Insulin increases plasma membrane content and reduces phosphorylation of Na(+)-K(+) pump alpha(1)-subunit in HEK-293 cells.
Insulin stimulates K(+) uptake and Na(+) efflux via the Na(+)-K(+) pump in kidney, skeletal muscle, and brain. The mechanism of insulin action in these tissues differs, in part, because of differences in the isoform complement of the catalytic alpha-subunit of the Na(+)-K(+) pump. To analyze specifically the effect of insulin on the alpha(1)-isoform of the pump, we have studied human embryonic kidney (HEK)-293 cells stably transfected with the rat Na(+)-K(+) pump alpha(1)-isoform tagged on its first exofacial loop with a hemagglutinin (HA) epitope. The plasma membrane content of alpha(1)-subunits was quantitated by binding a specific HA antibody to intact cells. Insulin rapidly increased the number of alpha(1)-subunits at the cell surface. This gain was sensitive to the phosphatidylinositol (PI) 3-kinase inhibitor wortmannin and to the protein kinase C (PKC) inhibitor bisindolylmaleimide. Furthermore, the insulin-stimulated gain in surface alpha-subunits correlated with an increase in the binding of an antibody that recognizes only the nonphosphorylated form of alpha(1) (at serine-18). These results suggest that insulin regulates the Na(+)-K(+) pump in HEK-293 cells, at least in part, by decreasing serine phosphorylation and increasing plasma membrane content of alpha(1)-subunits via a signaling pathway involving PI 3-kinase and PKC.
Sweeney
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G
Programme in Cell Biology, Hospital for Sick Children, Toronto M5G 1X8, Canada.
Niu
W
W
Canfield
V A
VA
Levenson
R
R
Klip
A
A
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006388:Hemagglutinins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology*; D008301:Maleimides / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D011188:Potassium / Q000378:metabolism; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction / Q000502:physiology; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*; D000077191:Wortmannin
D000730:Androstadienes; D004791:Enzyme Inhibitors; D006388:Hemagglutinins; D007211:Indoles; D007328:Insulin; D008301:Maleimides; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011188:Potassium; D020033:Protein Isoforms; D011493:Protein Kinase C; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D000077191:Wortmannin; C088060:bisindolylmaleimide