ERK1/2 mediates insulin stimulation of Na,K-ATPase by phosphorylation of the α-subunit in human skeletal muscle cells

Insulin stimulates Na+,K+-ATPase activity and induces translocation of Na+,K+-ATPase molecules to the plasma membrane in skeletal muscle. We determined the molecular mechanism by which insulin regulates Na+,K+-ATPase in differentiated primary human skeletal muscle cells (HSMCs). Insulin action on Na+,K+-ATPase was dependent on ERK1/2 in HSMCs. Sequence analysis of Na+,K+-ATPase alpha-subunits revealed several potential ERK phosphorylation sites. Insulin increased ouabain-sensitive Rb-86(+) uptake and [H-3]ouabain binding in intact cells. Insulin also increased phosphorylation and plasma membrane content of the Na+,K+-ATPase alpha(1)- and alpha(2)-subunits. Insulin-stimulated Na+,K+-ATPase activation, phosphorylation, and translocation of alpha-subunits to the plasma membrane were abolished by 20 muM PD98059, which is an inhibitor of MEK1/2, an upstream kinase of ERK1/2. Furthermore, inhibitors of phosphatidylinositol 3-kinase (100 nM wortmannin) and protein kinase C (10 muM GF109203X) had similar effects. Notably, insulin-stimulated ERK1/2 phosphorylation was abolished by wortmannin and GF109203X in HSMCs. Insulin also stimulated phosphorylation of alpha(1)- and alpha(2)-subunits on Thr-Pro amino acid motifs, which form specific ERK substrates. Furthermore, recombinant ERK1 and -2 kinases were able to phosphorylate alpha-subunit of purified human Na+,K+-ATPase in vitro. In conclusion, insulin stimulates Na+,K+-ATPase activity and translocation to plasma membrane in HSMCs via phosphorylation of the alpha-subunits by ERK1/2 mitogen-activated protein kinase.