ERK1/2 Mediates Insulin Stimulation of Na,K-ATPase by Phosphorylation of the α-Subunit in Human Skeletal Muscle Cells

• Published in: The Journal of biological chemistry Vol. 279; no. 24; pp. 25211 - 25218
• Main Authors: Al-Khalili, Lubna, Kotova, Olga, Tsuchida, Hiroki, Ehrén, Ingrid, Féraille, Eric, Krook, Anna, Chibalin, Alexander V.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 11.06.2004
• Subjects: Biotinylation; Cells, Cultured; Enzyme Activation; Humans; Insulin - pharmacology; Medicin och hälsovetenskap; Mitogen-Activated Protein Kinase 1 - physiology; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases - physiology; Muscle, Skeletal - enzymology; Ouabain - metabolism; Phosphorylation; Protein Kinase C - physiology; Protein Subunits; Sodium-Potassium-Exchanging ATPase - chemistry; Sodium-Potassium-Exchanging ATPase - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M402152200

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 α1- 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 nm 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 α1- 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.