Quantitative proteomics analysis of CaMKII phosphorylation and the CaMKII interactome in the mouse forebrain.
AUTHORS
ABSTRACT
Ca(2+)/calmodulin-dependent protein kinase IIα (CaMKIIα) autophosphorylation at Thr286 and Thr305/Thr306 regulates kinase activity, modulates subcellular targeting, and is critical for normal synaptic plasticity and learning and memory. Here, a mass spectrometry-based approach was used to identify Ca2+-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 wildtype (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 6 and 7 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 co-precipitated with WT CaMKII holoenzymes in the synaptic fraction compared to 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.
Ca(2+)/calmodulin-dependent protein kinase IIα (CaMKIIα) autophosphorylation at Thr286 and Thr305/Thr306 regulates kinase activity, modulates subcellular targeting, and is critical for normal synaptic plasticity and learning and memory. Here, a mass spectrometry-based approach was used to identify Ca2+-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 wildtype (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 6 and 7 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 co-precipitated with WT CaMKII holoenzymes in the synaptic fraction compared to 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.