Differential regulatory mechanism of Ca2+/calmodulin-dependent protein kinase kinase isoforms.
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.
Tokumitsu
H
H
Department of Chemistry, Kagawa Medical University, 1750-1 Miki-cho, Kita-gun, Kagawa 761-0793, Japan. tokumit@kms.ac.jp
Iwabu
M
M
Ishikawa
Y
Y
Kobayashi
R
R
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D004789:Enzyme Activation / Q000235:genetics; D004791:Enzyme Inhibitors / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000138:chemical synthesis / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D017346:Protein Serine-Threonine Kinases; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase