The sodium hydrogen exchanger isoform one (NHE1) plays a critical role coordinating asymmetric events at the leading edge of migrating cells and is regulated by a number of phosphorylation events influencing both the ion transport and cytoskeletal anchoring required for directed migration. Lysophosphatidic acid (LPA) activation of RhoA kinase (Rock) and the Ras-ERK growth factor pathway induces cytoskeletal reorganization, activates NHE1 and induces an increase in cell motility. We report that both Rock I and II stoichiometrically phosphorylate NHE1 at threonine 653 in vitro using mass spectrometry and reconstituted kinase assays. In fibroblasts expressing NHE1 alanine mutants for either Rock (T653A) or ribosomal S6 kinase (Rsk; S703A) we show each site is partially responsible for the LPA-induced increase in transport activity while NHE1 phosphorylation by either Rock or Rsk at their respective site is sufficient for LPA stimulated stress fiber formation and migration. Furthermore, mutation of either T653 or S703 leads to a higher basal pH level and a significantly higher proliferation rate. Our results identify the direct phosphorylation of NHE1 by Rock and suggest that both RhoA and Ras pathways mediate NHE1-dependent ion transport and migration in fibroblasts.
The sodium hydrogen exchanger (NHE1) plays a role in intracellular pH homeostasis and acts as scaffolding anchor for a diverse set of proteins. The extended carboxyl terminus is phosphorylated by seven known protein kinases. Phosphorylation site of NHE1 by RhoA Kinase (Rock) was recently defined and found critical for a number of cellular functions. However, the impact of Rock phosphorylation on NHE1‐related signaling for a range of agonists has not been determined. Here we examine three different signaling pathways and the role Rock phosphorylation of NHE1. Using stably expressing tagged NHE1 we identify proteins co‐immunoprecipitating with NHE1 in control and agonist stimulated cells. Cellular function of adhesion and proliferation is also presented. Finally the influence of Rock phosphorylation of NHE1 on cellular migration was determined for three signaling pathways using an impedance‐based assay. This work was supported with funds from NSF MCB‐0817784
The challenge of hypoxia in solid tumors induces several physiological changes in the cancer cell microenvironment including increased extracellular acidity. While proton exchangers including the sodium hydrogen exchanger (NHE1) support tumor cell invasion and metastasis, the relationship between hypoxia and increased acidity on migration/proliferation remains unclear. In this study we investigate the relationship between the sodium hydrogen exchanger isoform 1 (NHE1) and hypoxia inducible factor 1 (HIF‐1α) expression in H1299. HIF‐1α expression was observable after 24 hours of either 1% oxygen or chemically induced hypoxia and was maximally expressed after 72 hours of hypoxia. In parallel we found that NHE1 expression slightly increased after 72 hours in hypoxic conditions. We also show that hypoxia decreased cell proliferation in H1299 non small lung cancer cells. In addition, the impact of NHE1 inhibition on hypoxia induced MMP‐9 activity was altered. Hypoxia induced changes in cell motility and alkalization of intracellular pH was also observed. These results point to a relationship between hypoxia and NHE1 that influences cancer cell behavior. NIH Grant 1 R15 HL074924–01A1
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