Alpha-thrombin, epidermal growth factor, and okadaic acid activate the Na+/H+ exchanger, NHE-1, by phosphorylating a set of common sites.
The ubiquitous and amiloride-sensitive Na+/H+ exchanger (NHE-1), a plasma membrane phosphoglycoprotein that regulates intracellular pH, is rapidly activated by growth factors. We showed previously that epidermal growth factor (EGF), alpha-thrombin, or serum stimulates Na+/H+ exchange activity in growth-arrested Chinese hamster lung fibroblasts (ER22 cells) in a time-dependent manner which correlates with increased phosphorylation of NHE-1 at serine residues (Sardet, C., Counillon, L., Franchi, A., and Pouysségur, J. (1990) Science 247, 723-726). Here we show that the tumor promoter, okadaic acid, a potent in vivo inhibitor of serine/threonine protein phosphatases 1 (PP1) and 2A (PP2A), stimulates Na+/H+ exchange in G0-arrested ER22 cells and in exchanger-deficient fibroblasts transfected with the human NHE-1 cDNA. Okadaic acid effects are maximal at 1 microM (EC50 = 500 nM), detected in 2 min, complete within 15-20 min, and are additives when combined with EGF or alpha-thrombin. Parallel to the pHi-induced rise, okadaic acid alone or together with growth factors stimulated the phosphorylation of NHE-1. More importantly tryptic phosphopeptide maps of NHE-1, immunoprecipitated from cells treated with EGF, alpha-thrombin, or okadaic acid, show a common pattern of phosphorylation. This pattern consists of five major 32P-labeled peptides (P1-P5) present in lower amounts in resting cells. One of them, P5, barely detectable in resting cells is increased up to 15-fold in mitogen-stimulated cells. Taken together these results reinforce the notion that phosphorylation of NHE-1 controls the set point value of the exchanger and suggest that: (i) the proximate step in Na+/H+ exchange activation is mediated by as yet unidentified growth factor-activatable serine "NHE-1 kinase(s)" and (ii) this NHE-1 kinase(s), partly active in resting cells, integrate signals from receptor tyrosine kinases and G protein-coupled receptors.
Sardet
C
C
Centre de Biochimie-Centre National de la Recherche Scientifique, Université de Nice, France.
Fafournoux
P
P
Pouysségur
J
J
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D004988:Ethers, Cyclic / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D019319:Okadaic Acid; D010449:Peptide Mapping; D010766:Phosphorylation; D011233:Precipitin Tests; D017923:Sodium-Hydrogen Exchangers; D013917:Thrombin / Q000494:pharmacology*; D014357:Trypsin
D002352:Carrier Proteins; D004988:Ethers, Cyclic; D017923:Sodium-Hydrogen Exchangers; D019319:Okadaic Acid; D004815:Epidermal Growth Factor; D014357:Trypsin; D013917:Thrombin