TRAF4-mediated ubiquitination of NGF receptor TrkA regulates prostate cancer metastasis
Ramesh Singh, … , Bert W. O’Malley, Ping Yi
Ramesh Singh, … , Bert W. O’Malley, Ping Yi
Published May 1, 2018
Citation Information: J Clin Invest. 2018;128(7):3129-3143. https://doi.org/10.1172/JCI96060.
View: Text | PDF
Research Article Cell biology Genetics

TRAF4-mediated ubiquitination of NGF receptor TrkA regulates prostate cancer metastasis

Abstract

Receptor tyrosine kinases (RTKs) are important drivers of cancers. In addition to genomic alterations, aberrant activation of WT RTKs plays an important role in driving cancer progression. However, the mechanisms underlying how RTKs drive prostate cancer remain incompletely characterized. Here we show that non-proteolytic ubiquitination of RTK regulates its kinase activity and contributes to RTK-mediated prostate cancer metastasis. TRAF4, an E3 ubiquitin ligase, is highly expressed in metastatic prostate cancer. We demonstrated here that it is a key player in regulating RTK-mediated prostate cancer metastasis. We further identified TrkA, a neurotrophin RTK, as a TRAF4-targeted ubiquitination substrate that promotes cancer cell invasion and found that inhibition of TrkA activity abolished TRAF4-dependent cell invasion. TRAF4 promoted K27- and K29-linked ubiquitination at the TrkA kinase domain and increased its kinase activity. Mutation of TRAF4-targeted ubiquitination sites abolished TrkA tyrosine autophosphorylation and its interaction with downstream proteins. TRAF4 knockdown also suppressed nerve growth factor (NGF) stimulated TrkA downstream p38 MAPK activation and invasion-associated gene expression. Furthermore, elevated TRAF4 levels significantly correlated with increased NGF-stimulated invasion–associated gene expression in prostate cancer patients, indicating that this signaling axis is significantly activated during oncogenesis. Our results revealed a posttranslational modification mechanism contributing to aberrant non-mutated RTK activation in cancer cells.

Authors

Ramesh Singh, Dileep Karri, Hong Shen, Jiangyong Shao, Subhamoy Dasgupta, Shixia Huang, Dean P. Edwards, Michael M. Ittmann, Bert W. O’Malley, Ping Yi

×

Figure 4

TRAF4 interacted with TrkA and promoted its ubiquitination.

Options: View larger image (or click on image) Download as PowerPoint
TRAF4 interacted with TrkA and promoted its ubiquitination. (A) HA-TRAF4...
(A) HA-TRAF4 interacted with FLAG-TrkA in transiently transfected 293T cells. Shown is a co-IP experiment using an anti-FLAG antibody for immunoprecipitation. (B) WT TRAF4 but not the RING domain deletion mutant promoted TrkA ubiquitination. 293T cells were cotransfected with constructs as indicated. FLAG-TrkA was immunoprecipitated with an anti-FLAG antibody, and the ubiquitinated TrkA was visualized by Western blot analysis using an anti-HA antibody. (C) Endogenous TrkA interacted with endogenous TRAF4 in DU145 cells. Shown is a co-IP experiment using a TrkA-specific antibody or IgG control for immunoprecipitation. (D) TRAF4 knockdown abolished NGF-induced TrkA ubiquitination. DU145 cells were transfected with control siRNA or siTRAF4 and HA-ubiquitin. Cells were then treated with 50 ng/ml NGF for 15 minutes before harvest. Ubiquitinated TrkA was detected using an anti-ubiquitin antibody in a Western blot analysis from cell lysates immunoprecipitated with an anti-TrkA antibody. Ub, ubiquitin. (E) TRAF4 overexpression promoted TrkA ubiquitination at the cell membrane. 293T cells were cotransfected with TrkA and HA-Ub in the absence or presence of TRAF4 cotransfection. Cytosolic and membrane fraction were isolated and subjected to immunoprecipitation using an anti-FLAG antibody, and the ubiquitinated TrkA was visualized by Western blot analysis using an anti-HA antibody. (F) TRAF4-mediated TrkA polyubiquitination through K27- or K29-linked ubiquitin chain. K6–K63 represent the ubiquitin mutant with all lysine mutations except the indicated number of lysine.