Ubiquitination of the DNA-Damage Checkpoint Kinase CHK1 by TRAF4 Is Required for CHK1 Activation

Authors

Xinfang Yu
Wei Li
Haidan Liu
Qipan Deng
Xu Wang
Hui Hu
Zijun Y Xu-Monette
Wei Xiong
Zhongxin Lu
Ken H Young
Wei Wang
Yong Li

Publication Date

5-1-2020

Journal

Journal of Hematology & Oncology

DOI

10.1186/s13045-020-00869-3

PMID

32357935

PMCID

PMC7193419

PubMedCentral® Posted Date

5-1-2020

PubMedCentral® Full Text Version

Post-Print

Published Open-Access

yes

Keywords

Animals, Cell Line, Tumor, Checkpoint Kinase 1, Colorectal Neoplasms, DNA Damage, Enzyme Activation, Humans, Mice, Phosphorylation, TNF Receptor-Associated Factor 4, Ubiquitination, Colorectal cancer, Checkpoint kinase 1, Tumor necrosis factor receptor-associated factor 4, Ubiquitination, Chemoresistance

Abstract

BACKGROUND: Aberrant activation of DNA damage response (DDR) is a major cause of chemoresistance in colorectal cancer (CRC). CHK1 is upregulated in CRC and contributes to therapeutic resistance. We investigated the upstream signaling pathways governing CHK1 activation in CRC.

METHODS: We identified CHK1-binding proteins by mass spectrometry analysis. We analyzed the biologic consequences of knockout or overexpression of TRAF4 using immunoblotting, immunoprecipitation, and immunofluorescence. CHK1 and TRAF4 ubiquitination was studied in vitro and in vivo. We tested the functions of TRAF4 in CHK1 phosphorylation and CRC chemoresistance by measuring cell viability and proliferation, anchorage-dependent and -independent cell growth, and mouse xenograft tumorigenesis. We analyzed human CRC specimens by immunohistochemistry.

RESULTS: TRAF4 catalyzed the ubiquitination of CHK1 in multiple CRC cell lines. Following DNA damage, ubiquitination of CHK1 at K132 by TRAF4 is required for CHK1 phosphorylation and activation mediated by ATR. Notably, TRAF4 was highly expressed in chemotherapy-resistant CRC specimens and positively correlated with phosphorylated CHK1. Furthermore, depletion of TRAF4 impaired CHK1 activity and sensitized CRC cells to fluorouracil and other chemotherapeutic agents in vitro and in vivo.

CONCLUSIONS: These data reveal two novel steps required for CHK1 activation in which TRAF4 serves as a critical intermediary and suggest that inhibition of the ATR-TRAF4-CHK1 signaling may overcome CRC chemoresistance.

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