Requirements for calcium and calmodulin in the calmodulin kinase activation cascade.
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.
Tokumitsu
H
H
Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201, USA.
Soderling
T R
TR
D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D004789:Enzyme Activation; D004926:Escherichia coli; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010766:Phosphorylation; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D016324:Sequence Tagged Sites; D018411:Spodoptera; D013912:Threonine / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection
D002147:Calmodulin; D017931:DNA Primers; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D013912:Threonine; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D000409:Alanine; D002118:Calcium