cAMP-induced phosphorylation and inhibition of Na+/H+ exchanger 3 (NHE3) are dependent on the presence but not the phosphorylation of NHE regulatory factor

被引:101
作者
Zizak, M
Lamprecht, G
Steplock, D
Tariq, N
Shenolikar, S
Donowitz, M
Yun, CHC
Weinman, EJ
机构
[1] Johns Hopkins Univ, Sch Med, Dept Med, GI Div, Baltimore, MD 21205 USA
[2] W Virginia Univ, Sch Med, Dept Med, Morgantown, WV 26506 USA
[3] Dept Vet Affairs Med Ctr, Med Serv, Clarksburg, WV 26301 USA
[4] Duke Univ, Med Ctr, Dept Pharmacol & Canc Biol, Durham, NC 27710 USA
关键词
D O I
10.1074/jbc.274.35.24753
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 (NRE3). 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.
引用
收藏
页码:24753 / 24758
页数:6
相关论文
共 21 条
[1]   PARATHYROID-HORMONE AND PARATHYROID HORMONE-RELATED PEPTIDE INHIBIT THE APICAL NA+/H+ EXCHANGER NHE-3 ISOFORM IN RENAL-CELLS (OK) VIA A DUAL SIGNALING CASCADE INVOLVING PROTEIN-KINASE-A AND PROTEIN-KINASE-C [J].
AZARANI, A ;
GOLTZMAN, D ;
ORLOWSKI, J .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1995, 270 (34) :20004-20010
[2]  
Cabado AG, 1996, J BIOL CHEM, V271, P3590
[3]   G protein-coupled receptors in gastrointestinal physiology III.: Asymmetry in plasma membrane signal transduction:: lessons from brush-border Na+/H+ exchangers [J].
Donowitz, M ;
Khurana, S ;
Tse, CM ;
Yun, CHC .
AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY, 1998, 274 (06) :G971-G977
[4]   Ezrin is a cyclic AMP-dependent protein kinase anchoring protein [J].
Dransfield, DT ;
Bradford, AJ ;
Smith, J ;
Martin, M ;
Roy, C ;
Mangeat, PH ;
Goldenring, JR .
EMBO JOURNAL, 1997, 16 (01) :35-43
[5]   The β2-adrenergic receptor interacts with the Na+/H+-exchanger regulatory factor to control Na+/H+ exchange [J].
Hall, RA ;
Premont, RT ;
Chow, CW ;
Blitzer, JT ;
Pitcher, JA ;
Claing, A ;
Stoffel, RH ;
Barak, LS ;
Shenolikar, S ;
Weinman, EJ ;
Grinstein, S ;
Lefkowitz, RJ .
NATURE, 1998, 392 (6676) :626-630
[6]   NHE2 and NHE3 are human and rabbit intestinal brush-border proteins [J].
Hoogerwerf, WA ;
Tsao, SC ;
Devuyst, O ;
Levine, SA ;
Yun, CHC ;
Yip, JW ;
Cohen, ME ;
Wilson, PD ;
Lazenby, AJ ;
Tse, CM ;
Donowitz, M .
AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY, 1996, 270 (01) :G29-G41
[7]   Identification of sites required for down-regulation of Na+/H+ exchanger NHE3 activity by cAMP-dependent protein kinase - Phosphorylation-dependent and -independent mechanisms [J].
Kurashima, K ;
Yu, FH ;
Cabado, AG ;
Szabo, EZ ;
Grinstein, S ;
Orlowski, J .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1997, 272 (45) :28672-28679
[8]   The role of NHERF and E3KARP in the cAMP-mediated inhibition of NHE3 [J].
Lamprecht, G ;
Weinman, EJ ;
Yun, CHC .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1998, 273 (45) :29972-29978
[9]  
LEVINE SA, 1993, J BIOL CHEM, V268, P25527
[10]   ACTIVATION OF PROTEIN-KINASE-A ACUTELY INHIBITS AND PHOSPHORYLATES NA/H EXCHANGER NHE-3 [J].
MOE, OW ;
AMEMIYA, M ;
YAMAJI, Y .
JOURNAL OF CLINICAL INVESTIGATION, 1995, 96 (05) :2187-2194