Insulin- and glucose-induced phosphorylation of the Na+,K+-adenosine triphosphatase alpha-subunits in rat skeletal muscle

Phosphorylation of the alpha -subunits of Na+,K+-adenosine triphosphatase i n response to insulin, high extracellular glucose concentration, and phorbo l 12-myristate 13-acetate was investigated in isolated rat soleus muscle. A ll three stimuli increased alpha -subunit phosphorylation approximately 3-f old. Phorbol 12-myristate 13-acetate- and high glucose-induced phosphorylat ion of the alpha -subunit was completely abolished by the PKC inhibitor GF1 09203X, whereas insulin-stimulated phosphorylation was only partially reduc ed. Notably, insulin stimulation resulted in phosphorylation of the alpha - subunit on serine, threonine, and tyrosine residues, whereas high extracell ular glucose or phorbol 12-myristate 13-acetate stimulation mediated phosph orylation. only on serine and threonine residues. Insulin stimulation resul ted in translocation of Na+,K+-adenosine triphosphatase alpha (2)-subunit t o the plasma membrane and increased Na+,K+-adenosine triphosphatase activit y in the same membrane fraction. High glucose had no effect on alpha -subun its distribution. Immunoprecipitation with antiphosphotyrosine antibody and subsequent Western blot analysis with anti-alpha (1)- and -alpha (2)-subun it antibodies revealed that both alpha (1)- and alpha (2)-subunit isoforms underwent phosphorylation on tyrosine residues in response to insulin, alth ough with different time course and magnitude. Thus, we show that insulin-s timulated phosphorylation of Na+,K+-adenosine triphosphatase alpha -subunit occurs via a PKC- and tyrosine kinase-dependent mechanism, whereas high gl ucose-induced phosphorylation is only PKC-dependent. Phosphorylation of Na,K+-adenosine triphosphatase alpha (2)-subunits may be involved in regulati on of Na+,K+-adenosine triphosphatase activity by insulin or high extracell ular glucose in skeletal muscle.