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Distinct autophosphorylation sites sequentially produce autonomy and inhibition of the multifunctional
1Department of Pharmacology, Stanford University School of Medicine, California 94305.
Abstract:
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.
Insights
Autophosphorylation of Ca2+/calmodulin-dependent protein kinase (CaM kinase) initially enhances its activity independently of calcium. However, further autophosphorylation eventually suppresses this autonomous activity, revealing a dual regulatory role.
Area of Science:
- Neuroscience
- Molecular Biology
- Biochemistry
Background:
- Ca2+/calmodulin-dependent protein kinase (CaM kinase) acts as a mediator for neurotransmitters and hormones using Ca2+ as a second messenger.
- Autophosphorylation is a key regulatory mechanism for protein kinases, influencing their activity and signaling pathways.
Purpose of the Study:
- To investigate if autophosphorylation converts multifunctional CaM kinase to a Ca2+/calmodulin-independent form.
- To determine if autophosphorylation serves as a mechanism for short- or long-term enhancement of Ca2+ signaling.
Main Methods:
- Studied the effect of continuous Ca2+/calmodulin stimulation on CaM kinase activity.
- Analyzed Ca2+-independent autophosphorylation and its impact on substrate phosphorylation.
- Utilized tryptic phosphopeptide mapping and sequencing to identify phosphorylation sites.
Main Results:
- Autophosphorylation increases CaM kinase's ability to phosphorylate substrates autonomously.
- A critical level of phosphate incorporation leads to a burst of Ca2+-independent autophosphorylation.
- This autonomous activity is eventually suppressed, inhibiting both Ca2+-dependent and independent substrate phosphorylation.
- Threonine286 was identified as the critical site for autonomous activity; additional sites mediate inhibitory autophosphorylation.
Conclusions:
- Autophosphorylation has a dual role in modulating CaM kinase activity: initial enhancement followed by suppression.
- This mechanism allows for sustained kinase activity after Ca2+ levels decrease but also provides a feedback inhibition.
- Threonine286 is crucial for the autonomous function, while other sites regulate the inhibitory phase.