Direct interaction between ERK2 and NHE1 in vivo and in silico.
a Proximity ligase assay carried out in AP-1 WT hNHE1 cells treated with NHE1 and ERK1/2 primary antibodies. Proximity ligase signal appears as red dots and the merge image highlights the interaction between NHE1 and ERK. F-actin was stained with phalloidin488. Scale bars represent 10 μm. Data are representative of three independent replicates. b Proximity ligase assay carried out in AP-1 WT hNHE1 cells. As a negative control, cells were only treated with NHE1 primary antibodies and the appearance of the red dots indicates unspecific binding. F-actin was stained with phalloidin488. Scale bars represent 10 μm. Data are representative of three independent replicates. c Quantification of PLA data was carried out in ImageJ. PLA signal from at least ten different image areas in each experiment were counted by particle analysis and the average PLA signal per cell was plotted in the bar graph. Data are representative of three independent replicates. d ERK1/2 docking motifs, D-domain and F-site. Φ indicates hydrophobic amino acid residues (typically L, V, or I), and X any other amino acid. e Overall NHE1 topology and localization of putative D-domains and ERK2 phosphorylation sites in the hNHE1cdt. The positions of identified D-domains as well as predicted (S/T)P-phosphorylation sites are indicated by stars. Insert: alignment of the consensus ERK2 phosphorylation sites in hNHE1; (S/T)P sites indicated by grey background. f Alignment of hNHE1 D-domains to known D-domains. The consensus hydrophobic and positively charged residues are highlighted in yellow and blue, respectively. g Sequence conservation of putative D-domains in NHE1cdt in various species. D1, D2, and D3 are indicated with grey bars above the alignment. The solid horizontal line separates tetrapods (top) from teleosts (bottom)