Distinct autophosphorylation sites sequentially produce autonomy and inhibition of the multifunctional Ca2+/calmodulin-dependent protein kinase.
The multifunctional Ca2+/calmodulin-dependent protein kinase (multifunctional CaM kinase) may be an important mediator for neurotransmitters and hormones that utilize Ca2+ as a "second messenger." We examined the ability of autophosphorylation to convert the multifunctional CaM kinase to a Ca2+/calmodulin-independent (autonomous) form to determine whether autophosphorylation is a mechanism for short- or long-term enhancement of Ca2+ action. As the kinase incorporates phosphate during continuous stimulation by Ca2+/calmodulin, its ability to phosphorylate exogenous substrates becomes increasingly autonomous. Withdrawal of Ca2+ after a critical level of phosphate incorporation is reached leads to a "burst" or rapid increase in Ca2+-independent autophosphorylation. The "burst" of autophosphorylation is distinct from the initial Ca2+-dependent autophosphorylation, however, since it inhibits substrate phosphorylation. Both Ca2+-dependent and Ca2+-independent substrate phosphorylation are inhibited by this autonomous autophosphorylation. Thus, autophosphorylation has a dual role in modulating the activity of multifunctional CaM kinase. It initially enables the kinase to continue phosphorylating substrates after Ca2+ levels decline, but it eventually suppresses this autonomous activity. Tryptic phosphopeptide mapping demonstrates that appearance of phosphothreonine-containing peptides is common to several conditions used to generate an autonomous enzyme. Sequencing reveals the critical "autonomy" site to be threonine286. The inhibitory mode of autophosphorylation involves 3 additional phosphopeptides containing a serine and a threonine residue.
Lou
L L
LL
Department of Pharmacology, Stanford University School of Medicine, California 94305.
Schulman
H
H
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004591:Electrophoresis, Polyacrylamide Gel; D010449:Peptide Mapping; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000096:biosynthesis*
D047428:Protein Kinase Inhibitors; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium