Phosphorylation of Shank3 by Rho-Kinase regulates surface translocation of NMDA and AMPA receptors in PSD

[Speaker] Rijwan Uddin Ahammad:1,2
[Co-author] Yasuhiro Funahashi:2, Md. Omar Faruk:1,2, Emran Hossen:1,2, Kozo Kaibuchi.:2
1:Cell Pharmacology, Doctoral Student, Japan, 2:Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, Japan

The small GTPase RhoA and its downstream effector Rho-kinase (Rock) are considered as one of the key regulators in dendritic spine formation and synaptic plasticity. However, how RhoA/Rho-kinase signaling involved in modulating synaptic plasticity still remains unknown. In the excitatory synapses, postsynaptic density (PSD) located usually at the tip of dendritic spine containing glutamate receptors and other membrane bound scaffold proteins plays role in synaptic plasticity, but how they are regulated and what are the upstream signaling is not clearly understood yet. We have recently developed a phosphoproteomic analysis method that uses affinity beads coated with 14-3-3 proteins to enrich phosphorylated proteins and established the kinase-associated neural phosphosignaling (KANPHOS) database that provides the phosphorylated sites identified by our phosphoproteomic approaches (Nagai et al, Neuron, 2016; Nagai et al, Trends Pharmacol Sci, 2016). In this study, using our KANPHOS database, we identified Shank3, which is adapter protein of AMPA and NMDA receptors in PSD, as a novel Rho-kinase substrate. We also found that Shank3 interacted with 14-3-3 and this interaction was regulated by Rho-kinase. Phospho-deficient mutants of Shank3 (T551A, S694A and S781A) reduced its interaction with 14-3-3. To monitor the in vivo phosphorylation state of Shank3, we prepared antibodies that specifically recognized phosphorylated Shank3, and we identified Rho-Kinase phosphorylated Shank3 at T551, S694 and S781 in mouse neostriatum slice culture. Furthermore, NMDA activated Rho-kinase through CaMKII in striatal slices, which phosphorylated Shank3 and increased interaction of Shank3 with 14-3-3. Finally, we identified that NMDA-induced Rho-kinase activation increased the interaction of Shank3 with NMDA and AMPA receptors. These results indicate that the phosphorylation of Shank3 by Rho-kinase regulates its interaction with 14-3-3. This phosphorylation may regulate surface translocation of NMDA and AMPA receptors in PSD to orchestrate synaptic plasticity.
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