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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): Orbitrap Fusion ETD

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

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

Vachel Laura L Shcheynikov Nikolay N Yamazaki Osamu O Fremder Moran M Ohana Ehud E Son Aran A Shin Dong Min DM Yamazaki-Nakazawa Ai A Yang Chin-Rang CR Knepper Mark A MA Muallem Shmuel S

Science signaling 20181030 554

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 ...[more]

PMID: 30377224

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Project description:The mitogen-activated protein kinase (MAPK) p38 pathway is reported to regulate macrophage responses to lipopolysaccharide (LPS) at least partly via the phosphorylation of the mRNA-destabilizing factor tristetraprolin (TTP). LPS-activated MAPK p38 phosphorylates and activates the downstream kinase MAPK-activated protein kinase 2 (MK2), which then phosphorylates serines 52 and 178 of TTP, resulting in loss of mRNA-destabilizing activity. As a consequence, mRNAs that contain binding sites for TTP are stabilized in a manner that is acutely sensitive to the activity of the MAPK p38 pathway. Dual specificity phosphatase 1 (DUSP1) dephosphorylates and inactivates MAPK p38. Dusp1-/- macrophages overexpress a number of pro-inflammatory mediators, but their genome-wide responses to LPS have not yet been described in detail. Dusp1-/- mice are exceptionally sensitive to a wide variety of inflammatory challenges, including experimental models of endotoxemia or sepsis. It has been suggested (but not yet proven) that DUSP1 controls the inflammatory response of macrophages in part via the regulation of MAPK p38 activity and TTP phosphorylation status. We generated a mouse knock-in strain, in which codons 52 and 178 of the endogenous Zfp36 gene (which encodes TTP) were mutated to alanine codons. The mutation gives rise to a constitutively active form of TTP, which cannot be inactivated via the p38 MAPK pathway. The Zfp36aa/aa strain was back-crossed against C57/BL6 for > 10 generations. We also generated a double-targeted strain in which the Zfp36 mutation was combined with disruption of the Dusp1 gene. This expression array describes LPS responses of primary mouse bone marrow-derived macrophages of four genotypes, and closely matched genetic background: wild type (Dusp1+/+ : Zfp36+/+); TTP mutant (Zfp36aa/aa); DUSP1 knock out (Dusp1-/-); and double targeted (Dusp1-/- : Zfp36aa/aa). The data provide a comprehensive picture of the impact of Dusp1 deletion or TTP mutation on the responses of primary macrophages to LPS. They also demonstrate that the excessive inflammatory responses of Dusp1-/- macrophages are largely a consequence of the phosphorylation and inactivation of TTP. Genome wide expression profiles of wild type, Dusp1-/-, Zfp36aa/aa and Dusp1-/-/Zfp36aa/aa M-CSF derived macrophages, stimulated with LPS for 1 or 4 hours

Dataset Information

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

Publications

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

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