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

Abstract

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 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.

Functional expression of electrogenic sodium bicarbonate cotransporter 1 (NBCe1) in mouse cortical astrocytes is dependent on S255‐257 and regulated by mTOR

“…In an attempt to nevertheless obtain a proxy for phosphorylation abundance and thus functional relevance, we additionally calculated the phosphorylation site occupancy, that is, the fraction of a given protein that is phosphorylated at a given site (Olsen et al, ) as described in Section 2. Comparison of phosphorylation site occupancies on NBCe1 in control astrocytes revealed a phosphorylated fraction of on average 51% for the well‐known and IRBIT‐regulated phosphorylation sites at pS232‐233, pS235‐236 (Hong et al, ; Vachel et al, ), 70% for pS245 and 53% for pS255‐257 (Figure S3b). Comparison of pS255‐257 and pS245 occupancies across conditions did again not reveal any significant differences (Figure b and Figure S3c).…”

“…IRBIT‐controlled serine phosphorylation sites of NBCe1‐B in epithelia have been identified at positions 12, 65, 232, 233, and 235, whose differential phosphorylation status resulted in active or inactive conformations of the transporter and its sensitivity to intracellular chloride. The residues 255–257 were identified to be phosphorylated as well, however, not regulated by IRBIT (Vachel et al, ). In another study that has used a phosphoproteomic approach for global monitoring of native phosphorylation of plasma membrane proteins from a single mouse cerebellum, among the 41 proteins that fall into the category “transporters”, NBCe1 was found to be phosphorylated at several residues, among them S232‐233 and S255‐257 (Schindler, Ye, Jensen, & Nothwang, ).…”

“…Unfortunately, MS intensities cannot readily be used to estimate absolute abundances across different peptides due to differing, peptide sequence dependent response factors. In an attempt to nevertheless obtain a proxy for phosphorylation abundance and thus functional relevance, we additionally calculated the phosphorylation site occupancy, that is, the fraction of a given protein that is phosphorylated at a given site (Olsen et al, 2010) pS232-233, pS235-236 (Hong et al, 2013;Vachel et al, 2018), 70% for pS245 and 53% for pS255-257 ( Figure S3b). Comparison of pS255-257 and pS245 occupancies across conditions did again not reveal any significant differences (Figure 6b and Figure S3c).…”

“…Phosphorylation of NBCe1 has been identified as crucial determinant for regulation of NBCe1 trafficking and functional expression. In epithelial cells, phosphorylation of S1026 mediates the cAMP‐dependent shift in the stoichiometry of NBCe1‐B (Gross et al, ), SPAK‐dependent phosphorylation at S65 leads to internalization of NBCe1 and decreased membrane expression (Hong et al, ; Yang et al, ), and individual phosphorylation of S12, S65, and S232–235 results in various NBCe1‐B conformations with distinct transport properties (Vachel et al, ). Phosphoproteomic analysis in mice with conditional deletion of mTORC1 signaling from renal proximal tubule showed decreased phosphorylation of T3, S1060, and S232 of NBCe1 (Grahammer et al, ), but the functional significance of these phosphorylation sites on transporter activity has not been addressed.…”

STAT3‐dependent regulation of the electrogenic Na+/HCO3− cotransporter 1 (NBCe1) functional expression in cortical astrocytes

“…Astrocytic NBCe1 activity is shown to be regulated by several stimuli, among them acute extracellular pH changes, metabolic alkalosis, growth factors, and 4‐aminopyridine (4AP; Khakipoor et al, 2017; Salameh, Ruffin, & Boron, 2014; Schrödl‐Häußel, Theparambil, Deitmer, & Roussa, 2015; Theparambil et al, 2015, 2017). At the molecular level, NBCe1 associated cellular responses within or outside the CNS can be mediated either through direct phosphorylation (J. H. Hong et al, 2013; Khakipoor et al, 2019; Vachel et al, 2018; Yang et al, 2011) or/and by activation of several signaling pathways, such as mTOR, JNK, and Src/ERK signaling (Khakipoor et al, 2019; Namkoong et al, 2015; Schrödl‐Häußel et al, 2015).…”