In Vitro and in Vivo Phosphorylation of Myelin Basic Protein by Exogenous and Endogenous Adenosine 3′ : 5′-Monophosphate-dependent Protein Kinases in Brain

Abstract

Abstract Myelin basic protein, an encephalitogenic protein, was phosphorylated by adenosine 3' : 5'-monophosphate (cyclic AMP)-dependent protein kinase from bovine brain, and its phosphorylation was stimulated by cyclic AMP. The ability of the protein to serve as substrate for the protein kinase was comparable to that of histone fractions and better than that of casein, protamine, or phosvitin. The apparent Km of the enzyme for the protein was 2 x 10-5 m. The protein phosphorylated by the protein kinase showed a different migration on disc gel electrophoresis from that of the native protein, suggesting that the modification of the protein molecule by phosphorylation resulted in an alteration of physicochemical properties of the protein. The protein caused the activation of the protein kinase, and preincubation of the enzyme with the protein resulted in a substantial increase of cyclic AMP-independent activity. Protein kinase modulator inhibited the enzyme activity in the presence or absence of cyclic AMP when myelin basic protein was used as substrate. Amino acid residues of myelin basic protein which were phosphorylated by bovine brain protein kinase were seryl and threonyl. The maximum amount of phosphate incorporated into the protein was 3.80 moles per mole of protein, which was 3.6 times higher than that incorporated into histone on the basis of weight. Native myelin basic protein contained 0.20 mole of phosphorus per mole of protein, of which 0.07 and 0.09 mole were associated with phosphoserine and phosphothreonine, respectively. An extensively washed myelin fraction contained an endogenous protein kinase which catalyzed the endogenous phosphorylation of myelin basic protein in the presence of ATP and Mg2+. In vivo phosphorylation of myelin basic protein was observed by injection of [32P]orthophosphate into the ventricle of rat brain. Myelin basic protein fraction showed the highest specific radioactivity on the basis of protein amount, compared to that of crude homogenate and myelin fraction. Incorporated phosphate was released in the course of time. The results indicate that the phosphorylation and dephosphorylation of myelin basic protein may be in a dynamic state and that the turnover rate of phosphate in the protein may be relatively rapid.