Article Figures & Data
Figures
- Figure 1.
NLGN4X is phosphorylated by PKA at S712. A, Alignment of the transmembrane membrane domain and ICD of human NLGN4X and NLGN4Y. The PKC phosphorylation site, T707, is boxed in yellow; ASD mutations are boxed in gray; the transmembrane domain is boxed in blue; the PKA site on NLGN4X S712 is boxed in green; and the Cdk5 phosphorylation site on NLGN4X and NLGN4Y S712 is boxed in pink. B, GST-fusion proteins were incubated with [γ−32P] ATP and purified PKA, PKC, and CaMKII. Phosphorylation of NLGNs was analyzed by autoradiography. CBB protein staining was used as the loading control. The arrow indicates the size of the undegraded form of GST-fused NLGN4X C-tail. C, Sequence alignment of NLGN1–3, 4X, and 4Y. The NLGN4X phosphorylated S712 antibody epitope is underlined in red. D, GST-fusion proteins of NLGN1–3, 4X (WT, S712A), or 4Y were incubated with purified PKA. PKA phosphorylation was analyzed by immunoblot probing with pS712 antibody. E, Lysate from transfected primary cortical neurons was treated with lambda protein phosphatase and evaluated by immunoblotting with HA-Ab or pS712-Ab. α-Actin was used as a loading control.
- Figure 2.
NLGN4X is expressed and phosphorylated at S712 in human neurons. PKA and PKC are specific to phosphorylating NLGN4X S712 and NLGN4X T707, respectively. NLGN4X R704C ASD mutation disrupts NLGN4X S712 phosphorylation. A, Schematic for generating induced neurons from hiPS cells. B, Induced neurons from hiPS cells were immunoprecipitated with NLGN4X/4Y pS712 antibody. Phosphorylation of NLGN4X was evaluated by immunoblot, probing with NLGN4X antibody. C, E, GST-fusion proteins of NLGN4X (WT, R704C, T707A, S712A) and NLGN4Y (WT) were incubated with PKC or PKA, and phosphorylation was evaluated by immunoblotting with pT707-Ab or pS712-Ab. Total protein was evaluated with GST-Ab. D, F, Phosphorylation levels (mean ± SEM) were normalized to NLGN4X. Statistical significance was tested using a t test (n = 3).
- Figure 3.
Serine 712 phosphorylation is conserved in NLGN4Y but shows kinase specificity. A, C, E, GST-fusion proteins of NLGN4X (WT, T707A, R710H, S712A) and NLGN4Y (WT, H710R) were incubated with PKC, PKA, or Cdk5, and phosphorylation was evaluated by immunoblotting with pT707-Ab or pS712-Ab. Total protein was evaluated with GST-Ab. B, D, F, Phosphorylation levels (mean ± SEM) were normalized to NLGN4X. Statistical significance was tested using a t test (n = 4).
- Figure 4.
NLGN4X S712 phosphorylation affects spine density. A, Coexpression of NLmiRs with wild-type, phosphonull (S712A), and phosphomimetic (S712D) mutants in cultured hippocampal neurons. Surface and intracellular NLGN4X was labeled with HA antibody, which recognized a tag inserted downstream of the signal peptide. Scale bar, 10 μm. B, Mean ± SEM normalized to NLGN4X (n = 25), NLGN4X S712A (p > 0.05, n = 24), NLGN4X S712D (p > 0.05; n = 27). C, Coexpression of NLmiRs with NLGN4X WT, NLGN4X S712A, NLGN4X S712D, and NLGN4X R704C transfected into cultured hippocampal neurons. Staining was done for total HA and GFP. Reconstructed dendritic spines in the bottom panels are color coded according to the spine type (red, thin; blue, mushroom; green, stubby). D–G, Mean ± SEM of spine density (D), thin spine density (E), mushroom spine density (F), and stubby spine density (G) for NLGN4X (n = 44), NLGN4X S712A (n = 50), NLGN4X S712D (n = 57), and NLGN4X R704C (n = 44).
- Figure 5.
Phosphonull NLGN4X S712 enhances mEPSC frequency. A, The experimental design for transfecting and recording from primary hippocampal rat neurons, as well as representative traces of mEPSCs recorded from those cells (B). C, The mean of mEPSC frequency and cumulative probability plots of mEPSC interevent intervals and the mean of mEPSC frequency. p values were calculated using the Kolmogorov–Smirnov test with Dallal–Wilkinson–Lillie in WT (n = 12), NLmiRs (n = 14), NLGN4X WT (n = 11), NLGN4X S712A (n = 11), and NLGN4X S712D (n = 11) conditions. D, The mean of mEPSC amplitude and cumulative probability plots of mEPSC amplitude and the mean of mEPSC amplitude. p values were calculated using the Kolmogorov–Smirnov test with Dallal–Wilkinson–Lillie in WT (n = 12), NLmiRs (n = 14), NLGN4X WT (n = 11), NLGN4X S712A (n = 11), and NLGN4X S712D (n = 11) conditions.
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