Phosphorylation of NHE3-S719 regulates NHE3 activity through the formation of multiple signaling complexes.

Molecular Biology of the Cell
Rafiquel SarkerMark Donowitz

Abstract

Casein kinase 2 (CK2) binds to the NHE3 C-terminus and constitutively phosphorylates a downstream site (S719) that accounts for 40% of basal NHE3 activity. The role of CK2 in regulation of NHE3 activity in polarized Caco-2/bbe cells was further examined by mutation of NHE3-S719 to A (not phosphorylated) or D (phosphomimetic). NHE3-S719A but not -S719D had multiple changes in NHE3 activity: 1) reduced basal NHE3 activity-specifically, inhibition of the PI3K/AKT-dependent component; 2) reduced acute stimulation of NHE3 activity by LPA/LPA5R stimulation; and 3) reduced acute inhibition of NHE3 activity-specifically, elevated Ca2+ related (carbachol/Ca2+ ionophore), but there was normal inhibition by forskolin and hyperosmolarity. The S719A mutant had reduced NHE3 complex size, reduced expression in lipid rafts, increased BB mobile fraction, and reduced binding to multiple proteins that bind throughout the NHE3 intracellular C-terminus, including calcineurin homologous protein, the NHERF family and SNX27 (related PDZ domains). These studies show that phosphorylation of the NHE3 at a single amino acid in the distal part of the C-terminus affects multiple aspects of NHE3 complex formation and changes the NHE3 lipid raft distribution,...Continue Reading

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Citations

Sep 12, 2019·Physiological Reviews·S F Pedersen, L Counillon
Sep 3, 2019·Kidney Research and Clinical Practice·Ibrahim Kashoor, Daniel Batlle
Aug 11, 2020·Gastroenterology·Shabnam Abtahi, Jerrold R Turner

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Methods Mentioned

BETA
confocal microscopy
Assay
pull down
pull-down
immunoprecipitation
coIP

Software Mentioned

LI
Scaffold
AIM
Mascot
MS2 Processor
MetaMorph
Xtract
COR
Proteome Discoverer ( PD )
Proteomic

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