Differential regulatory mechanism of Ca2+/calmodulin-dependent protein kinase kinase isoforms

Biochemistry. 2001 Nov 20;40(46):13925-32. doi: 10.1021/bi010863k.


We have previously demonstrated that the alpha isoform of Ca(2+)/calmodulin-dependent protein kinase kinase (CaM-KKalpha) is strictly regulated by an autoinhibitory mechanism and activated by the binding of Ca(2+)/CaM [Tokumitsu, H., Muramatsu, M., Ikura, M., and Kobayashi, R. (2000) J. Biol. Chem. 275, 20090-20095]. In this study, we find that rat brain extract contains Ca(2+)/CaM-independent CaM-KK activity. This result is consistent with an enhanced Ca(2+)/CaM-independent activity (60-70% of total activity) observed with the recombinant CaM-KKbeta isoform. By using various truncation mutants of CaM-KKbeta, we have identified a region of 23 amino acids (residues 129-151) located at the N-terminus of the catalytic domain as an important regulatory element of the autonomous activity. A CaM-KKbeta deletion mutant of this domain shows a significant increase of Ca(2+)/CaM dependency for the CaM-KK activity as well as for the autophosphorylation activity. The activities of CaM-KKalpha and CaM-KKbeta chimera, in which autoinhibitory sequences were replaced by each other, were completely dependent on Ca(2+)/CaM, suggesting that the autoinhibitory regions of CaM-KKalpha and CaM-KKbeta are functional. These results establish for the first time that residues 129-151 of CaM-KKbeta participate in the release of the autoinhibitory domain from its catalytic core, resulting in generation of autonomous activity.

Publication types

  • Comparative Study

MeSH terms

  • Amino Acid Sequence
  • Animals
  • COS Cells
  • Calcium-Calmodulin-Dependent Protein Kinase Kinase
  • Enzyme Activation / genetics
  • Enzyme Inhibitors / metabolism
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Glutathione Transferase / genetics
  • Isoenzymes / antagonists & inhibitors
  • Isoenzymes / genetics
  • Isoenzymes / metabolism
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Protein-Serine-Threonine Kinases / antagonists & inhibitors
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism*
  • Rats
  • Recombinant Fusion Proteins / antagonists & inhibitors
  • Recombinant Fusion Proteins / chemical synthesis
  • Recombinant Fusion Proteins / metabolism
  • Signal Transduction / genetics


  • Enzyme Inhibitors
  • Isoenzymes
  • Peptide Fragments
  • Recombinant Fusion Proteins
  • Glutathione Transferase
  • Protein-Serine-Threonine Kinases
  • Calcium-Calmodulin-Dependent Protein Kinase Kinase