Fig. 6. CASK phosphorylates endogenous neurexins in cultured neurons.

A. Neurexin phosphorylation in neurons is reduced by synaptic activity, in contrast with total ³²P incorporation. Rat hippocampal cultures (DIV 14) were treated with 50 μM APV and 500 nM TTX (APV + TTX) or vehicle (−) overnight. Subsequently, in vivo ³²P-phosphorylation assay was performed in the presence of TTX and APV or the vehicle. Total lysate was separated on SDS-PAGE and quantified from phosphorimager scans. Compared to active neurons, total ³²P incorporated was significantly reduced in silenced neurons (≈ 58 ± 1.8 %). Neurexins were affinity-purified (Aff ppt) on immobilized CASK PDZ-domain, and the proteins were separated on SDS-PAGE followed by transfer onto a nitrocellulose membrane. A phosphorimager scan was used to visualize the phosphorylated β-neurexins (³²P, Nx), followed by immunoblotting (WB) for β-neurexins (Nx). Lysates were also probed for CASK expression and GDI as a control for total cellular protein. Phosphorylated total proteins under each condition were also detected by phosphorimager scanning (see Suppl. Fig. S9), with a notable reduction in the total ³²P incorporated by inactivated neurons. The bar graph depicts β-neurexin phosphorylation levels in the active and silenced neurons. Data are represented as means ± SEM, n = 6; asterisk, P = 0.0128.

B. Neurexin phosphorylation is reduced in CASK knockout neurons. Mouse hippocampal cultures from wild type and CASK conditional knockout mice were transduced with lentiviral vector expressing Cre recombinase-GFP fusion protein at DIV 4. Six days later (DIV 10), in vivo ³²P-phosphorylation assay was performed in the presence of TTX and APV. Total lysate was separated on SDS-PAGE and visualized with phosphorimager scanning (top panel). Neurexins were affinity-purified (Aff ppt) on immobilized CASK PDZ-domain, and the proteins were separated on SDS-PAGE, followed by transfer onto a nitrocellulose membrane. Phosphorylated β-neurexins were visualized with phosphorimager scanning (³²P, Nx), followed by immunoblotting (WB) for β-neurexins (Nx). Lysates were also probed for CASK and GDI. Bar graph depicts β-neurexin phosphorylation levels in the wild type (WT)_and CASK knockout (KO) neurons. Data are represented as means ± SEM, n = 6; asterisk, P = 0.000308.

C. CASK efficiently phosphorylates endogenous neurexin in neurons. Rat hippocampal cultures (DIV 6) were transfected at high efficiency with empty vector (−), wild-type EGFP-CASK fusion protein (CASK^wt), or SV-mutant EGFP-CASK fusion protein (CASK^SV), using a modified calcium-phosphate method. 72 h later, an in vivo phosphorylation assay was performed with ³²P_i in the presence of TTX and APV. Total lysate was separated on SDS-PAGE and visualized with phosphorimager scanning (top panel). Neurexins were affinity-purified (Aff ppt) on immobilized CASK PDZ-domain, and proteins were separated on SDS-PAGE and transferred onto a nitrocellulose membrane. A phosphorimager scan was used to visualize the phosphorylated β-neurexins (³²P, Nx), followed by immunoblotting (WB) for β-neurexins (Nx); the upper band correlates with the radioactive signal. Lysates were also probed for CASK (bottom panel); lower bands represent endogenous CASK and upper bands EGFP-CASK. Bar graph depicts β-neurexin phosphorylation levels in CASK transfected neurons as compared to the untransfected neurons. Data are represented as means ± SD, n = 3; single asterisk, P = 2.9 × 10⁻⁵; double asterisk, P = 0.0016.