Regulation of Cl- signaling and ion transport by IRBIT-mediated recruitment of multiple kinases and phosphatases

ABSTRACT: IRBIT is a multifunctional protein that controls the activity of IP3 receptors and the transporters NBCe1-B, CFTR and Slc26a6, by an unknown mechanism. IRBIT interacts with the NBCe1-B N terminus autoinhibitory domain (AID), exposing two cryptic Cl- sensing GXXXP sites to confer regulation of NBCe1-B by intracellular Cl- (Cl-in). Here, LC-MS/MS phosphoprotein analysis revealed that IRBIT controls five NBCe1-B phosphorylation sites that determine both NBCe1-B active conformation and regulation by Cl-in. Specifically, IRBIT-dependent combinatorial dephosphorylation of pSer232, pSer233 or pSer235 generated the conformations pSer233/pSer235, pSer232/pSer235 or pSer232/pSer233. The activity of the pSer232/pSer235 form is similar to IRBIT-activated NBCe1-B, but it is insensitive to inhibition by Cl-in. The pSer232/pSer235 form properties were similar to wild-type NBCe1-B, while the pSer232/pSer233 form was partially active, further activated by IRBIT, but retained inhibition by Cl-in. In addition, IRBIT recruited the phosphatase PP1 and the kinase SPAK to control Ser65 phosphorylation, which reciprocally affected Cl-in sensing by the 32GXXXP36 motif. IRBIT also recruited the phosphatase calcineurin and the kinase CaMKII to control phosphorylation of Ser12, which reciprocally affected Cl-in sensing by the 194GXXXP198 motif. Ser232/Ser233/Ser235 are conserved in all NBCe1 variants. Mutating the serines to alanines resulted in fully active NBCe1 transporters, while mutating the serines to the phosphomimetic aspartates resulted in inhibited transporters, suggesting that Ser232/Ser233/Ser235 determine whether NBC transporters are in active or inactive conformations. These findings reveal how multiple kinase/phosphatase pathways use multiple phosphorylation sites to fine tune a transport function, which have important implications for epithelial fluid and HCO3- secretion.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: CHIN-RANG YANG  

LAB HEAD: Shmuel Muallem

PROVIDER: PXD010598 | Pride | 2020-03-04

REPOSITORIES: Pride