Requirements for calcium and calmodulin in the calmodulin kinase activation cascade

J Biol Chem. 1996 Mar 8;271(10):5617-22. doi: 10.1074/jbc.271.10.5617.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alanine
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Binding Sites
  • Brain / enzymology
  • Calcium / metabolism*
  • Calcium-Calmodulin-Dependent Protein Kinases / metabolism*
  • Calmodulin / metabolism*
  • Cell Line
  • Cloning, Molecular
  • DNA Primers
  • Enzyme Activation
  • Escherichia coli
  • Kinetics
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Oligodeoxyribonucleotides
  • Phosphorylation
  • Point Mutation
  • Polymerase Chain Reaction
  • Rats
  • Recombinant Proteins / metabolism
  • Sequence Tagged Sites
  • Spodoptera
  • Threonine / metabolism
  • Transcriptional Activation*
  • Transfection

Substances

  • Calmodulin
  • DNA Primers
  • Oligodeoxyribonucleotides
  • Recombinant Proteins
  • Threonine
  • Calcium-Calmodulin-Dependent Protein Kinases
  • Alanine
  • Calcium