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Investigation of the Interaction and Role of CHP1 and CHP2 with NHE1 in Lung Fibroblasts
Author(s) -
Kiefer Lea,
Wallert Mark,
Provost Joseph
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.725.17
Subject(s) - microbiology and biotechnology , intracellular ph , chemistry , intracellular , cytosol , gene isoform , transfection , biochemistry , biology , enzyme , gene
Intracellular pH (pH i ) homeostasis is crucial for cell survival. The membrane protein Sodium‐Hydrogen exchanger 1 (NHE1) is one of the major contributors to a stable pH i . Additionally, NHE1 serves as an anchor for intracellular proteins. NHE1 also plays an essential role in Non‐small cell lung cancer (NSCLC) metastasis as a side effect of the transporter's increased activity to compensate the Warburg effect. Several phosphorylation sites and protein cofactors affect NHE1's activity. The two isoforms calcineurin homologous proteins CHP1 and CHP2 each bind to the C‐terminus of NHE1 and regulate its function. CHP1 is ubiquitous and is key for basel level NHE1 activity while CHP2 is primarily expressed in gut cells, detected in most cancer cells and increases proton transport. To better understand the role of NHE1 and CHP1/CHP2 we generated RFP‐CHP1 and GFP‐CHP2 and transfected each into lung fibroblasts expressing tagged NHE1 (PSN cells) and into lung fibroblasts lacking NHE1 (PS120 cells). Cellular distribution of each CHP was determined and showed significant translocation of CHP1 and CHP2 to the plasma membrane in PSN cells while no localization was observed in PS120 cells. The specific amino acid required for CHP‐NHE1 interaction is determined in cells expressing one of eight different NHE1 amino acid substitutions at the putative CHP‐NHE binding domain of NHE1. To investigate the role of CHP2 binding to NHE we expressed GFP‐CHP2 in PSN and PS120 cells and conducted wound‐healing assays. In addition to cell migration rates, we tracked the migration path of individual cells to assess the impact of each NHE1 mutation on CHP2‐influenced motility.