SynGAP regulates steady-state and activity-dependent phosphorylation of cofilin - PubMed (original) (raw)

Bath application of NMDA in neuronal cultures increases the phosphorylation of CaMKII and synGAP, the activation of Ras and Rac, and the phosphorylation of PAK, but causes a decrease in the phosphorylation of cofilin. A, The diagram depicts one postulated pathway through which the NMDA receptor regulates Ras activity (left) and the Rac-dependent pathway that regulates cofilin (right). The dotted line represents a hypothetical link between activated Ras and activated Rac that is suggested by our data. Wild-type cortical or hippocampal neurons were treated with NMDA (25 μ

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) for 0.25, 1, 3, or 5 min. Phosphorylation of CaMKII, synGAP, PAK, and cofilin, and activation of Ras and Rac were measured as described in Materials and Methods. B, E, Samples from cortical and hippocampal cultures that were immunoblotted with anti-phospho-Thr286 CaMKII antibody showed a significant increase in phosphorylation within 15 s of treatment with NMDA (15 s* = 209 ± 23%, p = 0.009; 1 min* = 225 ± 35%, p = 0.016; 3 min* = 212 ± 35%, p = 0.023; 5 min* = 167 ± 25% of untreated control, p = 0.046; one-sample t test, data from cortical and hippocampal cultures were combined, n = 6 independent experiments). C, E, Samples from cortical and hippocampal cultures immunoblotted with anti-phospho-Ser1123 synGAP antibody showed a significant increase in phosphorylation of ser-1123 on synGAP by CaMKII after 15 s, followed by rapid dephosphorylation (15 s* = 189 ± 28%, p = 0.01; 1 min = 146 ± 24%; 3 min = 104 ± 24%; 5 min* = 38.1 ± 16% of untreated control, p = 0.004; two-tailed one-sample t test; data from cortical and hippocampal cultures were combined, n = 10 independent experiments). D, E, Activated Ras was measured in homogenates of hippocampal and cortical cultures as described under Materials and Methods. Treatment with NMDA (25 μ

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) induced brief activation of Ras [control = 8.32 ± 2.88 arbitrary units (a.u.); 15 s* = 24.8 ± 8.0 a.u., p = 0.018; 1 min = 12.6 ± 2.4 a.u.; 3 min = 8.31 ± 1.71 a.u.; 5 min = 4.50 ± 0.64 a.u.; repeated measures ANOVA; n = 3 independent experiments]. F, E, Activated Rac measured from hippocampal and cortical cultures is significantly elevated after 1 min treatment with NMDA (25 μ

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) compared with untreated controls (0.25 min = 109 ± 19.8%; 1 min* = 183 ± 22.3%, p = 0.048, two-tailed one-sample t test; 3 min = 207 ± 58.7%; 5 min = 204 ± 54.3% of control; n = 5). G, E, Anti-phospho-Thr423 PAK was elevated more than twofold in the first 15 s of treatment with NMDA (15 s* = 267 ± 46.4%, p = 0.009; 1 min* = 294 ± 54.7%, p = 0.009; 3 min* = 286 ± 63.7%, p = 0.022; 5 min = 132 ± 67.5%; two-tailed, one sample t test; n = 8 independent experiments). H, E Anti-phospho-Ser3 cofilin was dephosphorylated in response to treatment with NMDA for 15 s to 5 min (15 s* = 56.2 ± 8.68%, p > 0.001; 1 min* = 64.2 ± 9.37%, p = 0.005; 3 min* = 64.8 ± 11.3%, p = 0.001; 5 min* = 78.3 ± 9.46%, p = 0.047; two-tailed, one-sample t test; n = 10 independent experiments). Representative immunoblots are shown in E. D shows means ± SEM B, C, F, G and H show GM ±SEs of the SEGM.