Osmotic stimulation of the Na+/H+ exchanger NHE1: relationship to the activation of three MAPK pathways (original) (raw)
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Cellular Physiology and Biochemistry, 2007
Osmotic stress modulates mitogen activated protein kinase (MAPK) activities, leading to altered gene transcription and cell death/survival balance, however, the mechanisms involved are incompletely elucidated. Here, we show, using a combination of biochemical and molecular biology approaches, that three MAPKs exhibit unique interrelationships with the Na + /H + exchanger, NHE1, after osmotic cell shrinkage: Extracellular Signal Regulated Kinase (ERK1/2) is inhibited in an NHE1-dependent, pH i -independent manner, c-Jun N-terminal kinase (JNK1/2) is stimulated, in part through NHE1-mediated intracellular alkalinization, and p38 MAPK is activated in an NHE1independent manner, and contributes to NHE1 activation and ERK inhibition. Shrinkage-induced ERK1/2 inhibition was attenuated in Ehrlich Lettre Ascites cells by NHE1 inhibitors (EIPA, cariporide) or removal of extracellular Na + , and mimicked by human (h) NHE1 expression in cells lacking endogenous NHE1 activity. The effect of NHE1 on ERK1/2 was pH i -independent and upstream of MEK1/2. Shrinkageactivation of JNK1/2 was attenuated by EIPA, augmented by hNHE1 expression, and abolished in the presence of HCO 3 -. Basal JNK activity was augmented at alkaline pH i . Shrinkage-activation of p38 MAPK was NHE1-independent, and p38 MAPK inhibition (SB203580) attenuated NHE1 activation and ERK1/2 inhibition. Long-term shrinkage elicited caspase-3 activation and a loss of cell viability, which was augmented by ERK1/2 or JNK1/2 inhibition, and attenuated by p38 MAPK inhibition.
Journal of Biological Chemistry, 1999
The signaling pathways by which cell volume regulates ion transporters, e.g. Na ؉ /H ؉ exchangers (NHEs), and affects cytoskeletal organization are poorly understood. We have previously shown that shrinkage induces tyrosine phosphorylation in CHO cells, predominantly in an 85-kDa band. To identify volume-sensitive kinases and their substrates, we investigated the effect of hypertonicity on members of the Src kinase family. Hyperosmolarity stimulated Fyn and inhibited Src. Fyn activation was also observed in nystatin-permeabilized cells, where shrinkage cannot induce intracellular alkalinization. In contrast, osmotic inhibition of Src was prevented by permeabilization or by inhibiting NHE-1. PP1, a selective Src family inhibitor, strongly reduced the hypertonicity-induced tyrosine phosphorylation. We identified one of the major targets of the osmotic stresselicited phosphorylation as cortactin, an 85-kDa actinbinding protein and well known Src family substrate. Cortactin phosphorylation was triggered by shrinkage and not by changes in osmolarity or pH i and was abrogated by PP1. Hyperosmotic cortactin phosphorylation was reduced in Fyn-deficient fibroblasts but remained intact in Src-deficient fibroblasts. To address the potential role of the Src family in the osmotic regulation of NHEs, we used PP1. The drug affected neither the hyperosmotic stimulation of NHE-1 nor the inhibition of NHE-3. Thus, members of the Src family are volumesensitive enzymes that may participate in the shrinkage-related reorganization of the cytoskeleton but are probably not responsible for the osmotic regulation of NHE.
PLoS ONE, 2011
The Na + /H + Exchanger isoform 1 (NHE1) is a highly versatile, broadly distributed and precisely controlled transport protein that mediates volume and pH regulation in most cell types. NHE1 phosphorylation contributes to Na + /H + exchange activity in response to phorbol esters, growth factors or protein phosphatase inhibitors, but has not been observed during activation by osmotic cell shrinkage (OCS). We examined the role of NHE1 phosphorylation during activation by OCS, using an ideal model system, the Amphiuma tridactylum red blood cell (atRBC). Na + /H + exchange in atRBCs is mediated by an NHE1 homolog (atNHE1) that is 79% identical to human NHE1 at the amino acid level. NHE1 activity in atRBCs is exceptionally robust in that transport activity can increase more than 2 orders of magnitude from rest to full activation. Michaelis-Menten transport kinetics indicates that either OCS or treatment with the phosphatase inhibitor calyculin-A (CLA) increase Na + transport capacity without affecting transport affinity (K m = 44 mM) in atRBCs. CLA and OCS act non-additively to activate atNHE1, indicating convergent, phosphorylation-dependent signaling in atNHE1 activation. In situ 32 P labeling and immunoprecipitation demonstrates that the net phosphorylation of atNHE1 is increased 4-fold during OCS coinciding with a more than 2-order increase in Na + transport activity. This is the first reported evidence of increased NHE1 phosphorylation during OCS in any vertebrate cell type. Finally, liquid chromatography and mass spectrometry (LC-MS/MS) analysis of atNHE1 immunoprecipitated from atRBC membranes reveals 9 phosphorylated serine/threonine residues, suggesting that activation of atNHE1 involves multiple phosphorylation and/or dephosphorylation events.
p38 MAP kinase modulates liver cell volume through inhibition of membrane Na+ permeability
Journal of Clinical Investigation, 2001
Introduction Mitogen-activated protein (MAP) kinases are ubiquitous serine/threonine protein kinases that play an important role in translating extracellular signals to the nucleus (1, 2). Among these, p38 MAP kinase represents a human homologue of the Saccharomyces cerevisiae HOG-1 gene product, a yeast MAP kinase required for cellular osmoregulation (2, 3). p38 MAP kinase is specifically regulated by changes in environmental osmolarity by dual tyrosine/threonine phosphorylation (4) mediated by two MAP kinase kinases (MEKs), MEK-3 and MEK-6 (5, 6). Members of the p38 signaling complex are also activated in response to lipopolysaccharides, proinflammatory cytokines, and ultraviolet radiation (4, 7, 8). It has been shown that p38 plays an important role in the cellular response to osmotic changes: inhibition of p38 by SB203580 prevents volume-sensitive induction of multiple mRNAs (2, 9, 10). In addition to the effects on p38, change in cell volume also appears to be an important stimulus for modulation of other members of the MAP kinase family, including JNK and ERK (2, 9). Cell volume homeostasis is mandatory for maintenance of cellular integrity, but also represents a means of coupling changes in membrane transport to other organ-specific functions. An increase in cell volume, for example, appears to serve as a signal regulating many liver functions, stimulating protein synthesis, secretion, and gene expression. In addition to these physiologic roles, failure to regulate cell volume has been implicated in liver cell injury associated with alcohol, ischemia/reperfusion, and organ preservation (11-13). Thus, one function of MAP kinases may be to mediate volume-sensitive changes in gene expression. Recent studies suggest that p38 MAP kinase is constitutively active in certain cells, and may also be capable of regulating membrane transport through direct effects on ion channels (14, 15). In hepatocytes, membrane Na + permeability is low under basal conditions, but decreases in cell volume stimulate an adaptive response that involves Na + influx through opening of nonselective cation (NSC) channels. The resulting water influx leads to restoration of cell volume toward basal values, a process referred to as regulatory volume increase (RVI) (16, 17). Since MAP kinases exhibit volume-sensitive changes in activity, the purpose of these studies was to assess the potential role of p38 and other MAP kinase signaling pathways in volume-dependent changes in membrane Na + permeability. The findings suggest that
Plant Journal, 1999
Plant growth is severely affected by hyper-osmotic salt conditions. Although a number of salt-induced genes have been isolated, the sensing and signal transduction of salt stress is little understood. We provide evidence that alfalfa cells have two osmo-sensing protein kinase pathways that are able to distinguish between moderate and extreme hyper-osmotic conditions. A 46 kDa protein kinase was found to be activated by elevated salt concentrations (above 125 mM NaCl). In contrast, at high salt concentrations (above 750 mM NaCl), a 38 kDa protein kinase, but not the 46 kDa kinase, became activated. By biochemical and immunological analysis, the 46 kDa kinase was identi®ed as SIMK, a member of the family of MAPKs (mitogen-activated protein kinases). SIMK is not only activated by NaCl, but also by KCl and sorbitol, indicating that the SIMK pathway is involved in mediating general hyper-osmotic conditions. Salt stress induces rapid but transient activation of SIMK, showing maximal activity between 8 and 16 min before slow inactivation. When inactive, most mammalian and yeast MAPKs are cytoplasmic but undergo nuclear translocation upon activation. By contrast, SIMK was found to be a constitutively nuclear protein and the activity of the kinase was not correlated with changes in its intracellular compartmentation, suggesting an intra-nuclear mechanism for the regulation of SIMK activity.
Hypertonicity Activates Na+/H+ Exchange through Janus Kinase 2 and Calmodulin
Journal of Biological Chemistry, 2003
The type 1 sodium-hydrogen exchanger (NHE-1) is a ubiquitous electroneutral membrane transporter that is activated by hypertonicity in many cells. NHE-1 may be an important pathway for Na ؉ entry during volume restoration, yet the molecular mechanisms underlying the osmotic regulation of NHE-1 are poorly understood. In the present study we conducted a screen for important signaling molecules that could be involved in hypertonicity-induced activation of NHE-1 in CHO-K1 cells. Hypertonicity rapidly activated NHE-1 in a concentration-dependent manner as assessed by proton microphysiometry and by measurements of intracellular pH on a FLIPR TM (fluorometric imaging plate reader). Inhibitors of Ca 2؉ /calmodulin (CaM) and Janus kinase 2 (Jak2) attenuated this activation, whereas neither calcium chelation nor inhibitors of protein kinase C, the Ras-ERK1/2 pathway, Src kinase, and Ca 2؉ /calmodulindependent enzymes had significant effects. Hypertonicity also resulted in the rapid tyrosine phosphorylation of Jak2 and STAT3 (the major substrate of Jak2) and CaM. Phosphorylation of Jak2 and CaM were blocked by AG490, an inhibitor of Jak2. Immunoprecipitation studies showed that hypertonicity stimulates the assembly of a signaling complex that includes CaM, Jak2, and NHE-1. Formation of the complex could be blocked by AG490. Thus, we propose that hypertonicity induces activation of NHE-1 in CHO-K1 cells in large part through the following pathway: hypertonicity 3 Jak2 phosphorylation and activation 3 tyrosine phosphorylation of CaM 3 association of CaM with NHE-1 3 NHE-1 activation.
Journal of Membrane Biology, 1996
Amiloride-sensitive, Na + -dependent, DIDSinsensitive cytoplasmic alkalinization is observed after hypertonic challenge in Ehrlich ascites tumor cells. This was assessed using the fluorescent pH-sensitive probe 2Ј,7Ј-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). A parallel increase in the amiloride-sensitive unidirectional Na + influx is also observed. This indicates that hypertonic challenge activates a Na + /H + exchanger. Activation occurs after several types of hypertonic challenge, is a graded function of the osmotic challenge, and is temperature-dependent. Observations on single cells reveal a considerable variation in the shrinkage-induced changes in cellular pH i , but the overall picture confirms the results from cell suspensions.