The Sodium-Hydrogen Exchanger NHE1 Is an Akt Substrate Necessary for Actin Filament Reorganization by Growth Factors

Abstract

The kinase Akt mediates signals from growth factor receptors for increased cell proliferation, survival, and migration, which contribute to the positive effects of Akt in cancer progression. Substrates are generally inhibited when phosphorylated by Akt; however, we show phosphorylation of the plasma membrane sodium-hydrogen exchanger NHE1 by Akt increases exchanger activity (H ؉ efflux). Our data fulfill criteria for NHE1 being a bona fide Akt substrate, including direct phosphorylation in vitro, using mass spectrometry and Akt phospho-substrate antibodies to identify Ser 648 as the Akt phosphorylation site and loss of increased exchanger phosphorylation and activity by insulin and platelet-derived growth factor in fibroblasts expressing a mutant NHE1-S648A. How Akt induces actin cytoskeleton remodeling to promote cell migration and tumor cell metastasis is unclear, but disassembly of actin stress fibers by platelet-derived growth factor and insulin and increased proliferation in growth medium are inhibited in fibroblasts expressing NHE1-S648A. We predict that other functions shared by Akt and NHE1, including cell growth and survival, might be regulated by increased H ؉ efflux.The serine/threonine kinase Akt/protein kinase B functions as a convergence site of cues that signal through the lipid kinase phosphoinositide 3-kinase (PI 3-kinase), 4 including activated integrin receptors, growth factor receptor tyrosine kinases, cytokine receptors, and G protein-coupled receptors. Akt confers signal relay from these upstream regulators to increase cell proliferation, cell survival, protein synthesis, and glycolytic flux (1). These actions contribute to the positive effects of Akt in cancer progression, and PI 3-kinase/Akt signaling is commonly aberrant in human tumors (2). Akt also regulates reorganization of the actin cytoskeleton, including disassembly of bundled actin stress fibers and formation of actin filament-rich membrane protrusions (3, 4), which likely enhance the positive effects of some Akt isoforms on cell migration (5) and tumor cell metastasis (6). However, compared with most cell processes regulated by Akt, our understanding of how Akt regulates cytoskeleton dynamics is least understood. Akt generally phosphorylates substrates at an RXRXX(S/T) recognition motif (7), and in most instances Akt phosphorylation inhibits activity or function of the substrate. Akt promotes cell proliferation by phosphorylating and inhibiting the negative cell cycle regulators p21Cip1/WAF1 (8) and p27 Kip1 (9 -11) and Wee1 kinase (12). It promotes cell survival by phosphorylating and inhibiting the function of proapoptosis regulators, including Bad (13,14) and forkhead transcription factors (15,16), and it increases protein synthesis by phosphorylating and inhibiting the negative regulators of mTOR, tubersclerosis sclerosis protein 2 (17) and proline-rich Akt substrate of 40 kDa (18). Additionally, Akt increases metabolism in part by phosphorylating and inhibiting glycogen synthase 3 (19). However, there are a few exceptions w...

Defining the Na+/H+ exchanger NHE1 interactome in triple-negative breast cancer cells

“…AKT phosphorylation of NHE1 is stimulatory, induces cytoskeletal remodeling, and is necessary for the activation of NHE1 by insulin and PDGF [59]. We confirmed that AKT interacts with NHE1 in MDA-MB-231 cells by co-immunoprecipitation experiments (Fig.…”

Na + /H + exchanger-mediated hydrogen ion extrusion as a carcinogenic signal in triple-negative breast cancer etiopathogenesis and prospects for its inhibition in therapeutics

“…Examples of kinase-mediated activators of NHE1 include: extracellular signal regulated kinases ERK1/2 which are important in the activation of NHE1 in response to intracellular acidosis, and for which NHE1 serves as a signal scaffold (54,55); AKT (protein kinase B) which activates NHE1 in response to insulin and platelet-derived growth factor in fibroblasts (56), but inhibits NHE1 activity in cardiomyocytes (57); -Raf which phosphorylates NHE1 at amino acid threonine 653 (58); and p90 ribosomal S6 kinase (p90 RSK ) which phosphorylates NHE1 at serine 703, activating the exchanger in response to serum (59). Adaptor protein 14-3-3 binds NHE1 at the phosphorylated serine 703 residue and prevents its dephosphorylation (60).…”

Ratiometric Imaging of pH Probes

“…Changes in the rate of pHi recovery can be used to determine the regulation of ion transporter activity, including by growth factors, oncogenes, and extracellular osmolarity. For example, platelet-derived growth factor increases the activity of the plasma membrane Na + /H + exchanger NHE1, as determined by increased pHi recoveries measured in a nominally HCO 3 À -free HEPES buffer that prevents the activity of anion exchangers (Meima, Webb, Witkowska, & Barber, 2009). Fluorescence intensities are used to determine ratios that are converted to pH values using a nigericin calibration.…”