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B-Raf associates with and activates the NHE1 isoform of the Na+/H+ exchanger

Pratap Karki1, Xiuju Li, David Schrama

  • 1Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.

The Journal of Biological Chemistry
|February 25, 2011
PubMed

Insights

B-Raf, a common cancer oncogene, directly interacts with and activates the sodium proton exchanger 1 (NHE1). This interaction is crucial in melanoma, where the B-Raf V600E mutation elevates intracellular pH via increased NHE1 activity.

Area of Science:

  • Oncology
  • Molecular Biology
  • Cell Physiology

Background:

  • B-Raf is a frequent oncogene in human cancers, particularly melanoma.
  • The V600E mutation in B-Raf leads to its constitutive activation.
  • Sodium proton exchanger 1 (NHE1) regulates crucial cellular functions including pH, volume, migration, and proliferation.

Purpose of the Study:

  • To investigate the interaction between B-Raf and NHE1.
  • To determine the role of B-Raf in regulating NHE1 activity, especially in melanoma.
  • To elucidate the impact of B-Raf mutations on cellular pH regulation.

Main Methods:

  • Protein kinase screening to identify B-Raf binding partners.
  • Immunoprecipitation assays to confirm in vivo association of B-Raf and NHE1.
  • In vitro binding assays using purified NHE1 protein fragments.
  • Enzyme activity assays and intracellular pH measurements in cell lines.
  • Pharmacological inhibition and genetic knockdown of B-Raf.

Main Results:

  • B-Raf was identified as a binding partner of NHE1, interacting with its cytosolic tail.
  • B-Raf and NHE1 associate in vivo in HeLa and HEK cells.
  • Melanoma cells with the B-Raf V600E mutation exhibit elevated resting intracellular pH and increased NHE1 activity.
  • Inhibition of B-Raf activity (using sorafenib, PLX4720, or siRNA) reduced NHE1 activity and ERK phosphorylation.
  • B-Raf V600E mutation increases NHE1 activity, leading to higher intracellular pH.

Conclusions:

  • B-Raf directly associates with and stimulates the activity of NHE1.
  • The oncogenic B-Raf V600E mutation enhances NHE1 activity, contributing to elevated intracellular pH in melanoma cells.
  • Targeting B-Raf may represent a therapeutic strategy for modulating NHE1 activity in relevant cancers.

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