Distinct autophosphorylation sites sequentially produce autonomy and inhibition of the multifunctional Ca2+/calmodulin-dependent protein kinase
Open accessAbstract
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.
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T286 autophosphorylation is primarily intersubunit within a holoenzyme, although at very high kinase concentration it can also occur by an interholoenzyme mechanism.
“This autophosphorylation is primarily intersubunit autophosphorylation within a holoenzyme, although at very high kinase concentration it can also occur by an interholoenzyme mechanism (FIGURE 2B4) (71, 79–81).”
From Synaptic memory and CaMKII (2023) · cites this paper as [80]
The discussion (page unavailable in our copy) identifies threonine 286 as the autophosphorylation site responsible for autonomous kinase activity. It does not establish that phosphorylation is primarily between subunits of one holoenzyme or that phosphorylation between holoenzymes occurs at very high kinase concentrations; searches for those mechanisms found no supporting passage. (LLM, conf=0.82)
Experimental — accuracy not yet measuredOnce Ca2+/CaM dissociates from a T286-phosphorylated kinase, autonomous activity phosphorylates T305 and T306, blocking Ca2+/CaM rebinding and braking further stimulation.
“However, once Ca2+/CaM dissociates from a kinase phosphorylated at T286, its autonomous activity quickly phosphorylates T305 and T306, blocking rebinding of Ca2+/CaM and exerting a brake on further Ca2+/CaM stimulation, in what is at times referred to as inhibitory autophosphorylation (80, 84–86).”
From Synaptic memory and CaMKII (2023) · cites this paper as [80]
The Results report that phosphorylation at T286 produces autonomous activity and that removal of Ca2+ triggers a rapid inhibitory burst of autonomous autophosphorylation. Figure 8 depicts phosphorylation of a serine and a threonine after Ca2+/calmodulin dissociates, reducing further Ca2+/calmodulin-dependent activity. Our copy does not identify those inhibitory residues as T305 and T306 or establish that inhibition occurs by blocking Ca2+/calmodulin rebinding. (LLM, conf=0.88)
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“This autophosphorylation is primarily intersubunit autophosphorylation within a what is at times referred to as inhibitory autophosphorylation (80, 84 – 86).”
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- Authors
- Lou, L., & Schulman, H.
- Affiliations
- Stanford Medicine (US)
- ORCID
- Schulman, H
- Venue
- The Journal of Neuroscience
- Publisher
- Society for Neuroscience
- Published
- 1989-06-01
- Volume
- 9
- Issue
- 6
- Pages
- 2020-2032
- ISSN
- 0270-6474 (ISSN-L)
- 1529-2401
- PMC
- PMC6569737
- OpenAlex
- W2144838091
- Cited by
- 161
- References
- 2
- Citations by year
- 2025: 4 · 2024: 3 · 2023: 5 · 2022: 7 · 2021: 1 · 2020: 5 · 2019: 4 · 2018: 4 · 2017: 4 · 2016: 1 · 2014: 1 · 2013: 2
- FWCI
- 3.35
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Journal of Neuroscience (publishedVersion, cc-by-nc-sa)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Institute of General Medical Sciences (GM 10686, GM 30179)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Signaling Pathways in Disease
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › S100 Proteins and Annexins
- Keywords
- CaMKII, autophosphorylation, phosphothreonine, phosphopeptide mapping, calcium signaling, second messengers
- MeSH terms
- Animals, Calcium, Electrophoresis, Polyacrylamide Gel, Peptide Mapping, Phosphorylation, Protein Kinases, Calcium-Calmodulin-Dependent Protein Kinases, Protein Kinase Inhibitors
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