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

Department of Cell and Tissue Biology, University of California, San Francisco, California 94143, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 08/2009; 284(39):26666-75. DOI: 10.1074/jbc.M109.019448
Source: PubMed


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.

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    • "Independent of its exchanger function, NHE1 is known to mediate migration in a number of cell types via its cytoplasmic tail binding to the actin cytoskeleton (Cardone et al., 2005; Denker and Barber, 2002; Meima et al., 2009; Schneider et al., 2009; Stock and Schwab, 2006; Stuwe et al., 2007)and MIA has been shown to only affect NHE1 exchanger function and not actin binding by NHE (Counillon et al., 1993; Slepkov et al., 2007). To support this statement, we noted no decrease in branching morphogenesis when we added MIA to mammary tissue at 48 hours. "
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    ABSTRACT: Regulation of intracellular pH (pHi) and protection against cytosolic acidification is primarily a function of the ubiquitous plasma membrane Na+/H+exchanger-1 (NHE1), which uses a highly conserved process to transfer cytosolic hydrogen ions (H+) across plasma membranes in exchange for extracellular sodium ions (Na+). Growth factors, which are essential regulators of morphogenesis, have also been found to be key activators of NHE1 exchanger activity; however, the crosstalk between both has not been fully evaluated during organ development. Here we report that mammary branching morphogenesis induced by transforming growth factor-alpha (TGFα) requires PI3K-dependent NHE1-activation and subsequent pHi alkalization. Inhibiting NHE1 activity after TGFα stimulation with 10 μM of the NHE1-specific inhibitor N-Methyl-N-isobutyl Amiloride (MIA) dramatically disrupted branching morphogenesis, induced extensive proliferation, ectopic expression of the epithelial hyper-proliferative marker Keratin-6 and sustained activation of MAPK. Together these findings indicate a novel developmental signaling cascade involving TGFα>PI3K>NHE1>pHi alkalization, which leads to a permissible environment for MAPK negative feedback inhibition and thus regulated mammary branching morphogenesis.
    Full-text · Article · Feb 2012 · Developmental Biology
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    • "Further analysis of the cortactin–NHE1 interaction is needed to elucidate this novel mechanism. The activation of NHE1 exchanger activity is also regulated downstream of growth factor receptors (Meima et al., 2009), which could represent a common EGF-dependent cortactin phosphorylation pathway. Recent work suggests that NHE1 localizes to invadopodia, where it acidifies the extracellular space, increasing ECM degradation (Busco et al., 2010). "
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    ABSTRACT: Invadopodia are invasive protrusions with proteolytic activity uniquely found in tumor cells. Cortactin phosphorylation is a key step during invadopodia maturation, regulating Nck1 binding and cofilin activity. The precise mechanism of cortactin-dependent cofilin regulation and the roles of this pathway in invadopodia maturation and cell invasion are not fully understood. We provide evidence that cortactin-cofilin binding is regulated by local pH changes at invadopodia that are mediated by the sodium-hydrogen exchanger NHE1. Furthermore, cortactin tyrosine phosphorylation mediates the recruitment of NHE1 to the invadopodium compartment, where it locally increases the pH to cause the release of cofilin from cortactin. We show that this mechanism involving cortactin phosphorylation, local pH increase, and cofilin activation regulates the dynamic cycles of invadopodium protrusion and retraction and is essential for cell invasion in 3D. Together, these findings identify a novel pH-dependent regulation of cell invasion.
    Full-text · Article · Nov 2011 · The Journal of Cell Biology
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    • "The role of NHE1 translocation and the relative importance of altered translocation compared with changes in intrinsic catalytic activity will need further investigation. Phosphorylation of NHE1 downstream of Akt has been reported to lead to both an increase [16] in catalytic activity and a decrease [25]. Phosphorylation of NHE1 downstream of protein kinase A and protein kinase G leads to decreased catalytic activity, whereas activation of ERK downstream of protein kinase C and reactive oxygen species leads to stimulation of catalytic activity [1,42]. "
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    ABSTRACT: The Na+/H+ exchanger NHE1 is a highly regulated membrane protein that is required for pH homoeostasis in cardiomyocytes. The activation of NHE1 leads to proton extrusion, which is essential for counteracting cellular acidity that occurs following increased metabolic activity or ischaemia. The activation of NHE1 intrinsic catalytic activity has been well characterized and established experimentally. However, we have examined in the present study whether a net translocation of NHE1 to the sarcolemma of cardiomyocytes may also be involved in the activation process. We have determined the distribution of NHE1 by means of immunofluorescence microscopy and cell-surface biotinylation. We have discovered changes in the distribution of NHE1 that occur when cardiomyocytes are stimulated with insulin that are PI3K (phosphoinositide 3-kinase)-dependent. Translocation of NHE1 also occurs when cardiomyocytes are challenged by hypoxia, or inhibition of mitochondrial oxidative metabolism or electrically induced contraction, but these responses occur through a PI3K-independent process. As the proposed additional level of control of NHE1 through translocation was unexpected, we have compared this process with the well-established translocation of the glucose transporter GLUT4. In immunofluorescence microscopy comparisons, the translocation of NHE1 and GLUT4 to the sarcolemma that occur in response to insulin appear to be very similar. However, in basal unstimulated cells the two proteins are mainly located, with the exception of some co-localization in the perinuclear region, in distinct subcellular compartments. We propose that the mechanisms of translocation of NHE1 and GLUT4 are linked such that they provide spatially and temporally co-ordinated responses to cardiac challenges that necessitate re-adjustments in glucose transport, glucose metabolism and cell pH.
    Full-text · Article · Dec 2010 · Biochemical Journal
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