Regulation of phosphate incorporation into four brain phosphoproteins that are affected by experience

Various regulators of protein kinase activities were tested for their effects on the in vitro transfer of phosphate from [gamma-32P]ATP to four proteins of rat brain synaptic particulate preparations. One protein, of apparent molecular weight 44,000, accepted 32P in the presence of 8 mM EDTA and no added Mg2+. It was the major phosphoprotein of brain mitochondria. Its phosphorylation was inhibited by pyruvate and stimulated by K+, and it comigrated in electrophoretic gels with authentic alpha-subunit of pyruvate: lipoamide oxidoreductase (decarboxylating) (EC 1.2.4.1) from bovine heart. The major kinase acting on three proteins of apparent molecular weights 24,000, 21,000, and 19,000 was stimulated by Ca2+, by preincubation with phospholipase C, and by 12-tetradecanoyl 4-beta-phorbol 13-acetate. Phosphorylation of these lower-molecular-weight proteins was inhibited by ACTH1-24, by cyclic 3',5'-adenosine monophosphate, and by 50 microM trifluoperazine. The stimulatory effect of Ca2+ was antagonized by calmodulin. The kinase in question appears to be B-50 protein kinase or protein kinase C.