14-3-3 Binding to Na+/H+ Exchanger Isoform-1 Is Associated with Serum-dependent Activation of Na+/H+ Exchange

Abstract

Na؉ /H ؉ exchanger isoform-1 (NHE1), the ubiquitous form of the Na ؉ /H ؉ exchanger, has increased activity in hypertensive patients and in animal models of hypertension. Furthermore, NHE1 is activated in cells stimulated with growth factors. We showed previously that activation of the exchanger is dependent on phosphorylation of serine 703 (Ser(P) 703 ) by p90 ribosomal S6 kinase (RSK). Because the NHE1 sequence at Ser(P) 703(RIGSDP) is similar to a consensus sequence (RSXSXP) specific for 14-3-3 ligands, we evaluated whether serum stimulated 14-3-3 binding to NHE1. Five different GST-NHE1 fusion proteins spanning amino acids 515-815 were phosphorylated by RSK and used as ligands in a far Western analysis; only those containing Ser(P) 703 exhibited high affinity 14-3-3 binding. In PS127A cells (NHE1-overexpressing Chinese hamster fibroblasts) stimulated with 20% serum, NHE1 co-precipitation with GST-14-3-3 fusion protein increased at 5 min (5.2 ؎ 0.4-fold versus control; p < 0.01) and persisted at 40 min (3.9 ؎ 0.3-fold; p < 0.01). We confirmed that binding occurs at the RIGSDP motif using PS120 (NHE1 null) cells transfected with S703A-NHE1 or P705A-NHE1 (based on data indicating that 14-3-3 binding requires phosphoserine and ؉2 proline). Serum failed to stimulate association of 14-3-3 with these mutants. A GST-NHE1 fusion protein was phosphorylated by RSK and used as a ligand to assess the effect of 14-3-3 on protein phosphatase 1-mediated dephosphorylation of Ser(P) 703 . GST-14-3-3 limited dephosphorylation (66% of initial state at 60 min) compared with GST alone (27% of initial state; p < 0.01). The protective effect of GST-14-3-3 was lost in the GST-NHE1 P705A mutant. Finally, the baseline rate of pH recovery in acid-loaded cells was equal in unstimulated cells expressing wild-type or P705A-NHE1. However, activation of NHE1 by serum was dramatically inhibited in cells expressing P705A-NHE1 compared with wild-type (0.13 ؎ 0.02 versus 0.48 ؎ 0.06 mmol of H ؉ /min/liter, p < 0.01). These data suggest that 14-3-3 binding to NHE1 participates in serum-stimulated exchanger activation, a new function for 14-3-3. NHE11 is the ubiquitous form of the Na ϩ /H ϩ exchanger, responsible for regulation of intracellular pH and cell volume. NHE1 has increased activity in hypertensive patients and in animal models of hypertension, and the exchanger can be stimulated by various exogenous factors. In several examples of increased NHE1 activity, the primary change is not in NHE1 expression but in post-translational regulation of activity. In immortalized cells from hypertensive patients, enhanced NHE1 activity appears to reflect increased V max rather than increased NHE1 mRNA (1). Similarly, vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) show an increased V max compared with their normotensive Wistar-Kyoto (WKY) counterparts but do not overexpress NHE1 mRNA or protein (2-4). Furthermore, there is no evidence for genetic linkage between the Na ϩ /H ϩ exchanger gene and human hypertension (5), a...

A Novel Membrane Protein Capable of Binding the Na+/H+ Antiporter (Nha1p) Enhances the Salinity-resistant Cell Growth of Saccharomyces cerevisiae

“…We have also shown that these conserved domains probably play similar functions in yeast and other fungi (28). The C1, C2, and C3 domains consist of 16,23, and 15 residues, respectively, and are located at the juxtamembrane area of the cytoplasmic tail region. Deletion of these domains decreases salinity-resistant growth, which suggests that these domains are important for growth during highly saline conditions, possibly because they influence the antiporter activity of Nha1p (28).…”

Solution Structure of the Cytoplasmic Region of Na+/H+ Exchanger 1 Complexed with Essential Cofactor Calcineurin B Homologous Protein 1

“…3B). The secondary structure consists of ␣A (residues [11][12][13][14][15][16][17][18][19][20][21][22], ␣B (residues 26 -37), ␣C (residues 48 -51), ␣D (residues 64 -70), ␣E (residues 80 -88), ␣F (residues 111-122), ␣G (residues 132-143), ␣H (residues 149 -162), ␣I (residues 174 -180), and ␣J (residues 185-188) (Figs. 1B and 3B).…”

Calcineurin Homologous Protein Isoform 2 (CHP2), Na+/H+ Exchangers-Binding Protein, Is Expressed in Intestinal Epithelium.

“…[16][17][18] This regulation has been suggested to involve multiple factors. [19][20][21][22][23][24][25][26] Ca 2ϩ -calmodulin and NHE3 regulatory factor (NHE-RF) have been shown to modulate NHE1 and NHE3 activity, respectively. 19,22) Calcineurin homologous protein (CHP), a 22-kDa protein closely related to the Ca 2ϩ -binding B subunit of the heterodimeric phosphatase calcineurin, 20) acts as a crucial cofactor for at least NHE1-3 activity through direct interaction with the cytoplasmic domain of NHEs.…”