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Phosphorylation of OTUB1 at Tyr 26 stabilizes the mTORC1 component, Raptor

Abstract

Raptor plays a critical role in mTORC1 signaling. High expression of Raptor is associated with resistance of cancer cells to PI3K/mTOR inhibitors. Here, we found that OTUB1-stabilized Raptor in a non-canonical manner. Using biochemical assays, we found that the tyrosine 26 residue (Y26) of OTUB1 played a critical role in the interaction between OTUB1 and Raptor. Furthermore, non-receptor tyrosine kinases (Src and SRMS kinases) induced phosphorylation of OTUB1 at Y26, which stabilized Raptor. Interestingly, phosphorylation of OTUB1 at Y26 did not affect the stability of other OTUB1-targeted substrates. However, dephosphorylation of OTUB1 destabilized Raptor and sensitized cancer cells to anti-cancer drugs via mitochondrial reactive oxygen species-mediated mitochondrial dysfunction. Furthermore, we detected high levels of phospho-OTUB1 and Raptor in samples of patients with renal clear carcinoma. Our results suggested that regulation of OTUB1 phosphorylation may be an effective and selective therapeutic target for treating cancers via down-regulation of Raptor.

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Fig. 1: OTUB1 deubiquitinase stabilized Raptor.
Fig. 2: Tyrosine 26 (Y26) residue of OTUB1 was critical for stabilization of Raptor.
Fig. 3: Mutation of OTUB1 at the Y26 residue increased mitochondrial dysfunction via down-regulation of Raptor.
Fig. 4: Src and SRMS kinase phosphorylated OTUB1 at Y26 residue, which determined substrate specificity.
Fig. 5: Phosphorylation of OTUB1 at tyrosine 26 by Src kinase played a critical role in stabilizing Raptor.
Fig. 6: UBC13 was important for OTUB1-mediated stabilization of Raptor.
Fig. 7: OTUB1 Y26 residue increased sensitivity to anti-cancer drug due to degradation of Raptor.
Fig. 8: Raptor expression contributed to poor prognosis of patients with RCC.

Data availability

All data generated or analyzed during this study are included in this article and its supplementary data files and all original data are available from the corresponding authors upon request.

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Acknowledgements

The biospecimens and data used for the present study were provided by the Biobank of Keimyung University Dongsan Hospital Biobank, a member of the Korea Biobank Network.

Funding

This work was supported by an NRF grant funded by the Korea Government (MSIP) [NRF-2021R1A4A1029238, NRF-2019R1A2C2005921 and NRF-2020R1C1C1009889]. This work was supported by a grant from the KRIBB Research Initiative Program.

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KM, EL and TKK conceived and designed the project; SUS, SMW, and MWK performed experiments and analyses, and acquired data; EL and TKK contributed technical/reagents materials, analytical tools, and/or grant support; KM, and TKK prepared, wrote the manuscript; SMW, KM, EL and TKK reviewed, and/or revised the manuscript. All authors discussed the results and commented on the manuscript. The author(s) read and approved the final manuscript.

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Correspondence to Eun-Woo Lee, Kyoung-jin Min or Taeg Kyu Kwon.

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Seo, S.U., Woo, S.M., Kim, M.W. et al. Phosphorylation of OTUB1 at Tyr 26 stabilizes the mTORC1 component, Raptor. Cell Death Differ 30, 82–93 (2023). https://doi.org/10.1038/s41418-022-01047-3

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