Quantitative Proteomics Analysis of CaMKII Phosphorylation and the CaMKII Interactome in the Mouse Forebrain

Ca2+/calmodulin-dependent protein kinase II alpha (CaMKII alpha) 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 Ca2+-dependent and -independent in vitro autophosphorylation sites in recombinant CaMKII alpha and CaMKII beta. CaMII 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 CaMKII alpha (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 CaMKII alpha and CaMKI1 beta, respectively, were detected in WT mice. Thr286-phosphorylated CaMKI1 alpha a and Thr287-phosphorylated CaMKII beta were selectively enriched in WT Triton-insoluble (synaptic) fractions compared to Triton-soluble (membrane) and cytosolic fractions. In contrast, Thr306-phosphorylated CaMKII alpha and Ser315- and Thr320/Thr321-phosphorylated CaMKII beta were selectively enriched in WT cytosolic fractions. The T286A-KI mutation significantly reduced levels of phosphorylation of CaMKII alpha at Ser275 across all subcellular fractions and of cytosolic CaMKII beta 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 CaMKII alpha, likely contributing to the diverse synaptic and behavioral deficits of T286A-KI mice.