Insulin- and Glucose-Induced Phosphorylation of the Na+,K+-Adenosine Triphosphatase -Subunits in Rat Skeletal Muscle

Phosphorylation of the ␣-subunits of Na ؉ ,K ؉ -adenosine triphosphatase in response to insulin, high extracellular glucose concentration, and phorbol 12-myristate 13-acetate was investigated in isolated rat soleus muscle.All three stimuli increased ␣-subunit phosphorylation approximately 3-fold.Phorbol 12-myristate 13-acetate-and high glucose-induced phosphorylation of the ␣-subunit was completely abolished by the PKC inhibitor GF109203X, whereas insulin-stimulated phosphorylation was only partially reduced.Notably, insulin stimulation resulted in phosphorylation of the ␣-subunit on serine, threonine, and tyrosine residues, whereas high extracellular glucose or phorbol 12-myristate 13-acetate stimulation mediated phosphorylation only on serine and threonine residues.Insulin stimulation resulted in translocation of Na ؉ ,K ؉ -adenosine triphosphatase ␣ 2 -subunit to the plasma membrane and increased Na ؉ ,K ؉ -adenosine triphosphatase activity in the same membrane fraction.High glucose had no effect on ␣-subunits distribution.Immunoprecipitation with antiphosphotyrosine antibody and subsequent Western blot analysis with anti-␣ 1 -and -␣ 2 -subunit antibodies revealed that both ␣ 1 -and ␣ 2 -subunit isoforms underwent phosphorylation on tyrosine residues in response to insulin, although with different time course and magnitude.Thus, we show that insulin-stimulated phosphorylation of Na ؉ ,K ؉ -adenosine triphosphatase ␣-subunit occurs via a PKC-and tyrosine kinase-dependent mechanism, whereas high glucose-induced phosphorylation is only PKC-dependent.Phosphorylation of Na ؉ ,K ؉ -adenosine triphosphatase ␣-subunits may be involved in regulation of Na ؉ ,K ؉ -adenosine triphosphatase activity by insulin or high extracellular glucose in skeletal muscle.