Insulin stimulates NHE1 activity by sequential activation of phosphatidylinositol 3-kinase and protein kinase C zeta in human erythrocytes.
The signaling cascade linking insulin receptor stimulation to the activation of Na/H exchanger (NHE) was investigated in human erythrocytes, a simple cell model expressing the NHE1 isoform and protein kinase C (PKC) alpha and zeta isoforms only. Our results demonstrate the presence of phosphatidylinositol (PtdIns) 3-kinase in these cells and its activation by insulin. With a similar time-course, insulin also promoted both the translocation and activation of PKC zeta, but had no effect on PKC alpha. Inhibition of PtdIns 3-kinase with wortmannin prevented the activation of PKC zeta by insulin. Stimulation of NHE1 was observed after 10 min of insulin treatment and persisted for at least 60 min. This effect was totally abolished by wortmannin or GF 109203X, an inhibitor of all PKC isoforms, but not by Gö 6976, a specific inhibitor of conventional and novel PKCs (e.g. PKC alpha). These data indicate that PKC zeta activation is mediated by a PtdIns 3-kinase-dependent mechanism and that NHE1 stimulation involves the sequential activation of PtdIns 3-kinase and PKC zeta. In addition, insulin stimulation of NHE1 occurred without altering the phosphorylation state of the exchanger, suggesting that the phosphorylation of an ancillary protein by PKC zeta would be responsible for activation of the transporter.
Sauvage
M
M
Laboratoire des Biomembranes et Messagers Cellulaires, Université Paris XI, Orsay, France.
Mazière
P
P
Fathallah
H
H
Giraud
F
F
D000730:Androstadienes / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002227:Carbazoles / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004912:Erythrocytes / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D008301:Maleimides / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D015398:Signal Transduction / Q000187:drug effects; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D013997:Time Factors; D000077191:Wortmannin; D000099221:Protein Kinase C zeta
D000730:Androstadienes; D002227:Carbazoles; D007211:Indoles; D007328:Insulin; D007527:Isoenzymes; D008301:Maleimides; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D019000:Phosphotyrosine; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D017923:Sodium-Hydrogen Exchangers; D000077191:Wortmannin; D000099221:Protein Kinase C zeta; C069139:growth factor-activatable Na-H exchanger NHE-1; C081021:Go 6976; C496628:PRKCA protein, human; C070515:bisindolylmaleimide I