Figure 5.

PDLIM1 associates with p75^NTR via the C-terminal PDZ-binding motif, an interaction regulated by phosphorylation of S425. (a) Using a biotinylated peptide to the C-terminus of either p75^NTR (p75^wt peptide, Biotin-N-LCSESTATSPV-COOH, residues 417-427) or a Valine deletion mutant of p75^NTR (p75^MT peptide, biotin-N- LCSESTATSP-COOH, residues 417-426) as affinity reagents, PDLIM1 was confirmed as a p75^NTR-interacting protein by western blot analysis. (b) To assess the physiological relevance of the phosphorylation of S425 within the PDZ-binding motif, biotin-labeled peptides corresponding to the p75^NTR wild-type C-terminal (SPV), the S425 phosphorylated C-terminal (^PhosphoSPV) and the PDZ-binding motif deleted C-terminal (ΔSPV) were used as affinity reagents. PDLIM1 was shown to interact with maximum affinity to the wild-type unphosphorylated peptide. (c) Biotin-labeled p75^NTR peptides (SPV, ^PhosphoSPV, ΔSPV, ΔV, SP and SPM) were incubated with His-tagged recombinant PDLIM1 protein, respectively. Pull-down assays were performed to determine the amount of PDLIM1 protein bound to each peptide. Flow-through shows unbound proteins after incubation. (d) Immunoprecipitation with PDLIM1 antibodies co-precipitated p75^NTR and PDLIM1 while the mutant alleles of p75^NTR (S303G, ΔSPV and SPM) or p75^NTR cells treated with the PKA inhibitor KT5720 (KT; 200 nM) were severely compromised for PDLIM1 binding. (e) Interaction of PDLIM1 with p75^NTR was confirmed in patient-derived glioma stem cells/tumor-initiating cells (BT42, BT134, BT147) by immunoprecipitation using anti-PDLIM1. Input cell lysates were assessed by western blot analysis for expression of p75^NTR and PDLIM1.