We propose that PH domain phosphorylation by Plk2 leads to detachment from Selleck Obeticholic Acid membranes, potentially
increasing accessibility to proteasomal degradation. (2) Phosphorylation of both PDZGEF1 and SynGAP induced large gel mobility shifts suggestive of extensive conformational changes. Because these alterations were associated with increased enzymatic activity, we suggest phosphorylation at these sites locks SynGAP or PDZGEF1 in an open, active conformation. (3) Additional phosphosites within or near the GAP domain of SynGAP (S326, S390) did not appear to be involved in conformational changes but did interfere with Plk2 ability to modulate SynGAP enzymatic activity, suggesting an independent mode of regulation that may involve direct GAP domain control. Importantly, expression of Plk2 phosphorylation-deficient mutants of RasGRF1, SynGAP, and PDZGEF1 abolished specific aspects of PTX-induced spine remodeling generally consistent with knockdown and overexpression studies, demonstrating that Plk2 phosphorylation of these Ras/Rap regulators
is required for full homeostatic regulation of dendritic spines. Ribociclib mouse Overactivity-induced removal of sGluA1 was restricted to proximal dendrites and dependent on Plk2 kinase activity, mirroring RasGRF1/SPAR expression and dendritic spine loss. In contrast, hyperexcitation reduced sGluA2 in both proximal and distal dendrites through a Plk2 kinase-dependent and -independent mechanism, respectively. These results confirm and extend our previous findings that a kinase-independent interaction of Plk2 with NSF dislodges GluA2, causing loss of surface expression in secondary dendrites (Evers et al., 2010). Although it is currently unclear how these two mechanisms act on different dendritic subregions, these findings may suggest that GluA1 and GluA2 subserve distinct functions during homeostatic adaptation to overexcitation and support the idea that proximal dendrites employ a different or additional homeostatic mechanism from distal dendrites (Figure S7J). Multiple mechanisms of homeostatic synaptic plasticity exist based
on mode of activity only manipulation, developmental stage, and cell type (Pozo and Goda, 2010). Here we elucidated two distinct and complementary mechanisms of homeostasis depending on dendritic locus as well as Plk2 kinase activity (Figure S7J), with the following lines of evidence: Plk2 is induced in a proximal-to-distal gradient by chronic overactivity (Pak and Sheng, 2003). Plk2 kinase activity was required for depletion of RasGRF1/SPAR, PSD scaffold proteins, dendritic spines, as well as sGluA1/A2 specifically within the proximal dendrite. In contrast, PTX-induced sGluA2 removal in distal dendrites was kinase independent. These results may reflect a need to regulate distal AMPARs via a graded, linear response in proportion to the level of synaptic activity experienced, but to control proximal dendritic synapses in an all-or-none fashion, potentially in response to more traumatic or persistent insults.