(A–D) Sequence analysis of regulated phosphosites using the Motif-X tool (Schwartz and Gygi, 2005). The frequency of residues surrounding the phosphorylated serine is indicated by the size of the letters. Among the upregulated sites, the motif RXXS is overrepresented, whereas the SP motif is conspicuously present among downregulated motifs. (E) In vitro kinase assays evaluating the ability of selected kinases to phosphorylated phosphosites identified on active zone proteins. Five phosphosites were selected from four active zone proteins for testing against CaMKII, PKA, and PKC. For each site, two 13-mer peptides were synthesized as substrates (for sequences of the peptide see Figure 6—source data 1) that correspond to the sequence surrounding the phosphorylated serine, with one of them containing an alanine instead of the serine as control. The following peptides were used as positive controls: (i) synapsin S603 that is known to be phosphorylated by CaMKII (Jovanovic et al., 2001) and (ii) autocamide-2, kemptide, and neurogranin were used as known substrates of CaMKII, PKA, and PKC, respectively. Background was determined by omitting peptide from the assay. The data show means of three replicates, with the bars indicating the range of values.