Epidermal growth factor-induced vacuolar (H+)-atpase assembly: a role in signaling via mTORC1 activation.
Using proteomics and immunofluorescence, we demonstrated epidermal growth factor (EGF) induced recruitment of extrinsic V(1) subunits of the vacuolar (H(+))-ATPase (V-ATPase) to rat liver endosomes. This was accompanied by reduced vacuolar pH. Bafilomycin, an inhibitor of V-ATPase, inhibited EGF-stimulated DNA synthesis and mammalian target of rapamycin complex 1 (mTORC1) activation as indicated by a decrease in eukaryotic initiation factor 4E-binding 1 (4E-BP1) phosphorylation and p70 ribosomal S6 protein kinase (p70S6K) phosphorylation and kinase activity. There was no corresponding inhibition of EGF-induced Akt and extracellular signal-regulated kinase (Erk) activation. Chloroquine, a neutralizer of vacuolar pH, mimicked bafilomycin effects. Bafilomycin did not inhibit the association of mTORC1 with Raptor nor did it affect AMP-activated protein kinase activity. Rather, the intracellular concentrations of essential but not non-essential amino acids were decreased by bafilomycin in EGF-treated primary rat hepatocytes. Cycloheximide, a translation elongation inhibitor known to augment intracellular amino acid levels, prevented the effect of bafilomycin on amino acids levels and completely reversed its inhibition of EGF-induced mTORC1 activation. In vivo administration of EGF stimulated the recruitment of Ras homologue enriched in brain (Rheb) but not mammalian target of rapamycin (mTOR) to endosomes and lysosomes. This was inhibited by chloroquine treatment. Our results suggest a role for vacuolar acidification in EGF signaling to mTORC1.
Polypeptide Hormone Laboratory, Faculty of Medicine, McGill University, Montreal, Quebec H3A 2B2, Canada.
Yanqing
Y
Xu
Amanda
A
Parmar
Emmanuelle
E
Roux
Alejandro
A
Balbis
Victor
V
Dumas
Stephanie
S
Chevalier
Barry I
BI
Posner
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D002738:Chloroquine / Q000494:pharmacology; D011992:Endosomes / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000502:physiology*; D066246:ErbB Receptors / Q000378:metabolism; D005260:Female; D022781:Hepatocytes / Q000378:metabolism; D018942:Macrolides / Q000494:pharmacology; D008297:Male; D000076222:Mechanistic Target of Rapamycin Complex 1; D008938:Mitosis; D046912:Multiprotein Complexes; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D055503:Protein Multimerization; D021122:Protein Subunits / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects; D058570:TOR Serine-Threonine Kinases; D025262:Vacuolar Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000596:Amino Acids; D018942:Macrolides; D046912:Multiprotein Complexes; D010750:Phosphoproteins; D021122:Protein Subunits; D011506:Proteins; D020543:Proteome; C508191:proline-rich Akt substrate, 40 kDa protein, mouse; D004815:Epidermal Growth Factor; D002738:Chloroquine; C040929:bafilomycin A1; D066246:ErbB Receptors; D000076222:Mechanistic Target of Rapamycin Complex 1; D058570:TOR Serine-Threonine Kinases; D025262:Vacuolar Proton-Translocating ATPases