G. Sweeney et al., Insulin increases plasma membrane content and reduces phosphorylation of Na+-K+ pump alpha(1)-subunit in HEK-293 cells, AM J P-CELL, 281(6), 2001, pp. C1797-C1803
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 tissue
s differs, in part, because of differences in the isoform complement of the
catalytic alpha -subunit of the Na+-K+ pump. To analyze specifically the e
ffect of insulin on the alpha (1)-isoform of the pump, we have studied huma
n embryonic kidney (HEK)-293 cells stably transfected with the rat Na+-K+ p
ump alpha (1)-isoform tagged on its first exofacial loop with a hemagglutin
in (HA) epitope. The plasma membrane content of alpha (1)-subunits was quan
titated by binding a specific HA antibody to intact cells. Insulin rapidly
increased the number of alpha (1)-subunits at the cell surface. This gain w
as sensitive to the phosphatidylinositol (PI) 3-kinase inhibitor wortmannin
and to the protein kinase C (PKC) inhibitor bisindolylmaleimide. Furthermo
re, the insulin-stimulated gain in surface alpha -subunits correlated with
an increase in the binding of an antibody that recognizes only the nonphosp
horylated form of alpha (1) (at serine-18). These results suggest that insu
lin regulates the Na+-K+ pump in HEK-293 cells, at least in part, by decrea
sing serine phosphorylation and increasing plasma membrane content of alpha
(1)-subunits via a signaling pathway involving PI 3-kinase and PKC.