Figure 5. RSK-induced NHE-1 phosphorylation increases its activity in hypoxia.

(A) HT-1080 cells were cultured for the indicated periods of time under normoxic or hypoxic conditions or for 30 min under hypoxia with the addition of the MEK1/2 inhibitor PD98059 (50 µM). Representative immunoblots showing p90RSK Ser³⁸⁰ phosphorylation and NHE-1 Ser⁷⁰³ phosphorylation. NHE-1 phosphorylation was revealed using antibodies directed against the 14-3-3 binding motif, following NHE-1 immunoprecipitation. Ratios of pRSK to total RSK and pNHE-1 to total NHE-1 are shown (n = 3). (B-D) Na⁺/H⁺ exchange rates were measured in cells exposed to normoxia (empty bars) or hypoxia (filled bars) in (B) the presence or absence of the p90RSK inhibitor BI-D1870 (10 µM) (n = 3), or (C) cells transfected with a dominant negative p90RSK (n = 3), or (D) cells transfected with WT NHE-1 or a S703A substitution NHE-1 mutant (n = 3). Columns represent the mean ± SEM indicated by the horizontal bars. The asterisks correspond to, ** p<0.001; *** p<0.0001.