Purification and characterization of calmodulin-dependent protein kinase III from rabbit reticulocytes and rat pancreas.
Calmodulin-dependent protein kinase III (CaM kinase III) phosphorylates and thereby inactivates eukaryotic elongation factor-2 (EF-2). This enzyme, purified to homogeneity from either rabbit reticulocytes or rat pancreas, had similar properties: it migrated with an apparent M(r) of 140,000 by gel filtration, was comprised of a major polypeptide of M(r) 95,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a high affinity for CaM (half-maximal activation < 1 nM). The M(r) 95,000 polypeptide was autophosphorylated by an intramolecular mechanism on seryl residues in the presence of Ca2+, CaM, and ATP, and phosphopeptide mapping indicated that several sites were phosphorylated. Autophosphorylation of CaM kinase III resulted in the generation of a partially Ca2+/calmodulin-independent activity. The enzyme could also be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. Amino acid sequencing of CaM kinase III indicated that it is distinct from other known proteins including the heat-shock protein hsp90, which was recently suggested to be identical to CaM kinase III (Nygärd, O., Nilsson, A., Carlberg, U., Nilsson, L., and Amons, R. (1991) J. Biol. Chem. 266, 16425-16430). Furthermore, hsp90 did not copurify with CaM kinase III, and the M(r) 95,000 protein did not cross-react with antibodies to hsp90. CaM kinase III exhibited Michaelis-Menten kinetics toward its substrates ATP and EF-2, with Km values of 15 and 2 microM, respectively. CaM kinase III was able to phosphorylate yeast EF-2 with an Km of 2 microM, but the enzyme did not significantly phosphorylate a variety of other substrates, confirming its identification as a novel protein kinase.
Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York, New York 10021.
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D000595:Amino Acid Sequence; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002147:Calmodulin / Q000378:metabolism; D002417:Cattle; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D054736:Elongation Factor 2 Kinase; D006360:Heat-Shock Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology*; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000378:metabolism; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D012156:Reticulocytes / Q000201:enzymology*; D013379:Substrate Specificity
D002147:Calmodulin; D006360:Heat-Shock Proteins; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D010750:Phosphoproteins; D011494:Protein Kinases; C517691:Eef2k protein, rat; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D054736:Elongation Factor 2 Kinase