In:
Science Signaling, American Association for the Advancement of Science (AAAS), Vol. 11, No. 554 ( 2018-10-30)
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 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 HCO 3 − secretion.
Type of Medium:
Online Resource
ISSN:
1945-0877
,
1937-9145
DOI:
10.1126/scisignal.aat5018
Language:
English
Publisher:
American Association for the Advancement of Science (AAAS)
Publication Date:
2018
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