Characterization of a soluble Mr-30,000 catalytic fragment of the neuronal calmodulin-dependent protein kinase II.
Chymotryptic digestion of postsynaptic densities releases a soluble, catalytically active fragment of the alpha (Mr 50,000) subunit of the neuronal cytoskeletal calmodulin-dependent protein kinase II. The purified soluble form of the kinase likewise yields the fragment. Denaturation of the enzyme results in more extensive proteolytic degradation. 125I-Iodopeptide maps of the isolated catalytic portions of both forms of the enzyme are similar and are contained within the map of the isolated alpha subunit. Catalytic fragments of both forms of the enzyme comigrate on two-dimensional SDS-PAGE/isoelectric focusing with pI 6.7-7.2. The fragment phosphorylates microtubule-associated protein (MAP-2) but is not activated by Ca+2/calmodulin nor is it inhibited by trifluoperazine. Km values for MAP-2 and ATP are indistinguishable from those of the holoenzyme, while the Vmax is similar to that of the holoenzyme activated with Ca+2/calmodulin. Overlays of Western blots of fragment with 125I-calmodulin shows a loss of calmodulin binding. Both the number of phosphorylation sites and the ability to autophosphorylate are markedly reduced in the catalytic fragment. Evaluation of the hydrodynamic parameters of the purified fragment yielded Mr value of 25,600 with a frictional ratio (f/f0) of 1.12; the Mr value determined by SDS-PAGE was 30,000. Thus, the catalytic fragment appears to represent an activated form of the kinase with a monomeric, globular structure unlike the native enzyme which exhibits oligomerization and cytoskeletal association. These results are consistent with a tertiary structure for the calmodulin-dependent protein kinase that contains distinct domains responsible for catalytic activity, regulation by calmodulin, cytoskeletal association and the multimeric organization of enzyme subunits.
Levine
H
H
Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709.
Sahyoun
N E
NE
D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D002147:Calmodulin / Q000502:physiology*; D002384:Catalysis; D003599:Cytoskeleton / Q000201:enzymology; D004789:Enzyme Activation; D007525:Isoelectric Focusing; D009474:Neurons / Q000201:enzymology*; D010446:Peptide Fragments / Q000302:isolation & purification* / Q000502:physiology; D010447:Peptide Hydrolases; D010766:Phosphorylation; D011489:Protein Denaturation; D011494:Protein Kinases / Q000032:analysis*; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility
D002147:Calmodulin; D010446:Peptide Fragments; D011494:Protein Kinases; D010447:Peptide Hydrolases