NMDAR Phosphoproteome Controls Synaptic Growth and Learning

Yasuhiro Funahashi; Rijwan Uddin Ahammad; Xinjian Zhang; Emran Hossen; Masahiro Kawatani; Shinichi Nakamuta; Akira Yoshimi; Minhua Wu; Huanhuan Wang; Mengya Wu; Li Xu; Md. Omar Faruk; Md Hasanuzzaman Shohag; You‐Hsin Lin; Daisuke Tsuboi; Tomoki Nishioka; Keisuke Kuroda; Mutsuki Amano; Yukihiko Noda; Kiyofumi Yamada; Kenji Sakimura; Taku Nagai; Takayuki Yamashita; Shigeo Uchino; Kozo Kaibuchi

doi:10.1101/2023.12.15.571360

NeurosciencebioRxivDec 15, 2023

Preprint

NMDAR Phosphoproteome Controls Synaptic Growth and Learning

Yasuhiro Funahashi iD, Rijwan Uddin Ahammad iD, Xinjian Zhang iD, Emran Hossen iD, Masahiro Kawatani iD, Shinichi Nakamuta iD, Akira Yoshimi iD, Minhua Wu iD, Huanhuan Wang iD, Mengya Wu iD, Li Xu iD, Md. Omar Faruk iD, Md Hasanuzzaman Shohag iD, You‐Hsin Lin, Daisuke Tsuboi iD, Tomoki Nishioka iD, Keisuke Kuroda iD, Mutsuki Amano iD, Yukihiko Noda iD, Kiyofumi Yamada iD, Kenji Sakimura iD, Taku Nagai iD, Takayuki Yamashita iD, Shigeo Uchino, Kozo Kaibuchi iD

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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