For SynGAP, either of two phosphomutants (S781A or S783A) largely blocked the gel mobility shift induced by Plk2 (Figure S6D), implying a critical role of these adjacent phosphosites for conformational changes in SynGAP.

Active Ras pull-down assays demonstrated that these sites, as well as S326 and S390, were also crucial for Plk2 to stimulate SynGAP activity against Ras (Figures S6E and S6F; Table S2). In the case of PDZGEF1, we observed no differences in Plk2-dependent gel mobility shift or alterations in Rap GEF activity with PD0332991 chemical structure any single Plk2 phosphosite mutant (Figure S6G). Various double, triple, and quadruple phosphomutant combinations of PDZGEF1 also yielded no effect on mobility shift or GEF activity (data not shown). However, loss of all five Plk2 phosphosites (5xA mutant) substantially blocked the mobility Selleckchem Small molecule library shift of PDZGEF1 by Plk2 (Figure S6G, right panel) and the Plk2-mediated increase in enzymatic activity of PDZGEF1 toward Rap (Figures S6H and S6I; Table S2). Next, to evaluate the functional importance of these phosphosites for overactivity-dependent spine remodeling, we performed quantitative spine analysis in proximal dendrites of neurons

expressing the most severe mutants of RasGRF1 (S71A), SynGAP (S390A or S783A), and PDZGEF1 (5xA) (Figure 6A). Transfection of either WT or RasGRF1 (S71A) increased spine density compared to GFP control (Figures 6B–6D and 6J; Table S1). PTX application significantly reduced spine density in neurons expressing WT RasGRF1, but this effect was partially blocked in neurons expressing RasGRF1 (S71A) (Figures 6C, 6D, and 6J). WT RasGRF1 also increased spine head size, which was reversed in the presence of PTX (Figure 6K).

In contrast, PTX treatment failed to reduce spine head width in neurons expressing RasGRF1 (S71A) (Figure 6K). Expression of WT or phosphomutants of SynGAP strongly reduced spine head size (Figures 6E–6G and 6L; Table S1) without changing spine density. PTX treatment of neurons expressing WT SynGAP led to even Org 27569 further reduction of head width as well as spine loss (perhaps due to some spine sizes falling below the cutoff threshold for detection) (Figures 6E, 6J, and 6L). In contrast, these PTX effects were abolished in neurons expressing either SynGAP phosphomutant (Figures 6F, 6G, 6J, and 6L). Lastly, either WT or PDZGEF1 (5xA) mutant decreased spine density without changing head size (Figures 6H–6J and 6M; Table S1). PTX treatment further decreased spine density in neurons expressing WT PDZGEF1, but not in neurons expressing the quintuple phosphomutant (Figures 6H–6J). However, neither WT nor PDZGEF1 (5xA) mutant affected PTX-dependent reduction of spine head size (Figure 6M). There was no significant difference in spine length in any condition (Table S1). Together, these results suggested that phosphorylation of Ras/Rap regulators by Plk2 is required for homeostatic regulation of dendritic spines following chronic overactivity.