Substrate-directed Function of Calmodulin in Autophosphorylation of Ca2+/Calmodulin-dependent Protein Kinase II
Open accessAbstract
Autophosphorylation of Thr286 in Ca2+/calmodulin-dependent protein kinase II occurs within each holoenzyme by an intersubunit reaction and is essential for kinase function in vivo. In addition to a kinase-directed function of calmodulin to activate the kinase, a second calmodulin is required for the autophosphorylation of each Thr286 (Hanson, P. I., Meyer, T., Stryer, L., and Schulman, H. (1994) Neuron 12, 943-956). We have engineered heteromeric holoenzymes comprising distinct "kinase" and "substrate" subunits to test for kinase- and substrate-directed functions of calmodulin. The obligate kinase subunits have aspartate residues substituted for threonine at positions 286, 305, and 306 (the autophosphorylation and calmodulin-binding sites), making it constitutively active but unable to bind calmodulin. Obligate substrate subunits are catalytically inactive (K42M mutation) but are able to bind calmodulin. Phosphorylation of substrate subunits occurs specifically at Thr286 and is completely dependent upon the presence of calmodulin. Blocking the ability of the substrate subunit to bind calmodulin, either with inhibitor KN-93 or by mutagenesis of the calmodulin-binding domain of the substrate subunit, prevents its phosphorylation, consistent with a substrate-directed function of calmodulin that requires its direct binding to the subunit being phosphorylated.
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Phosphorylation of T286 requires that the subunit being phosphorylated have Ca2+/CaM bound.
“The phosphorylation of T286 requires that the subunit being phosphorylated have Ca2+/CaM bound (82, 83), resulting in autophosphorylation.”
From Synaptic memory and CaMKII (2023) · cites this paper as [83]
The Results (Fig. 4) show that substrate subunits unable to bind Ca2+/calmodulin were not phosphorylated, supporting the requirement for the subunits tested there. But the Results (Fig. 5) also show that a C289P substrate subunit was autophosphorylated without Ca2+/calmodulin, so the claim is too absolute. Page numbers are unavailable in our copy. (LLM, conf=0.88)
Experimental — accuracy not yet measuredAutophosphorylation occurs by transphosphorylation between subunits within a holoenzyme, but the substrate subunit needs to be bound to Ca2+/CaM because that is what exposes T286.
“Although autophosphorylation does occur by transphosphorylation between subunits within a holoenzyme, the substrate subunit needs to be bound to Ca2+/CaM because that is what exposes T286 (82, 83), thus challenging the model of perpetuating CaMKII activity.”
From Synaptic memory and CaMKII (2023) · cites this paper as [83]
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“Although autophosphorylation does occur by transphosphorylation between subunits within a holoenzyme, the substrate subunit needs to be bound to Ca 2 1 /CaM because that is what exposes T286 (82, 83), thus challenging the model of perpetuating CaMKII activity.”
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- Authors
- Rich, R. C., & Schulman, H.
- Affiliations
- Stanford Medicine (US)
- Corresponding author
- Schulman, Howard
- ORCID
- Schulman, Howard
- Venue
- Journal of Biological Chemistry
- Publisher
- Elsevier BV
- Published
- 1998-10
- Volume
- 273
- Issue
- 43
- Pages
- 28424-28429
- ISSN
- 0021-9258 (ISSN-L)
- OpenAlex
- W1989361508
- Cited by
- 162
- References
- 3
- Citations by year
- 2026: 3 · 2025: 3 · 2024: 5 · 2023: 4 · 2022: 10 · 2021: 3 · 2020: 8 · 2019: 2 · 2018: 9 · 2017: 5 · 2016: 4 · 2015: 4 · 2014: 5 · 2013: 4 · 2012: 9
- FWCI
- 2.21
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Journal of Biological Chemistry (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Funders
- National Institutes of Health (5t32ns007280-35)
- U.S. Public Health Service
- National Institute of Neurological Disorders and Stroke (5T32-NS07280-11)
- 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
- calmodulin, calcium/calmodulin-dependent protein kinase II, autophosphorylation, calmodulin binding, KN-93, calmodulin-binding domain, kinase activation
- MeSH terms
- Calmodulin, Peptides, Phosphorylation, Protein Binding, Protein Conformation, Substrate Specificity, Protein Engineering, Calcium-Calmodulin-Dependent Protein Kinases, Calcium Signaling, Holoenzymes, Calcium-Calmodulin-Dependent Protein Kinase Type 2
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