(A) TRAF4 overexpression increased the phosphorylation level of WT TrkA but not the K523_544_547R mutant in the presence of NGF. Specific tyrosine phosphorylation antibodies were used in the Western blot analysis in cells treated with 50 ng/ml NGF for 15 minutes. (B) Mutation of the K523, K544, and K547 residues reduced the association of Shc protein with TrkA. FLAG-TrkA WT or its mutant was transfected into DU145 cells, and the interaction between Shc protein and TrkA was determined in a co-IP experiment using a FLAG antibody for immunoprecipitation. (C) TRAF4 knockdown substantially reduced NGF-induced p38 MAPK phosphorylation (pT180/Y182). PC3 cells were treated with and without NGF (50 ng/ml) for 10 minutes after 18 hours of serum starvation. (D) The level of TRAF4 expression correlated with Slug gene expression in a prostate cancer patient cDNA array. (E) The level of TRAF4 expression correlated with the IL-6 gene expression in a prostate cancer patient cDNA array. Pearson’s correlations for D and E for fold change (log₂) in gene expression as determined by qRT-PCR (r = 0.852, P < 0.0001 and r = 0.890, P = 2.53 × 10^–14, respectively). (F) Working model of the role of TRAF4 in TrkA signaling and prostate cancer cell invasion. In low-TRAF4-expressing cells, NGF induce limited TrkA ubiquitination, resulting in low levels of TrkA kinase signaling. In TRAF4-overexpressing cells, high levels of TRAF4 significantly increase TrkA K27- and K29-linked ubiquitination at K523, K544, and K547 in the kinase domain upon NGF stimulation. This ubiquitination enhances TrkA kinase activity, its tyrosine phosphorylation levels, and the recruitment of downstream adaptor proteins, resulting in a hyperactivated TrkA signaling cascade. Consequently, NGF-responsive invasion-associated targeted gene transcription is upregulated to promote cell migration and invasion.