Phosphorylation of microtubule-associated proteins by a Ca2+/calmodulin-dependent protein kinase.
In an earlier study I demonstrated that rat brain cytosol contains a Ca2+/calmodulin-dependent protein kinase activity that phosphorylates microtubule-associated protein 2 (MAP-2) but not MAP-1. Comparison of sites of phosphate incorporated in MAP-2 catalyzed by the Ca2+/calmodulin-dependent kinase activity and the cyclic AMP-dependent protein kinase activity in cytosolic extracts revealed distinct sites of phosphorylation (Schulman, H., 1984, Mol. Cell. Biol., 4:1175-1178; abstract by me and J.A. Kuret and K.H. Spitzer [1983, Fed. Proc., 42:2250]. I have now used MAP-2 as a substrate to purify the Ca2+/calmodulin-dependent protein kinase responsible for MAP-2 phosphorylation. The brain appears to contain a single predominant Ca2+/calmodulin-dependent protein kinase that phosphorylates MAP-2. The enzyme was purified to apparent homogeneity by column chromatography using DEAE-cellulose, phosphocellulose, hydroxylapatite, Sepharose 6B, and a calmodulin-Sepharose affinity column. The 580,000-dalton holoenzyme consists of 51,000- and 60,000-dalton subunits. The purified enzyme phosphorylates MAP-2 at the same "sites" that are phosphorylated in cytosolic extracts and thus has the same specificity as the activity present in cytosol. Analysis of phosphorylated MAP-2.1 and MAP-2.2, the two components of MAP-2, suggests considerable homology in their phosphorylated domains.
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D007700:Kinetics; D008297:Male; D008869:Microtubule-Associated Proteins; D008970:Molecular Weight; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D002147:Calmodulin; D008869:Microtubule-Associated Proteins; D011506:Proteins; D011494:Protein Kinases; D002118:Calcium