Identification and characterization of delta B-CaM kinase and delta C-CaM kinase from rat heart, two new multifunctional Ca2+/calmodulin-dependent protein kinase isoforms.
We have identified, expressed and characterized two new isoforms of the multifunctional Ca2+/calmodulin-dependent kinase (CaM kinase) cloned from rat heart. Both isoforms are variants of the neuronal delta-CaM kinase (termed delta A-CaM kinase), and are designated as delta B-CaM kinase and delta C-CaM kinase. The new isoforms differ from delta A-CaM kinase in its isoform-specific insert region, between nucleotides 984 to 1087 of the delta A-CaM kinase cDNA. Replacing these 102 nucleotides, a sequence of 33 nucleotides which code for 11 amino acids (KRKSSSSQMM) are introduced in delta B-CaM kinase. The delta C-CaM kinase lacks all 102 nucleotides and the corresponding 34 amino acids which they encode. The predicted molecular masses of the delta B- and delta C-CaM kinase isoforms are 57,697 Da and 56,446 Da, respectively. Recombinant delta-CaM kinases purified from transfected COS-7 cells were found to associate into a larger holoenzyme estimated to contain 8 to 10 subunits. The relative subunit molecular masses on SDS-polyacrylamide gel electrophoresis are 59 kDa, 54 kDa and 52 kDa for delta A-, delta B- and delta C-CaM kinase, respectively. All three isoforms showed a strict dependence on Ca2+/calmodulin for activity and exhibited the Ca(2+)-dependent autophosphorylation and resultant conversion to Ca(2+)-independent kinase activity, characteristic features of multifunctional CaM kinase. Phosphopeptide analysis after partial CNBr digestion suggests that delta B-CaM kinase is the predominant soluble CaM kinase species purified from rat heart.
Edman
C F
CF
Department of Pharmacology, Stanford University Medical Center, CA 94305-5332.
Schulman
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D002147:Calmodulin / Q000494:pharmacology; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D014644:Genetic Variation; D007527:Isoenzymes / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000201:enzymology*; D010748:Phosphopeptides / Q000302:isolation & purification; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D051381:Rats; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D002147:Calmodulin; D017931:DNA Primers; D007527:Isoenzymes; D046911:Macromolecular Substances; D010748:Phosphopeptides; D017871:Calcium-Calmodulin-Dependent Protein Kinases