Regulation of the Na+/H+ Exchanger 1 by BRAF Kinase

• Author / Creator

  Augustine, Aruna M

• Mammalian Na+/H+ Exchanger 1 is a ubiquitously expressed membrane protein, which is dedicated to maintaining the acid-base balance in the cell. This balance is brought about by the exchange of one intracellular proton in exchange for extracellular sodium ion. NHE1 is the predominant isoform found in the myocardium and has been implicated in several cardiac diseases including cardiac hypertrophy and ischemic reperfusion induced injury. The NHE1 C-terminal domain is made up of 315 amino acids and is subject to regulation by several protein and protein kinases including the BRAF kinase. In our study we have demonstrated that the BRAF kinase binds to NHE1 C-terminal tail within a region encompassed by amino acids 634 to 710. We also demonstrate that BRAF kinase co-localizes with NHE1 in both AP1 cells as well as HeLa cells, in a serum-dependent manner and the withdrawal of serum results in a loss of co-localization between the two proteins. We further evaluated the role of the threonine 653 amino acid (a target of BRAF kinase phosphorylation) on the NHE1 C-terminal tail with respect to cell proliferation. Mutation of the residue to a non-phosphorylatable amino acid led to a reduction in the rate of cell proliferation. Additionally, we examined the role of BRAF-NHE1 interaction in melanoma and its effects on various cellular processes. We used specific inhibitors of wild type BRAF (SB590885), mutant BRAF (PLX4720) and NHE1 (EMD87580 and 5-(N,N-Hexamethylene) amiloride (HMA)) either alone or in combination to elucidate the role of BRAF-NHE1 interaction in M19, IF6, FM82 and Mel2A human melanoma cell lines. NHE1 did not affect the melanoma cell lines’ ability to proliferate. Migration was significantly reduced in the presence of inhibitors. A combination of BRAF inhibitor and HMA led to a small but significant effect on the rate of migration when compared to NHE1 or BRAF inhibition alone. Invasion was also significantly reduced in the presence of NHE1 or BRAF inhibitors. We also observed varying levels of vimentin among the four cell lines with M19 and Mel2A having high levels of this protein. Mature NHE1 protein was observed only in FM82 and Mel2A but not in M19 or IF6 cell line. These results highlight the importance of NHE1 in the various cellular events associated with tumorigenesis and give a better insight into the underlying mechanism. We further investigated the role played by stop codon polymorphisms (SCP) in NHE1 localization. We looked at four different SCPs namely, 735STOP, 543STOP, 449STOP and 321STOP. 735STOP localized to the plasma membrane (similar to wild type NHE1) while the other three mutants had cytosolic localization. This study provides us with a better understanding of the vital role played by NHE1 C-terminal tail and how these SCPs can cause deleterious effects within the cell.

• Subjects / Keywords

  • BRAF
  • NHE1
  • Melanoma

• Graduation date

  Fall 2017

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R3PK07G03

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.