Mutagenesis of Thr-286 in monomeric Ca2+/calmodulin-dependent protein kinase II eliminates Ca2+/calmodulin-independent activity.
We have examined the role of Thr-286 autophosphorylation in the autoregulation of Ca2+/calmodulin-dependent protein kinase II. Using site-directed mutagenesis, we have substituted alanine or serine for Thr-286, or isoleucine for Arg-283, in the 50-kDa subunit of the kinase and expressed each protein in bacteria. Activation and autophosphorylation of all four enzymes were stringently dependent on Ca2+/calmodulin, indicating that neither Arg-283 nor Thr-286 is an absolute requirement for the pseudosubstrate inhibition of the enzyme. Autophosphorylation of the Ile-283 or Ala-286 enzyme generated little, if any, Ca2+/calmodulin-independent kinase activity, unlike the parent (Thr-286) or Ser-286 enzyme. The enzymes expressed in bacteria are predominantly monomeric, indicating that the generation of Ca2+/calmodulin-independent activity does not require the cooperative interactions of subunits normally present in the brain holoenzyme.
Waxham
M N
MN
Department of Neurology, University of Texas Medical School, Houston 77225.
Aronowski
J
J
Westgate
S A
SA
Kelly
P T
PT
D001120:Arginine; D002118:Calcium / Q000494:pharmacology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002147:Calmodulin / Q000494:pharmacology*; D003001:Cloning, Molecular; D007532:Isoleucine; D007700:Kinetics; D046911:Macromolecular Substances; D009154:Mutation; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013912:Threonine
D002147:Calmodulin; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D007532:Isoleucine; D013912:Threonine; D001120:Arginine; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium