NMDAR Phosphoproteome Controls Synaptic Growth and Learning

Funahashi, Y.; Ahammad, R. U.; Zhang, X.; Hossen, E.; Kawatani, M.; Nakamuta, S.; Yoshimi, A.; Wu, M.; Wang, H.; Li, X.; Faruk, M. O.; Shohag, M. H.; Lin, Y.-H.; Tsuboi, D.; Nishioka, T.; Kuroda, K.; Amano, M.; Noda, Y.; Yamada, K.; Sakimura, K.; Nagai, T.; Yamashita, T.; Uchino, S.; Kaibuchi, K.

doi:10.1101/2023.12.15.571360

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bioRxiv · 10.1101/2023.12.15.571360

NMDAR Phosphoproteome Controls Synaptic Growth and Learning

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Abstract

In the mammalian brain, NMDA receptors (NMDARs) activation triggers a calcium-dependent signal transduction cascade resulting in postsynaptic remodeling and behavioral learning. However, the phosphoprotein signal flow through this transduction network is poorly understood. Here, we show that NMDAR-dependent phosphorylation drives the assembly of protein signaling complexes that regulate synaptic morphology and behavior. We performed large-scale phosphoproteomic analyses of protein kinase target proteins in successive layers of the signaling network in mouse striatal/accumbal slices. NMDARs activation resulted in the phosphorylation of 194 proteins, including Rho GTPase regulators. CaMKII-mediated phosphorylation of ARHGEF2 increased its RhoGEF activity, thereby activating the RhoA-Rho-kinase pathway. Subsequent phosphoproteomics of Rho-kinase revealed 221 protein targets, including SHANK3. Experimental validation revealed a pathway from NMDAR-dependent calcium influx through CaMKII, ARHGEF2, Rho-kinase, and SHANK3 to coordinate assembly of an actin-tethered postsynaptic complex of SHANK3/NMDAR/PSD95/DLGAP3 for spine growth and aversive learning. These findings show that NMDARs initiate metabolic phosphorylation for learning.

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Funahashi, Y., Ahammad, R. U., Zhang, X., Hossen, E., Kawatani, M., Nakamuta, S., Yoshimi, A., Wu, M., Wang, H., Li, X., Faruk, M. O., Shohag, M. H., Lin, Y.-H., Tsuboi, D., Nishioka, T., Kuroda, K., Amano, M., Noda, Y., Yamada, K., Sakimura, K., Nagai, T., Yamashita, T., Uchino, S., Kaibuchi, K.. 2023-12-15. NMDAR Phosphoproteome Controls Synaptic Growth and Learning. https://doi.org/10.1101/2023.12.15.571360

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