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

Summary, in English

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 α-subunits revealed several potential ERK phosphorylation sites. Insulin increased ouabain-sensitive ⁸⁶Rb⁺ uptake and [³H]ouabain binding in intact cells. Insulin also increased phosphorylation and plasma membrane content of the Na⁺,K⁺-ATPase α ₁- and α₂-subunits. Insulin-stimulated Na ⁺,K⁺-ATPase activation, phosphorylation, and translocation of α-subunits to the plasma membrane were abolished by 20 μM PD98059, which is an inhibitor of MEK1/2, an upstream kinase of ERK1/2. Furthermore, inhibitors of phosphatidylinositol 3-kinase (100 wortmannin) and protein kinase C (10 μM GF109203X) had similar effects. Notably, insulin-stimulated ERK1/2 phosphorylation was abolished by wortmannin and GF109203X in HSMCS. Insulin also stimulated phosphorylation of α ₁- and α₂-subunits on Thr-Pro amino acid motifs, which form specific ERK substrates. Furthermore, recombinant ERK1 and -2 kinases were able to phosphorylate α-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 α-subunits by ERK1/2 mitogen-activated protein kinase.