B-Raf Associates with and Activates the NHE1 Isoform of the Na+/H+ Exchanger

Abstract

The serine/threonine kinase B-Raf is the second most frequently occurring human oncogene after Ras. Mutations of B-Raf occur with the highest incidences in melanoma, and the most common mutant, V600E, renders B-Raf constitutively active. The sodium proton exchanger isoform 1 (NHE1) is a ubiquitously expressed plasma membrane protein responsible for regulating intracellular pH, cell volume, cell migration, and proliferation. A screen of protein kinases that bind to NHE1 revealed that B-Raf bound to the cytosolic regulatory tail of NHE1. Immunoprecipitation of NHE1 from HeLa and HEK cells confirmed the association of B-Raf with NHE1 in vivo. The expressed and purified C-terminal 182 amino acids of the NHE1 protein were also shown to associate with B-Raf protein in vitro. Because treatment with the kinase inhibitor sorafenib decreased NHE1 activity in HeLa and HEK cells, we examined the role of B-Raf in regulating NHE1 in malignant melanoma cells. Melanoma cells with the B-RafV600E mutation demonstrated increased resting intracellular pH that was dependent on elevated NHE1 activity. NHE1 activity after an acute acid load was also elevated in these cell lines. Moreover, inhibition of B-Raf activity by either sorafenib, PLX4720, or siRNA reduction of B-Raf levels abolished ERK phosphorylation and decreased NHE1 activity. These results demonstrate that B-Raf associates with and stimulates NHE1 activity and that B-Raf V600E also increases NHE1 activity that raises intracellular pH.

Potential Role of Sodium-Proton Exchangers in the Low Concentration Arsenic Trioxide-Increased Intracellular pH and Cell Proliferation

“…Interestingly, PD-98059 also blocked the increase in the pHi recovery rate as well as the increase in HMA−s J H + suggesting that p42/44 mapk activation is required for ATO stimulation of NHEs-like mediated transport in MDCK cells. This possibility confirms results reporting that p42/44 mapk activation associates with intracellular alkalization due to NHE1 activation in HeLa and HEK cell lines [24]. In the latter study a key role of the serine/threonine kinase B-raf, a protein expressed in MDCK cells, in p42/44 mapk -mediated activation of NHE1 is proposed.…”

“…In addition, there is also evidence that protein kinases C (PKC) are activated in response to ATO [21], [22]. Since activation of PKC [23] and p42/44 mapk [24] also associates with an increase in the pHi value due to activation of NHE1, we hypothesize that ATO will increase cell proliferation via a mechanism involving NHE-like activity. The results show that the lowest concentration of ATO used in this study (i.e., 0.05 µmol/L, equivalent to ∼0.01 ppm or ∼10 µg/L), which corresponds to the maximal recommended arsenic concentration of this molecule in the drinking-water to avoid health problems in humans by the World Health Organization [25], caused an increase in the proliferation of Madin-Darby canine kidney (MDCK) cells.…”

Physiology, Pharmacology and Pathophysiology of the pH Regulatory Transport Proteins NHE1 and NBCn1: Similarities, Differences, and Implications for Cancer Therapy

“…Notably, NHE1 was also found to interact with kinases in three of the upstream tiers in the MAPK cascade, specifically MAP4K4, the MAP3Ks Raf1 and TGF 1-activated kinase-1 (TAK1/ MAP3K7), and MAP2K2 [174]. A subsequent study reported the binding (direct or indirect) of B-Raf and the ERK effector Ribosomal S Kinase (p90RSK) to NHE1 [175]. Collectively, these data indicate that NHE1 may act as a scaffold for signaling via the MAPK complex and its downstream effector(s).…”

Upregulation of NHE1 protein expression enables glioblastoma cells to escape TMZ-mediated toxicity via increased H+extrusion, cell migration and survival

“…Human NHE1 protein consists of 815 amino acids with a calculated molecular weight of 85 kDa. However, because of the N-linked and O-linked glycosylation in its extracellular loop 1, the actual molecular weight of NHE1 can be varied from 85 to ~110 kDa in different cell types (19)(20)(21). Compared with NHE1 expression level in NSC, NHE1 protein was 9.8 ± 0.3 fold higher in GC#99, 15.6 ± 0.2 fold higher in GC#22 and 14.5 ± 1.2 fold in U87.…”