Expression of a multifunctional Ca2+/calmodulin-dependent protein kinase and mutational analysis of its autoregulation.
Autophosphorylation of multifunctional Ca2+/calmodulin-dependent protein kinase converts it from a Ca2(+)-dependent to a Ca2(+)-independent or autonomous kinase, a process that may underlie some long-term enhancement of transient Ca2+ signals. We demonstrate that the neuronal alpha subunit clone expressed in COS-7 cells (alpha-CaM kinase) is sufficient to encode the regulatory phenomena characteristic of the multisubunit kinase isolated from brain. Activity of alpha-CaM kinase is highly dependent on Ca2+/calmodulin. It is converted by autophosphorylation to an enzyme capable of Ca2(+)-independent (autonomous) substrate phosphorylation and autophosphorylation. Using site-directed mutagenesis, we separately eliminate five putative autophosphorylation sites within the regulatory domain and directly examine their individual roles. Ca2+/calmodulin-dependent kinase activity is fully retained by each mutant, but Thr286 is unique among the sites in being indispensable for generation of an autonomous kinase.
Hanson
P I
PI
Department of Pharmacology, Stanford University School of Medicine, California 94305-5332.
Kapiloff
M S
MS
Lou
L L
LL
Rosenfeld
M G
MG
Schulman
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000494:pharmacology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002147:Calmodulin / Q000494:pharmacology*; D002460:Cell Line; D003001:Cloning, Molecular; D006706:Homeostasis; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*
D002147:Calmodulin; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium