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

Ca2+/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 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 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.