TY - JOUR
T1 - Cyclic GMP kinase II (cGKII) inhibits NHE3 by altering its trafficking and phosphorylating NHE3 at three required sites
T2 - Identification of a multifunctional phosphorylation site
AU - Chen, Tiane
AU - Kocinsky, Hetal S.
AU - Cha, Boyoung
AU - Murtazina, Rakhilya
AU - Yang, Jianbo
AU - Tse, C. Ming
AU - Singh, Varsha
AU - Cole, Robert
AU - Aronson, Peter S.
AU - De Jonge, Hugo
AU - Sarker, Rafiquel
AU - Donowitz, Mark
N1 - Publisher Copyright:
© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2015/1/23
Y1 - 2015/1/23
N2 - The epithelial brush-border Na+/H+ exchanger NHE3 is acutely inhibited by cGKII/cGMP, but how cGKII inhibits NHE3 is unknown. This study tested the hypothesis that cGMP inhibits NHE3 by phosphorylating it and altering its membrane trafficking. Studies were carried out in PS120/NHERF2 and in Caco-2/Bbe cells overexpressing HA-NHE3 and cGKII, and in mouse ileum. NHE3 activity was measured with 2',7'-bis(car-boxyethyl)-S-(and 6)carboxyfluorescein acetoxy methylester/fluorometry. Surface NHE3 was determined by cell surface biotinylation. Identification of NHE3 phosphorylation sites was by iTRAQ/LC-MS/MS with TiO2 enrichment and immunoblotting with specific anti-phospho-NHE3 antibodies. cGMP/cGKII rapidly inhibited NHE3, which was associated with reduced surface NHE3. cGMP/cGKII increased NHE3 phosphorylation at three sites (rabbit Ser554, Ser607, and Ser663, equivalent to mouse Ser552, Ser605, and Ser659), all of which had to be present at the same time for cGMP to inhibit NHE3. NHE3-Ser663 phosphorylation was not necessary for cAMP inhibition of NHE3. Dexamethasone (4 h) stimulated wild type NHE3 activity and increased surface expression but failed to stimulate NHE3 activity or increase surface expression when NHE3 was mutated to either S663A or S663D. We conclude that 1) cGMP inhibition of NHE3 is associated with phosphorylation of NHE3 at Ser554, Ser607, and Ser663, all of which are necessary for cGMP/cGKII to inhibitNHE3.2)DexamethasonestimulatesNHE3byphosphorylation of a single site, Ser663. The requirement for three phosphorylation sites in NHE3 for cGKII inhibition, and for phosphorylation of one of these sites for dexamethasone stimulation of NHE3, is a unique example of regulation by phosphorylation.
AB - The epithelial brush-border Na+/H+ exchanger NHE3 is acutely inhibited by cGKII/cGMP, but how cGKII inhibits NHE3 is unknown. This study tested the hypothesis that cGMP inhibits NHE3 by phosphorylating it and altering its membrane trafficking. Studies were carried out in PS120/NHERF2 and in Caco-2/Bbe cells overexpressing HA-NHE3 and cGKII, and in mouse ileum. NHE3 activity was measured with 2',7'-bis(car-boxyethyl)-S-(and 6)carboxyfluorescein acetoxy methylester/fluorometry. Surface NHE3 was determined by cell surface biotinylation. Identification of NHE3 phosphorylation sites was by iTRAQ/LC-MS/MS with TiO2 enrichment and immunoblotting with specific anti-phospho-NHE3 antibodies. cGMP/cGKII rapidly inhibited NHE3, which was associated with reduced surface NHE3. cGMP/cGKII increased NHE3 phosphorylation at three sites (rabbit Ser554, Ser607, and Ser663, equivalent to mouse Ser552, Ser605, and Ser659), all of which had to be present at the same time for cGMP to inhibit NHE3. NHE3-Ser663 phosphorylation was not necessary for cAMP inhibition of NHE3. Dexamethasone (4 h) stimulated wild type NHE3 activity and increased surface expression but failed to stimulate NHE3 activity or increase surface expression when NHE3 was mutated to either S663A or S663D. We conclude that 1) cGMP inhibition of NHE3 is associated with phosphorylation of NHE3 at Ser554, Ser607, and Ser663, all of which are necessary for cGMP/cGKII to inhibitNHE3.2)DexamethasonestimulatesNHE3byphosphorylation of a single site, Ser663. The requirement for three phosphorylation sites in NHE3 for cGKII inhibition, and for phosphorylation of one of these sites for dexamethasone stimulation of NHE3, is a unique example of regulation by phosphorylation.
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U2 - 10.1074/jbc.M114.590174
DO - 10.1074/jbc.M114.590174
M3 - Article
C2 - 25480791
AN - SCOPUS:84921683215
SN - 0021-9258
VL - 290
SP - 1952
EP - 1965
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 4
ER -