cAMP-induced phosphorylation and inhibition of Na(+)/H(+) exchanger 3 (NHE3) are dependent on the presence but not the phosphorylation of NHE regulatory factor.
The members of the regulatory factor (RF) gene family, Na(+)/H(+) exchanger (NHE)-RF and NHE3 kinase A regulatory factor (E3KARP) are necessary for cAMP to inhibit the epithelial brush border NHE isoform 3 (NHE3). The mechanism of their action was studied using PS120 fibroblasts stably transfected with rabbit NHE3 and wild type rabbit NHE-RF or wild type human E3KARP. 8-Bromo-cAMP (8-Br-cAMP) had no effect on Na(+)/H(+) exchange activity in cells expressing NHE3 alone. In contrast, in cells co-expressing NHE-RF, 8-Br-cAMP inhibited NHE3 by 39%. In vivo phosphorylation of NHE3 demonstrated that cAMP increased phosphorylation in two chymotrypsin-generated phosphopeptides of NHE3 in cells containing NHE-RF or E3KARP but not in cells lacking these proteins. The requirement for phosphorylation of NHE-RF in this cAMP-induced inhibition of NHE3 was examined by studying a mutant NHE-RF in which serines 287, 289, and 290 were mutated to alanines. Wild type NHE-RF was a phosphorylated protein under basal conditions, but treatment with 8-Br-cAMP did not alter its phosphorylation. Mutant NHE-RF was not phosphorylated either under basal conditions or after 8-Br-cAMP. 8-Br-cAMP inhibited NHE3 similarly in PS120/NHE3 cells containing wild type or mutant NHE-RF. NHE-RF and NHE3 co-precipitated and did so similarly with and without cAMP. Mutant NHE-RF also similarly immunoprecipitated NHE3 in the presence and absence of 8-Br-cAMP. This study shows that members of the regulatory factor gene family, NHE-RF and E3KARP, are necessary for cAMP inhibition of NHE3 by allowing NHE3 to be phosphorylated. This inhibition is not dependent on the phosphorylation of NHE-RF.
Zizak
M
M
Department of Medicine, Gl Division, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Lamprecht
G
G
Steplock
D
D
Tariq
N
N
Shenolikar
S
S
Donowitz
M
M
Yun
C H
CH
Weinman
E J
EJ
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002918:Chymotrypsin; D000242:Cyclic AMP / Q000494:pharmacology*; D006801:Humans; D008871:Microvilli / Q000378:metabolism; D009154:Mutation; D010748:Phosphopeptides / Q000032:analysis; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011817:Rabbits; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors*
D010748:Phosphopeptides; D010750:Phosphoproteins; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000242:Cyclic AMP; D002918:Chymotrypsin