CaMKII to control phosphorylation of Ser¹², which affected Cl^- _in sensing by the ¹⁹⁴GXXXP¹⁹⁸ 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. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

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