| PSD-95 is a protein associated with the sub-synaptic scaffold, where one of its functions is to form receptor clusters through PDZ-PDZ (PSD95/Dlg/ZO-1) binding domain interactions. δ-catenin is a neuron-specific adherens junction-associated scaffolding protein of the Armadillo family that has previously been shown to interact directly with classical cadherins as well as presenilin-1 (PS-1), and appears to play a role in establishing and/or modifying neuronal morphology.;Deletion mapping of the interaction between PSD-95 and δ-catenin, performed by co-immunoprecipitation, GST pulldown, and yeast two-hybrid analysis, shows that the interaction is direct and mediated by the PDZ 2 domain of PSD-95 interacting with the PDZ-binding domain of δ-catenin.;The PSD-95 interaction with δ-catenin is dynamic. Co-immunoprecipitation of PSD-95 and δ-catenin from neuronal cultures was performed at multiple time points following stimulation by glutamate in the presence of receptor blockers. Two kinetically distinct receptor-mediated pathways dissociate PSD-95 from δ-catenin. One is a rapidly occurring response that takes place between 75–120 seconds of glutamate stimulation, which can be mediated by α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors. A slower response, mediated by Class I metabotropic glutamate receptors (mGluRs), takes place at >25 minutes of stimulation. Both responses are transient, with the PSD-95/S-catenin interaction decreasing to less than 50% of control and then recovering to near-baseline levels. Co-immunoprecipitation with δ-catenin antibody was performed to probe for an interaction with the following kinases: Calcium-calmodulin-dependent protein Kinase II (CaMKII), Protein Kinase A (PKA), Protein Kinase C (PKC) and Casein Kinase I (CKI). CKI was the only kinase to co-immunoprecipitate with δ-catenin.;To show that CKI can phosphorylate δ-catenin, a 32P in vitro kinase assay was performed, showing that a δ-catenin C-terminal peptide is phosphorylated by CKI. Treatment with CKI inhibitor before glutamate stimulation resulted in a statistically significant difference in the association of PSD-95 with δ-catenin, indicating that CKI inhibition affects the PSD-95/δ-catenin interaction in neurons. |
