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. 2022 Dec 26;24(1):404.
doi: 10.3390/ijms24010404.

Rho-Kinase/ROCK Phosphorylates PSD-93 Downstream of NMDARs to Orchestrate Synaptic Plasticity

Emran Hossen  1   2 , Yasuhiro Funahashi  2 , Md Omar Faruk  1   3 , Rijwan Uddin Ahammad  1   4 , Mutsuki Amano  1 , Kiyofumi Yamada  5 , Kozo Kaibuchi  1   2
Affiliations

Rho-Kinase/ROCK Phosphorylates PSD-93 Downstream of NMDARs to Orchestrate Synaptic Plasticity

Emran Hossen et al. Int J Mol Sci. .

Abstract

The N-methyl-D-aspartate receptor (NMDAR)-mediated structural plasticity of dendritic spines plays an important role in synaptic transmission in the brain during learning and memory formation. The Rho family of small GTPase RhoA and its downstream effector Rho-kinase/ROCK are considered as one of the major regulators of synaptic plasticity and dendritic spine formation, including long-term potentiation (LTP). However, the mechanism by which Rho-kinase regulates synaptic plasticity is not yet fully understood. Here, we found that Rho-kinase directly phosphorylated discs large MAGUK scaffold protein 2 (DLG2/PSD-93), a major postsynaptic scaffold protein that connects postsynaptic proteins with NMDARs; an ionotropic glutamate receptor, which plays a critical role in synaptic plasticity. Stimulation of striatal slices with an NMDAR agonist induced Rho-kinase-mediated phosphorylation of PSD-93 at Thr612. We also identified PSD-93-interacting proteins, including DLG4 (PSD-95), NMDARs, synaptic Ras GTPase-activating protein 1 (SynGAP1), ADAM metallopeptidase domain 22 (ADAM22), and leucine-rich glioma-inactivated 1 (LGI1), by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Among them, Rho-kinase increased the binding of PSD-93 to PSD-95 and NMDARs. Furthermore, we found that chemical-LTP induced by glycine, which activates NMDARs, increased PSD-93 phosphorylation at Thr612, spine size, and PSD-93 colocalization with PSD-95, while these events were blocked by pretreatment with a Rho-kinase inhibitor. These results indicate that Rho-kinase phosphorylates PSD-93 downstream of NMDARs, and suggest that Rho-kinase mediated phosphorylation of PSD-93 increases the association with PSD-95 and NMDARs to regulate structural synaptic plasticity.

Keywords: LTP; PSD-93; Rho-kinase; dendritic spine; phosphorylation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Rho-kinase phosphorylates PSD-93 in vitro. (A) PSD-93 domain organization: three PDZ domains (PDZ1 PDZ2 and PDZ3), one SH3 domain and one GK domain. The numbers represent amino acids. (B) Constitutively active Rho-kinase phosphorylated PSD-93 at the PDZ3-SH3 domain. Purified GST and GST-PSD-93 domain proteins were incubated with [γ32P] ATP in the presence or absence of Rho-kinase-cat in vitro. Samples were subjected to SDS-PAGE and silver staining followed by autoradiography. Asterisks denote intake GST-PSD-93 fusion domain proteins. The arrows denote recombinant Rho-kinase-cat and GST. Third bracket represents PSD-93 domain proteins. (C) The bar diagram shows the relative phosphorylation (%) of PSD-93 domain proteins. (D) Schematic presentation of PSD-93 phosphorylation sites. The PSD-93-PDZ3-SH3 domain contains four putative phosphorylation sites based on Rho-kinase consensus motifs of R/KXXpS/T and R/KXpS/T (R, arginine; K, lysine; X, any amino acid; S, serine and T, threonine). P denotes phosphate group. (E) Rho-kinase phosphorylated PSD-93-PDZ3-SH3 domain at four different sites. Purified wild-type and phospho-deficient mutants of PSD-93-PDZ3-SH3 were incubated with Rho-kinase-cat along with [γ32P] ATP in vitro. Next, the SDS-boiled samples were subjected to SDS-PAGE and silver staining followed by autoradiography. The upper panel shows autoradiography, and the lower panel shows a silver staining image. (F) Bar diagram showing the relative phosphorylation (%) of PSD-93 domain proteins. The horizontal lines indicate the mean ± SEM of three independent experiments.*, **, *** and **** represent p < 0.05, p < 0.01, p < 0.001 and p < 0.0001, respectively, for Dunnett’s multiple comparisons test. “ns” denotes “not significant”. (G) The amino acid sequence homology of PSD-93 phosphorylation sites (Thr585 and Thr612) in different species (humans, rats, and mice) is shown in a schematic graph. The numbers represent the amino acid position.
Figure 2
Figure 2
Rho-kinase phosphorylates PSD-93 at Thr612 in striatal slices. (A) Striatal slices were incubated with calyculin A (250 nM) for 60 min and/or Y-27632 (20 μM) for 60 min. The samples were analyzed by immunoblot analysis using anti-pT612 PSD-93, anti-PSD-93, anti-pT853 MYPT1, and anti-MYPT1 antibodies. (B,C) The bar diagram shows the quantification of the immunoblot data for pT612 PSD-93 and pT853 MYPT1, respectively. The horizontal lines indicate the mean ± SEM of three independent experiments. ** and *** represent p < 0.01 and p < 0.001, respectively, for Tukey’s multiple comparisons test.
Figure 3
Figure 3
Rho-kinase phosphorylates PSD-93 at Thr612 in striatal slices downstream of NMDARs. (A) Striatal slices were treated with or without the Rho-kinase inhibitor Y-27632 (20 μM for 60 min) and then treated with DMSO, or high K+ (KCl, 40 mM) for 15 sec, or NMDA (100 μM) for 15 s. The samples were analyzed with immunoblot analysis with anti-pT612 PSD-93, anti-PSD-93, anti-pT286 CaMKII, anti-CaMKII, anti-pT853 MYPT1, and anti-MYPT1 antibodies. (B–D) The bar diagram shows the quantification of the immunoblot data for pT612 PSD-93, pT286 CaMKII, and pT853 MYPT1, respectively. The horizontal lines indicate the mean ± SEM of three independent experiments. *, ** and **** represent, p < 0.05, p < 0.01, p < 0.0001, respectively, and “ns” denotes “not significant”, for Tukey’s multiple comparisons test.
Figure 4
Figure 4
Identification of PSD-93-binding proteins by LC-MS/MS. (A) Schematic presentation of the LC-MS/MS method using striatal slices. Coronal striatal slices were taken from mice brain with vibratome and treated with calyculin A (250 nM) for 60 min and/or Y-27632 (20 µM) for 60 min—and then subjected to immunoprecipitation using an anti-PSD-93 antibody. The Trypsin/Lys-C was used to digest immunoprecipitated proteins, which were then subjected to LC-MS/MS to identify PSD-93 interacting proteins. (B) The input lysate from striatal slices was analyzed by immunoblot analysis with anti-pT853 MYPT1 and anti-MYPT1 antibodies. (C) The bar diagrams show the quantitative analysis results of immunoblotting.
Figure 5
Figure 5
Rho-kinase positively regulates the interaction of PSD-93 with PSD-95, NMDARs and AMPARs. (A) PSD-93 phosphorylation by Rho-kinase increases its interaction with PSD-95, NR1 and GluR1 in striatal slices. Striatal slices were treated with calyculin A (250 nM) for 60 min after they were pretreated with Y-27632 (20 µM) for 60 min and then immunoprecipitated using an anti-PSD-93 antibody. The precipitated proteins were subjected to immunoblot analysis with antibodies against PSD-93, PSD-95, NR1, GluR1, pT853 MYPT1, and MYPT1 antibodies. (B–D) The bar diagram shows the statistical analysis results for the immunoblot data. The horizontal lines indicate the mean ± SEM of three independent experiments. * and ** represent p < 0.05 and p < 0.01, respectively, for Tukey’s multiple comparisons test. (E) Rho-kinase increased the interaction of PSD-93 with PSD-95 in COS7 cells: COS7 cells were cotransfected with Myc-PSD-95 and GST-PSD-93-PDZ3-SH3 (WT, phosphodeficient and phosphomimetic mutants) and then treated with calyculin A (50 nM) for 12 min with or without pretreatment with Y-27632 (20 µM). The samples were subjected to a GST pull-down assay. (F) The bar diagram shows the quantification of the immunoblot data. The horizontal lines indicate the mean ± SEM of three independent experiments. *, ** and *** represent p < 0.05, p < 0.01 and p < 0.001, respectively, for Tukey’s multiple comparisons test.
Figure 6
Figure 6
Chemically induced-LTP increases the Rho-kinase-mediated phosphorylation of PSD-93 and the colocalization of PSD-93 with PSD-95. (A) Chemical-LTP induces the PSD-93 phosphorylation in cultured striatal neurons. Striatal neurons were cultured until DIV21 and then neurons were treated with glycine (200 µM) after pretreatment with the Rho-kinase inhibitor Y-27632 (20 µM). The samples were analyzed by immunoblot analysis of anti-pT612 PSD-93, anti-PSD-93, anti-pT853 MYPT1, and anti-MYPT1 antibodies. (B,C) The horizontal lines represent the mean ± SEM of three independent experiments. *, ** and *** represent p < 0.05, p < 0.01, p < 0.001, respectively, and “ns” denotes “not significant”, for Tukey’s multiple comparisons test. (D) Colocalization of PSD-93 with PSD-95 during chemical-LTP in cultured primary striatal neurons. The neurons were cultured until DIV14 and infected with AAV-EGFP and AAV-Cre virus. After DIV21, the neurons were treated with glycine to induce chemical-LTP and the immunostaining was performed with anti-GFP (green), anti-PSD-93 (red) and anti-PSD-95 (white) antibodies. The scale bar is 10 μM. (E,F) The horizontal lines represent the mean ± SEM of five independent experiments. *, **, *** and **** represent p < 0.05, p < 0.01, p < 0.001 and p < 0.001, respectively, for Tukey’s multiple comparisons test.
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