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Quantitative proteomics analysis of CaMKII phosphorylation and the CaMKII interactome in the mouse forebrain
Anthony J Baucum1, Brian C Shonesy, Kristie L Rose
1⊥Department of Biology and Stark Neurosciences Research Institute, Indiana University-Purdue University Indianapolis, Indianapolis, Indiana 46202, United States.
Abstract:
Ca(2+)/calmodulin-dependent protein kinase IIα (CaMKIIα) autophosphorylation at Thr286 and Thr305/Thr306 regulates kinase activity and modulates subcellular targeting and is critical for normal synaptic plasticity and learning and memory. Here, a mass spectrometry-based approach was used to identify Ca(2+)-dependent and -independent in vitro autophosphorylation sites in recombinant CaMKIIα and CaMKIIβ. CaMKII holoenzymes were then immunoprecipitated from subcellular fractions of forebrains isolated from either wild-type (WT) mice or mice with a Thr286 to Ala knock-in mutation of CaMKIIα (T286A-KI mice) and analyzed using the same approach in order to characterize in vivo phosphorylation sites in both CaMKII isoforms and identify CaMKII-associated proteins (CaMKAPs). A total of six and seven autophosphorylation sites in CaMKIIα and CaMKIIβ, respectively, were detected in WT mice. Thr286-phosphorylated CaMKIIα and Thr287-phosphorylated CaMKIIβ were selectively enriched in WT Triton-insoluble (synaptic) fractions compared to Triton-soluble (membrane) and cytosolic fractions. In contrast, Thr306-phosphorylated CaMKIIα and Ser315- and Thr320/Thr321-phosphorylated CaMKIIβ were selectively enriched in WT cytosolic fractions. The T286A-KI mutation significantly reduced levels of phosphorylation of CaMKIIα at Ser275 across all subcellular fractions and of cytosolic CaMKIIβ at Ser315 and Thr320/Thr321. Significantly more CaMKAPs coprecipitated with WT CaMKII holoenzymes in the synaptic fraction compared to that in the membrane fraction, with functions including scaffolding, microtubule organization, actin organization, ribosomal function, vesicle trafficking, and others. The T286A-KI mutation altered the interactions of multiple CaMKAPs with CaMKII, including several proteins linked to autism spectrum disorders. These data identify CaMKII isoform phosphorylation sites and a network of synaptic protein interactions that are sensitive to the abrogation of Thr286 autophosphorylation of CaMKIIα, likely contributing to the diverse synaptic and behavioral deficits of T286A-KI mice.
Insights
Ca(2+)/calmodulin-dependent protein kinase IIα (CaMKIIα) autophosphorylation at Thr286 is crucial for synaptic plasticity and memory. Disrupting this site alters CaMKIIα and CaMKIIβ phosphorylation and protein interactions, impacting synaptic function and potentially contributing to neurodevelopmental disorders.
Area of Science:
- Neuroscience
- Molecular Biology
- Biochemistry
Background:
- Ca(2+)/calmodulin-dependent protein kinase IIα (CaMKIIα) autophosphorylation at Thr286 is essential for synaptic plasticity, learning, and memory.
- Understanding CaMKIIα and CaMKIIβ phosphorylation patterns and their associated proteins is critical for elucidating mechanisms of synaptic function.
Purpose of the Study:
- To identify in vitro and in vivo autophosphorylation sites of CaMKIIα and CaMKIIβ.
- To characterize the impact of abrogating CaMKIIα Thr286 autophosphorylation on CaMKII phosphorylation and associated protein interactions in vivo.
- To investigate the role of CaMKII-associated proteins (CaMKAPs) in synaptic function and their regulation by CaMKII phosphorylation.
Main Methods:
- Mass spectrometry-based proteomics was employed to identify CaMKII autophosphorylation sites in vitro and in vivo.
- Subcellular fractions from wild-type and CaMKIIα Thr286 to Ala knock-in (T286A-KI) mice forebrains were analyzed.
- Immunoprecipitation was used to identify CaMKII-associated proteins (CaMKAPs).
Main Results:
- Six and seven autophosphorylation sites were identified in CaMKIIα and CaMKIIβ, respectively.
- Phosphorylation at CaMKIIα Thr286 and CaMKIIβ Thr287 was enriched in synaptic fractions, while other sites (CaMKIIα Thr306, CaMKIIβ Ser315/Thr320/Thr321) were enriched in cytosolic fractions.
- The T286A-KI mutation reduced CaMKIIα Ser275 phosphorylation and altered CaMKIIβ cytosolic phosphorylation, alongside changing CaMKAP interactions, including those with proteins linked to autism spectrum disorders.
Conclusions:
- CaMKII isoform-specific phosphorylation sites regulate subcellular localization and protein interactions.
- Abrogation of CaMKIIα Thr286 autophosphorylation significantly alters the synaptic proteome and CaMKII interactions.
- These findings highlight the importance of CaMKII autophosphorylation in synaptic plasticity and suggest potential links to neurodevelopmental disorders.
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