Adenosine 3',5-monophosphate-dependent phosphorylation of a specific protein in synaptic membrane fractions from rat cerebrum.

SUMMARY The endogenous phosphorylation of protein in various membranous subcellular fractions from rat cerebrum has been studied in vitro. The endogenous phosphorylation of one minor protein component of synaptic membrane fractions showed a marked dependence upon the presence of added adenosine 3',5'-monophosphate (cyclic AMP). The apparent molecular weight of this phosphoprotein was approximately 100,000. Under the experimental conditions used, the selective stimulation of phosphorylation of a membrane protein by cyclic AMP was observed only in those subcellular fractions enriched in synaptic membranes. The results support other evidence suggesting that certain types of synaptic transmission may involve cyclic AMP-dependent phosphorylation of a specific protein constituent of synaptic membranes. Recent evidence suggests that adenosine 3') 5'-monophosphate may play an important role in certain types of synaptic transmission (l-4). The hypothesis has been proposed that the various diverse effects of cyclic AMP,' including those in the mammalian nervous system, are mediated through regulation of cyclic AMP-dependent protein kinases (5, 6). Those sub-cellular fractions of rat cerebrum which are enriched in synaptic membranes contain cyclic AMP-dependent protein kinase (7) as well as protein phosphatase (8), the latter being capable of removing phosphate from membrane protein which had been phosphorylated previously by cyclic AMP-dependent protein kinase. In addition, synaptic membrane fractions were found to be extremely effective as substrates for exogenous cyclic AMP-dependent protein kinase (9), being at least comparable to the histones in effectiveness as substrate for the enzyme. The high ability of these synaptic membrane fractions to serve as substrate for protein kinase was quite intriguing, since such 6 To whom corresoondence shoul d be addressed. iThe abbreviations used are: cyclic AMP, cyclic adenosi ne 3'5'-monophosphate; SDS, sodium dodecyl sulfate. membrane fractions possibly contain many distinct types of protein. It seemed plausible that one or a few of the protein components of the synaptic membrane might be exceptionally effective as substrate for protein kinase, and thereby account for the overall effect of cyclic AMP on synaptic membrane phos-phorylation. It was therefore of interest to examine the effect of cyclic AMP on the endogenous phosphorylation of individual protein components of the membrane. Subcellular fractions of rat cerebrum, prepared as described previously (7, 9), were suspended in 0.32 M sucrose. Immediately after preparation, aliquots of the various fractions, con taining 0.4 to 1.2 mg of membrane protein, were incubated for 5 s to 2 min at 30 " in a total volume of 0.2 ml …