Resolving the effect of Shank3 S1510 phosphorylation on Shank3 functioning

Abstract

Shank3 is an important scaffolding protein in the postsynaptic density (PSD). It functions in forming multiple complexes that anchor receptors, but also in connecting the PSD to the Actin cytoskeleton. Shank3 has many phosphorylation sites. Recently, it has been shown in a double CaMKIIa/b knockout that the phosphorylation levels of Shank3 are strongly decreased, suggesting that CaMKII is an important kinase in Shank3 phosphorylation. In this phospho-proteomics study, Serine 1510 came forward as an important phosphorylation site on Shank3, likely phosphorylated by CaMKII. Here, by using a phospho-dead (S1510A) and phospho-mimic (S1510D) mutation, we attempted to find the effect of S1510 phosphorylation on Shank3 functioning. As it is likely that CaMKII is involved in this phosphorylation and since it is known that CaMKII is highly involved in long term potentiation (LTP), we tested the effects of S1510 phosphorylation in LTP. We show that S1510 phosphorylation does not affect spine number, spine morphology, or GluA2 membrane levels after inducing LTP. A small trend in Shank3 recruitment after LTP, where S1510A has an increased Shank3 size and intensity in the spine, whereas in S1510D these are both decreased, suggests that S1510 phosphorylation possibly affects Shank3 recruitment after LTP. Lastly, we also show that S1510 phosphorylation likely does not affect Shank3 oligomerization nor the interaction between Shank3 and Homer. The interaction between Shank3 and Cortactin, Rich2, and GKAP, are possibly affected. All together we show that, based on our data, S1510 phosphorylation is presumably not as important as previously thought for the regulation of Shank3.