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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.
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Research Article Cell biology Genetics Article has an altmetric score of 6

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

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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

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Figure 6

TRAF4 ubiquitinated 3 lysine residues present in the kinase domain of TrkA.

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TRAF4 ubiquitinated 3 lysine residues present in the kinase domain of Tr...
(A) Deletion of the tyrosine kinase domain (TK) of TrkA abolished its ubiquitination. Upper panel: Schematic representation of TrkA and its deletion mutants. Lower panels: Ubiquitination levels of different TrkA deletion mutants. FLAG-TrkA and the mutants were cotransfected with TRAF4 and HA-Ub into 293T cells. The ubiquitinated TrkA was immunoprecipitated using a FLAG antibody and then detected using an anti-HA antibody in the Western blot. (B) Mutation of K523, K544, and K547 residues at the TK domain abolished TrkA ubiquitination. (C) TRAF4 hyperactivated TrkA WT but not the K523_544_547R mutant in an in vitro kinase assay. Left: Purified FLAG-TrkA in vitro kinase activity using a poly(Glu4, Tyr1) synthetic peptide as a substrate. The activity was measured through an ADP-Glo Kinase assay. Right: Protein levels of purified TrkA, its mutant, and PKCδ used in the kinase assay with or without TRAF4 overexpression as demonstrated by Western blotting using an anti-FLAG antibody. Data are presented as mean ± SEM. n = 3. *P < 0.05 by 1-way ANOVA.

Copyright © 2025 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)

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