CaMKII Phosphorylation Regulates Synaptic Enrichment of Shank3

SHANK3 is a large scaffolding protein in the postsynaptic density (PSD) that organizes protein networks, which are critical for synaptic structure and function. The strong genetic association of SHANK3 with autism spectrum disorder (ASD) emphasizes the importance of SHANK3 in neuronal development. SHANK3 has a critical role in organizing excitatory synapses and is tightly regulated by alternative splicing and posttranslational modifications. In this study, we examined basal and activity-dependent phosphorylation of Shank3 using mass spectrometry (MS) analysis from in vitro phosphorylation assays, in situ experiments, and studies with cultured neurons. We found that Shank3 is highly phosphorylated, and we identified serine 782 (S782) as a potent CaMKII phosphorylation site. Using a phosphorylation state-specific antibody, we demonstrate that CaMKII can phosphorylate Shank3 S782 in vitro and in heterologous cells on cotransfection with CaMKII. We also observed an effect of a nearby ASD-associated variant (Shank3 S685I), which increased S782 phosphorylation. Notably, eliminating phosphorylation of Shank3 with a S782A mutation increased Shank3 and PSD-95 synaptic puncta size without affecting Shank3 colocalization with PSD-95 in cultured hippocampal neurons. Taken together, our study revealed that CaMKII phosphorylates Shank3 S782 and that the phosphorylation affects Shank3 synaptic properties. Significance Statement The precise regulation of scaffolding proteins is important for neuronal development and dysregulation underlies some neurodevelopmental disorders. As an excitatory synapse scaffolding protein, SHANK3 plays a critical role in synapse structure and function and has a strong genetic linkage to autism spectrum disorder (ASD). In this report, we examined the fine regulation of Shank3 by phosphorylation. Our study characterizes a CaMKII phosphorylation site on Shank3 (S782) in vitro, in situ, and in neurons. The Shank3 phosphorylation is modulated by a neighboring ASD-associated mutation. Furthermore, S782 phosphorylation is involved in Shank3 synaptic enrichment. These findings reveal molecular mechanisms of SHANK3 function at excitatory synapses and a potential role in ASD etiology.