Multifunctional Ca2+/calmodulin-dependent protein kinase made Ca2+ independent for functional studies.
Multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) that is transiently expressed in COS-7 cells is essentially inactive when assayed without Ca2+. Physiological activation of the kinase occurs by binding of Ca2+/calmodulin near a putative autoinhibitory subdomain that contains the sequence His282-Arg-Gln-Glu-Thr286. We have markedly increased the Ca2(+)-independent activity of CaM kinase by altering the charge of this sequence by site-directed mutagenesis. The mutant containing Asp282-Gly-Glu-Glu-Thr286 is 67% Ca2+ independent. We also mimicked the effect of autophosphorylation at Thr286 by the mutant containing His282-Arg-Gln-Glu-Asp286, which is 36% Ca2+ independent. In addition to delineating the autoinhibitory domain by use of mutations that disable it, these constructs are of immediate practical value for simulating CaM kinase action in vivo without elevating Ca2+. To this end, we show that nuclear microinjection of cDNA of a constitutive mutant, but not of the wild-type kinase, initiates maturation of Xenopus oocytes.
Waldmann
R
R
Department of Pharmacology, Stanford University School of Medicine, California 94025-5332.
Hanson
P I
PI
Schulman
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002460:Cell Line; D005260:Female; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D009865:Oocytes / Q000201:enzymology; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014981:Xenopus
D015345:Oligonucleotide Probes; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium