Differential phosphorylation of multiple sites in purified protein I by cyclic AMP-dependent and calcium-dependent protein kinases.
Protein I, a specific neuronal phosphoprotein, has previously been shown, using rat brain synaptosome preparations, to contain multiple sites of phosphorylation which were differentially regulated by cAMP and calcium. In the present study, Protein I was purified to homogeneity from rat brain and its phosphorylation was investigated using homogeneous cAMP-dependent protein kinase and a partially purified calcium-calmodulin-dependent protein kinase from rat brain. Employing various peptide mapping techniques, a minimum of three phosphorylation sites could be distinguished in Protein I; the phosphorylated amino acid of each site was serine. One phosphorylation site was located in the collagenase-resistant portion of Protein I and was the principal target for phosphorylation by the catalytic subunit of cAMP-dependent protein kinase. This site was also phosphorylated by calcium-calmodulin-dependent protein kinase. The other two phosphorylation sites were located in the collagenase-sensitive portion of Protein I. These latter sites were markedly phosphorylated by calcium-calmodulin-dependent protein kinase, but not by cAMP-dependent protein kinase in concentrations sufficient to phosphorylate maximally the site in the collagenase-resistant portion. Thus, the phosphorylation of purified Protein I by purified cAMP-dependent and calcium-calmodulin-dependent protein kinases provides an enzymological explanation for the regulation of phosphorylation of endogenous Protein I in synaptosome preparations by cAMP and by calcium observed previously. The studies suggest that certain of the synaptic actions of two distinct second messengers, cAMP and calcium, are expressed through the distinct specificities of cAMP- and calcium-dependent protein kinases for the multiple phosphorylation sites in one neuron-specific protein, Protein I.
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology*; D002918:Chymotrypsin; D000242:Cyclic AMP / Q000494:pharmacology*; D007700:Kinetics; D008297:Male; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D010447:Peptide Hydrolases; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D013211:Staphylococcus aureus / Q000201:enzymology; D016704:Synapsins; D013574:Synaptosomes / Q000378:metabolism; D014357:Trypsin
D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D010748:Phosphopeptides; D016704:Synapsins; D000242:Cyclic AMP; D011494:Protein Kinases; D010447:Peptide Hydrolases; D002918:Chymotrypsin; D014357:Trypsin; D002118:Calcium