Journal:

Article Title: SynGAP regulates steady state and activity-dependent phosphorylation of cofilin

doi: 10.1523/JNEUROSCI.4695-08.2008

Figure Lengend Snippet: Steady-state activity of PAK and cofilin is altered and regulation cofilin activity by the NMDA receptor is deranged in synGAP mutant hippocampal cultures. Wild type, synGAP+/-, and synGAP-/- hippocampal cultures (DIV 15-18) were treated with 25 μM NMDA for 15 sec - 5 min and then immunoblotted with anti-phospho-Thr423 PAK(1-3) or anti-phospho-Ser3 cofilin antibody. Phosphoprotein levels were measured and quantified as described under methods. A, The levels of phospho-PAK and phospho-cofilin were basally elevated in untreated synGAP-/- hippocampal cultures. Changes in phosphorylation of PAK and cofilin are expressed as a percentage of the wild type untreated control values (synGAP+/- phospho-PAK = 153 ± 29.2% of wt, n = 7 litters; synGAP-/- phospho-PAK* = 202 ± 43.4% of wt, P = 0.006, n = 5 litters; synGAP+/- phospho-cofilin = 121 ± 18.6% of wt, n = 11 litters; synGAP-/- phopho-cofilin* =141 ± 12.9% of wt, P = 0.012, n = 10 litters). B, Despite the basal increase in phosphorylated PAK in the mutants, treatment with NMDA still induces an additional increase in phospho-PAK, similar to that in wild types, when each genotype is normalized to its own untreated genotype control (wild type; 15 sec = 267 ± 46.4%, 1 min = 294 ± 54.7%, 3 min = 286 ± 63.7%, 5 min = 132 ± 67.5% of wild type control; N = 8 litters) (SynGAP+/-; 15 sec = 316 ± 58.5%, 1 min = 315 ± 58.4%, 3 min = 256 ± 45.7%, 5 min = 134 ± 40.6% of synGAP+/- control; N = 8 litters) (SynGAP-/-; 15 sec = 252 ± 40.1%; 1 min = 415 ± 130%; 3 min = 322 ± 95.5%; 5 min = 224 ± 75.7% of synGAP-/- control; N = 8 litters). C, NMDA induces significantly less dephosphorylation of cofilin in synGAP mutant compared to wild-type cultures (wild type: 15 sec = 59.7 ± 7.37%; 1 min = 67.4 ± 7.05%; 3 min = 68.7 ± 6.68%; 5 min = 73.4 ± 78.91% of wild type control; N = 6 litters) (SynGAP+/-: 15 sec = 83.5 ± 5.64%; 1 min = 77.8 ± 5.29%; 3 min = 77.2 ± 7.04%; 5 min = 80.4 ± 5.16% of SynGAP+/- control; N = 4 litters) (SynGAP-/-: 15 sec* = 96.9 ± 1.07%, P = 0.006; 1 min* = 94.6 ± 4.87%, P = 0.37; 3 min = 81.0 ± 7.48%; 5 min = 78.0 ± 5.85% of SynGAP-/- control; N = 5 litters; one-way ANOVA comparing the three genotypes at each time point, followed by Tukey-Kramer multiple comparison test). All time points were significantly less than 100% in the wild type neurons; the 1, 3, and 5 min time points were significantly less that 100% in the synGAP+/- neurons; and only the 5 min time point was significantly less than 100% in the synGAP-/- neurons (P < 0.05, 2-tailed, one-sample t-test). Example blots are shown on right. Graphs represent geometric means ± SEGM.

Article Snippet: B , Despite the basal increase in phosphorylated PAK in the mutants, treatment with NMDA still induces an additional increase in phospho-PAK, similar to that in wild types, when each genotype is normalized to its own untreated genotype control (wild type; 15 sec = 267 ± 46.4%, 1 min = 294 ± 54.7%, 3 min = 286 ± 63.7%, 5 min = 132 ± 67.5% of wild type control; N = 8 litters) ( SynGAP +/- ; 15 sec = 316 ± 58.5%, 1 min = 315 ± 58.4%, 3 min = 256 ± 45.7%, 5 min = 134 ± 40.6% of synGAP +/- control; N = 8 litters) ( SynGAP -/- ; 15 sec = 252 ± 40.1%; 1 min = 415 ± 130%; 3 min = 322 ± 95.5%; 5 min = 224 ± 75.7% of synGAP -/- control; N = 8 litters).

Techniques: Activity Assay, Mutagenesis, Phospho-proteomics, Control, De-Phosphorylation Assay, Comparison