Brain protein phosphorylation in vitro: Selective substrate action of insulin

Insulin action on [32P]-phosphate incorporation into brain membranes was determined. Hippocampal homogenate tissue was phosphorylated with [32P]-ATP, and insulin was introduced at various times before or after ATP addition. With 50μM Mg⁺⁺ in the medium, insulin selectively stimulated the phosphorylation of a 47kD phosphoprotein, Protein Fl. This effect required the prior presence of ATP. No effect of insulin on other phosphoproteins, or on [32P]-phosphate incorporation into TCA-precipitated material, was observed under these conditions. At 1mM Mg⁺⁺, insulin selectively decreased the phosphorylation of the alpha-subunit of pyruvate dehydrogenase. Insulin had no effect on other phosphoproteins, or on [32P]-phosphate incorporation into TCA-precipitated material under these conditions. The present study suggests a role for insulin in the modulation of brain protein phosphorylation. Since Protein Fl is phosphorylated by exogenous C kinase, and is likely the CNS-specific B-50 protein, these data also indicate a brain-specific function for insulin, possibly by action on a Ca⁺⁺/phospholipid protein kinase.