Requirements for calcium and calmodulin in the calmodulin kinase activation cascade

Hiroshi Tokumitsu, Thomas R. Soderling

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110 Citations (Scopus)


We have previously purified and cloned rat brain Ca2+/calmodulin- dependent protein kinase kinase (CaM-KK), and the 68-kDa recombinant CaM-KK activates in vitro both CaM-kinase IV (CaM-K IV) and CaM-K I (Tokumitsu, H., Enslen, H., and Soderling, T. R. (1995) J. Biol. Chem. 270, 19320-19324). In the present study we have determined that activation of CaM-K IV through phosphorylation of Thr196 by CaM-KK is triggered by elevated intracellular Ca2+ in intact cells and requires binding of Ca2+/CaM to both enzymes. An expressed fragment of CaM-K IV (CaM-K IV178-246), which contains the activating phosphorylation site (Thr196) but not the autoinhibitory domain or the CaM-binding domain, still required Ca2+/CaM for phosphorylation by wild-type CaM-KK. A truncated mutant of CaM-KK (CaM-KK1-434) phosphorylated CaM-K IV178-246 in a Ca2+/CaM-independent manner, but this constitutively active CaM-KK1-434 required Ca2+/CaM for phosphorylation and activation of wild-type CaM-K IV. These results demonstrate that binding of Ca2+/CaM to both CaM-K IV and CaM-KK is required for the CaM-kinase cascade. Both CaM-KK and CaM-K IV appear to have similar Ca2+/CaM requirements with EC50 values of approximately 100 nM. Studies using co-expression of CaM-K IV with CaM-KK in COS-7 cells demonstrated that CaM-KK rapidly activated both total and Ca2+/CaM- independent activities of wild-type CaM-K IV, but not the Thr196 → Ala mutant, upon ionomycin stimulation.

Original languageEnglish
Pages (from-to)5617-5622
Number of pages6
JournalJournal of Biological Chemistry
Issue number10
Publication statusPublished - Mar 8 1996
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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