TY - JOUR
T1 - Phosphorylation of NHE3-S719 regulates NHE3 activity through the formation of multiple signaling complexes
AU - Sarker, Rafiquel
AU - Cha, Boyoung
AU - Kovbasnjuk, Olga
AU - Cole, Robert
AU - Gabelli, Sandra
AU - Tse, Chung Ming
AU - Donowitz, Mark
N1 - Funding Information:
ACKNOWLEDGMENTS This work was supported by National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases Grants R01DK26523, R01DK61765, P01DK072084, and P30DK089502. We acknowledge use of the Kudsi Imaging Core Facility of the Hopkins Digestive Disease Basic and Translational Research Core Center.
Publisher Copyright:
© 2017 Schutt and Moseley.
PY - 2017/7/1
Y1 - 2017/7/1
N2 - 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, which cause changes in specific aspects of basal as well as acutely stimulated and inhibited Na+/H+ exchange activity.
AB - 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, which cause changes in specific aspects of basal as well as acutely stimulated and inhibited Na+/H+ exchange activity.
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U2 - 10.1091/mbc.E16-12-0862
DO - 10.1091/mbc.E16-12-0862
M3 - Article
C2 - 28495796
AN - SCOPUS:85021666674
SN - 1059-1524
VL - 28
SP - 1754
EP - 1767
JO - Molecular Biology of the Cell
JF - Molecular Biology of the Cell
IS - 13
ER -