Identification of the modulatory kinase and downstream effectors associated with phosphorylation of delta-catenin’s PDZ-binding motif. (A) Representation of fold change in levels of phosphorylated delta-catenin relative to total delta-catenin in response to exogenous coexpression of the indicated kinases in HEK293 cells. Coexpression of constitutively active CDK5 and p25c with delta-catenin resulted in a 15-fold increase in the relative amount of phosphorylated delta-catenin in cell lysates (P = 0.0113); n = 3 independent cultures/experiments. (B) Quantification of fold change in relative levels of phosphorylated delta-catenin in primary rat hippocampal neurons (7 DIV) following DHPG treatment with and without pretreatment with Butyrolactone I. Pretreatment of cultures with Butyrolactone I largely prevented the DHPG-induced increase (P = 0.0404) in delta-catenin phosphorylation levels relative to controls (P = 0.5205); n = 3 independent cultures/experiments. (C) Quantification of levels of active RhoA in HEK293 cells expressing WT delta-catenin (baseline), phospho-null delta-catenin (102% of baseline; P = 0.8814), phospho-mimic delta-catenin (85% of baseline; P = 0.0332), Magi1 + delta-catenin (88% of baseline; P = 0.3152), or Pdlim5 + delta-catenin (69% of baseline; P = 0.0059); n = 4 independent experiments. (D) Representative ratiometric FRET confocal images of 4-DIV primary rat hippocampal neurons expressing a FRET-based (CFP–YFP) RhoA biosensor in combination with phospho-null or phospho-mimic delta-catenin mutants. (E) Quantification of ratio of dendrite-to-soma RhoA activity (Fr) in biosensor-expressing neurons, as observed via ratiometric FRET. Relative to controls (Fr = 2.462 ± 0.142), expression of phospho-null delta-catenin produced no change in dendritic RhoA activity (Fr = 2.713 ± 0.074; P = 0.2098). Expression of phospho-mimic delta-catenin produced a significant decrease in dendritic RhoA activity (Fr = 1.312 ± 0.048) compared with both control (P < 0.0001) and phospho-null delta-catenin -expressing neurons (P < 0.0001). (F) Quantification of RhoA activity in the soma of biosensor-expressing neurons, as observed via ratiometric FRET. Expression of either phospho-null or phospho-mimic delta-catenin in primary rat hippocampal neurons produced no changes in RhoA activity in the soma of neurons (P = 0.8906; P = 0.2903). For D–F, n = 4 neurons from independent cultures. (G) Immunoblot of HA-delta-catenin coimmunoprecipitation with Flag-Cortactin from HEK293 cell lysates. IP, immunoprecipitation. WCE, whole cell extract. (H) Densitometric quantification (normalized to total delta-catenin and antibody) of relative (to phospho-mimic) amounts of delta-catenin mutants pulled down by Cortactin. Phospho-null delta-catenin failed to coimmunoprecipitate with Cortactin, while phospho-mimic delta-catenin successfully immunoprecipitated with Cortactin (P = 0.0077). This association was further stabilized by coexpression of Pdlim5 (P = 0.0301). For H, n = 3 independent cultures/experiments. *, P < 0.05; **, P ≤ 0.001; ***, P ≤ 0.0001. For A–C, E, F, and H, significance was determined using a one-way ANOVA followed by Tukey’s test. Error bars indicate SEM. Scale bars, 10 µm.