Arginine methylation of USP9X promotes its interaction with TDRD3 and its anti-apoptotic activities in breast cancer cells

Abstract

The Tudor domain-containing proteins are characterized by their specific interactions with methylated protein motifs, including methyl-arginines and methyl-lysines. The Tudor domain-containing protein 3 (TDRD3) is one of the major methyl-arginine effector molecules that recognizes methylated arginine residues on histones and the C-terminal domain of RNA polymerase II, and activates transcription. However, majority of the cellular TDRD3 localizes to the cytoplasm and its functions there are still elusive. Here, we have identified ubiquitin-specific protease 9 X-linked (USP9X) as a TDRD3-interacting protein by GST (glutathione S-transferase) pull-down and co-immunoprecipitation. Detailed characterization suggests that the interaction between TDRD3 and USP9X is mediated through the Tudor domain of TDRD3 and the arginine methylation of USP9X. This interaction plays a critical role in TDRD3 protein stability, as knockdown of USP9X expression leads to increased TDRD3 ubiquitination. We also found that USP9X co-localizes with TDRD3 in cytoplasmic stress granules and this localization is diminished in Tdrd3-null mouse embryonic fibroblast cells, suggesting that TDRD3 is essential for USP9X stress granule localization. Furthermore, we found that one of the USP9X de-ubiquitination targets, myeloid cell leukemia protein 1, is regulated by TDRD3, indicating that TDRD3 potentially regulates USP9X de-ubiquitinase activity. Finally, we show that knockdown of TDRD3 expression sensitizes breast cancer cells to chemotherapeutic drug-induced apoptosis, likely due to its regulation of USP9X. This study provides a novel candidate strategy for targeting apoptosis pathways in cancer therapy.

Activation of the ERK/Creb/Bcl‑2 pathway protects periodontal ligament stem cells against hydrogen peroxide‑induced oxidative stress

“…Elevated ROS levels are associated with apoptosis and Bcl-2 is a critical regulator in ROS-associated apoptosis (47–49), suggesting that apoptotic cell death caused at least a portion of the PDLSCs cell death under the treatment with H 2 O 2 . Multiple results have demonstrated the role of Bcl-2 in apoptosis and there is a need to identify the upstream regulators of Bcl-2 (50–52). The present results demonstrated that the induction of Creb expression attenuated the H 2 O 2 -induced ROS elevation, suggesting that Creb protects PDLSCs from apoptosis by activating Bcl-2.…”

Arginine methylation of the C-terminus RGG motif promotes TOP3B topoisomerase activity and stress granule localization

“…The human PRMT1, PRMT3, CARM1/PRMT4, PRMT6 and PRMT8 cDNAs were cloned into a pGEX-6P-1 vector (GE Healthcare Life Sciences). The GST-Tudor (wild-type) and GST-Tudor (E691K) of TDRD3 have been described before ( 19 ). Human TOP3B cDNA and its truncated cDNAs (1-707 and 708–862) were cloned into pGEX-6P-1 and p3xFLAG-CMV vector (Sigma).…”

Structural plasticity of the TDRD3 Tudor domain probed by a fragment screening hit

“…It is hence of high interest to uncover small molecule regulators of stress granules. In addition, a recent research disclosed that TDRD3 binds the methylated USPX9 and such interaction promotes the antiapoptotic activities of breast cancer cells . Hence, TDRD3 might serve as a new target for cancer therapy.…”

“…TDRD3 has also been identified as a regulator of proliferation and invasion of breast cancer cells . The Tudor‐mediated interaction between TDRD3 and ubiquitin‐specific protease 9 X‐linked suppresses the ubiquitination of TDRD3 and colocalizes this complex into stress granules, and such interaction promotes the antiapoptotic activities of breast cancer cells . These findings suggest TDRD3 as a new therapeutic target for breast cancers.…”