Mechanism of the generation of autonomous activity of Ca 2+/calmodulin-dependent protein kinase IV

Hiroshi Tokumitsu, Naoya Hatano, Hiroyuki Inuzuka, Shigeyuki Yokokura, Naohito Nozaki, Ryoji Kobayashi

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Abstract

Ca2+/calmodulin-dependent protein kinase IV (CaM-KIV) is phosphorylated at Thr196 by Ca2+/calmodulin-dependent protein kinase kinase (CaM-KK), resulting in induction of both autonomous activity and a high level of Ca2+/CaM-dependent activity. We have shown that the kinetics of Thr196 phosphorylation of CaM-KIV by CaM-KK is well correlated with the generation of its autonomous activity, although Thr177 phosphorylation of CaM-KI does not induce its autonomous activity. The activities of CaM-KI chimera mutants fused with C-terminal regions (residues 296-469 and 296-350) of CaM-KIV are completely dependent on Ca2+/CaM, which is also the case for CaM-KI. Unlike wild-type CaM-KI, however, phosphorylation of Thr177 in the chimera mutants by CaM-KK resulted in generation of significant autonomous activities, indicating that the phosphorylation of Thr in the activation loop is sufficient to partially release the autoinhibitory region of CaM-KIV from the catalytic core. Indeed, the CaM-KIV peptide (residues 304-325) containing minimum autoinhibitory sequences (residues 314-321) suppressed the activity of non-phosphorylated CaM-KIV with an IC50 of ∼50 μM, and this suppression was competitive with respect to the peptide substrate; however, the CaM-KIV peptide was not capable of inhibiting Thr196-phosphorylated CaM-KIV. Taken together, these results indicated that the Thr196 phosphorylation of CaM-KIV by CaM-KK reduced the interaction of the catalytic core with the autoinhibitory region, resulting in generation of the autonomous activity.

Original languageEnglish
Pages (from-to)40296-40302
Number of pages7
JournalJournal of Biological Chemistry
Volume279
Issue number39
DOIs
Publication statusPublished - Sep 24 2004
Externally publishedYes

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Calcium-Calmodulin-Dependent Protein Kinase Type 4
Calcium-Calmodulin-Dependent Protein Kinases
Phosphorylation
Phosphotransferases
Peptides
Catalytic Domain
Inhibitory Concentration 50
Chemical activation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mechanism of the generation of autonomous activity of Ca 2+/calmodulin-dependent protein kinase IV. / Tokumitsu, Hiroshi; Hatano, Naoya; Inuzuka, Hiroyuki; Yokokura, Shigeyuki; Nozaki, Naohito; Kobayashi, Ryoji.

In: Journal of Biological Chemistry, Vol. 279, No. 39, 24.09.2004, p. 40296-40302.

Research output: Contribution to journalArticle

Tokumitsu, Hiroshi ; Hatano, Naoya ; Inuzuka, Hiroyuki ; Yokokura, Shigeyuki ; Nozaki, Naohito ; Kobayashi, Ryoji. / Mechanism of the generation of autonomous activity of Ca 2+/calmodulin-dependent protein kinase IV. In: Journal of Biological Chemistry. 2004 ; Vol. 279, No. 39. pp. 40296-40302.
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abstract = "Ca2+/calmodulin-dependent protein kinase IV (CaM-KIV) is phosphorylated at Thr196 by Ca2+/calmodulin-dependent protein kinase kinase (CaM-KK), resulting in induction of both autonomous activity and a high level of Ca2+/CaM-dependent activity. We have shown that the kinetics of Thr196 phosphorylation of CaM-KIV by CaM-KK is well correlated with the generation of its autonomous activity, although Thr177 phosphorylation of CaM-KI does not induce its autonomous activity. The activities of CaM-KI chimera mutants fused with C-terminal regions (residues 296-469 and 296-350) of CaM-KIV are completely dependent on Ca2+/CaM, which is also the case for CaM-KI. Unlike wild-type CaM-KI, however, phosphorylation of Thr177 in the chimera mutants by CaM-KK resulted in generation of significant autonomous activities, indicating that the phosphorylation of Thr in the activation loop is sufficient to partially release the autoinhibitory region of CaM-KIV from the catalytic core. Indeed, the CaM-KIV peptide (residues 304-325) containing minimum autoinhibitory sequences (residues 314-321) suppressed the activity of non-phosphorylated CaM-KIV with an IC50 of ∼50 μM, and this suppression was competitive with respect to the peptide substrate; however, the CaM-KIV peptide was not capable of inhibiting Thr196-phosphorylated CaM-KIV. Taken together, these results indicated that the Thr196 phosphorylation of CaM-KIV by CaM-KK reduced the interaction of the catalytic core with the autoinhibitory region, resulting in generation of the autonomous activity.",
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