14-3-3 binding to Na+/H+ exchanger isoform-1 is associated with serum-dependent activation of Na+/H+ exchange

Na+/H+ exchanger isoform-1 (NHE1), the ubiquitous form of the Na+/H+ exchan ger, has increased activity in hypertensive patients and in animal models o f hypertension. Furthermore, NHE1 is activated in cells stimulated with gro wth factors. We showed previously that activation of the exchanger is depen dent on phosphorylation of serine 703 (Ser(P)(703)) by p90 ribosomal S6 kin ase (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 whe ther serum stimulated 14-3-3 binding to NHE1. Five different GST-NHE1 fusio n 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) ex hibited high affinity 14-3-3 binding. In PS127A cells (NHE1-overexpressing Chinese hamster fibroblasts) stimulated with 20% serum, NHE1 co-precipitati on with GST-14-3-3 fusion protein increased at 5 min (5.2 +/- 0.4-fold vers us control; p < 0.01) and persisted at 40 min (3.9 +/- 0.5-fold; p < 0.01). We confirmed that binding occurs at the RIGSDP motif using PS120 (NHE1 nul l) cells transfected with S703A-NHE1 or P705A-NHE1 (based on data indicatin g 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 pr otein was phosphorylated by RSK and used as a ligand to assess the effect o f 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) com pared with GST alone (27% of initial state; p < 0.01). The protective effec t of GST-14-3-3 was lost in the GST-NHE1 P705A mutant. Finally, the baselin e 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 wa s 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). T hese data suggest that 14-3-3 binding to NHE1 participates in serum-stimula ted exchanger activation, a new function for 14-3-3.