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.

Fig. 1. OTUB1 deubiquitinase stabilized Raptor.

A Caki cells transfected with the GFP siRNA or 49 types of deubiquitinase siRNA were examined using western blotting for the Raptor proteins. The band intensity of Raptor was analyzed using ImageJ. B Caki cells were co-transfected with Flag-HA-DUB (OTUB1, USP53, and STAMBP) and pRK5-Myc-Raptor plasmids. The interaction was demonstrated using immunoprecipitation (IP). C HEK293T cells were co-transfected with Flag-HA-DUB and pRK5-Myc-Raptor plasmids in the presence of HA-Ub and treated with 0.5 µM MG132 for 6 h. Raptor ubiquitination was detected using an in vitro ubiquitination assay. D To analyze the ubiquitination of endogenous Raptor, Caki cells were co-transfected with HA-Ub and OTUB1 siRNA (siOTUB1) for the indicated time points. E–G Caki cells were transiently transfected with control siRNA (siCont) and siOTUB1 (E, F), and then incubated with 0.5 µM MG132 for 6 h (G). H Caki cells were transfected with different concentrations of the Flag-HA-OTUB1 plasmid. I, J Caki cells were transfected with siCont, two different sets of siOTUB1 (I) or pcDNA3.1(+), and Flag-HA-OTUB1 plasmid (J) and then treated with 20 µg/mL cycloheximide (CHX) for the indicated time points. The band intensity of Raptor was analyzed using ImageJ. The protein and mRNA expression levels were measured using western blotting and RT-PCR/qPCR, respectively. The band intensity of all proteins was analyzed using ImageJ. The values in graphs represent the mean ± SD of three independent experiments. ^*P < 0.01 compared to the Cont siRNA-transfected cells. ^#P < 0.05 compared to the MG132 in treatment OTUB1 siRNA-transfected cells. ^**P < 0.01 compared to the compared to vector-transfected cells. ^&P < 0.01 compared to the CHX treatment siCont-transfected cells. ^†P < 0.01 compared to the CHX treatment vector-transfected cells.

Fig. 2. Tyrosine 26 (Y26) residue of OTUB1 was critical for stabilization of Raptor.

A–D HEK293 cells (A, C) and Caki cells (B, D) were co-transfected with Flag-OTUB1 WT, mutant (OTUB1 C91S and ASA) (A, B) or Flag-OTUB1/Δ1-45 (C, D) plasmids in the presence of HA-Ub plasmid, following which the cells were treated with 0.5 µM MG132 for 6 h. The ubiquitination of Raptor was detected using in vitro (A, C) /endogenous (B, D) ubiquitination assay. E Caki cells were transfected with Flag-OTUB1 WT and four different sets of OTUB1 domain deletions (Δ1-10, Δ1-20, Δ1-30 and Δ1-45) in the presence of HA-Ub. Then, the cells were treated with 0.5 µM MG132 for 6 h. The ubiquitination of endogenous Raptor was detected using the ubiquitination assay. F Caki cells were transfected with Flag-OTUB1 WT, Flag-OTUB1 2 mutants (N22A/C23A), and 3 mutants (Y26A/D27A/E28A) in the presence of HA-Ub, following which the cells were treated with MG132 for 6 h. The ubiquitination of endogenous Raptor was detected using the ubiquitination assay. G, H Caki cells were transfected with plasmids harboring Flag-OTUB1 WT and each single mutant of Flag-OTUB1 (Y26A, D27A and E28A) (G) or Flag-OTUB1 (Y26A and Y26F) (H) in the presence of HA-Ub. The ubiquitination of endogenous Raptor was detected using the ubiquitination assay. The band intensity of all proteins was analyzed using ImageJ. The values in graphs represent the mean ± SD of three independent experiments. ^*P < 0.05 compared to the vector-transfected cells. ^#P < 0.05 compared to the OTUB1 WT-transfected cells.

Fig. 3. Mutation of OTUB1 at the Y26 residue increased mitochondrial dysfunction via down-regulation of Raptor.

A Representative confocal images of Caki/pDsRed2-mito cells co-transfected with Flag-OTUB1 WT, Flag-OTUB1 Y26A and Myc-Raptor. The nuclei were stained with DAPI, and the length of the mitochondria was measured using ZEN3.4. B–D Caki cells were transiently co-transfected with Flag-OTUB1 WT, Flag-OTUB1 Y26A and Myc-Raptor plasmids. Protein expression was detected using western blotting (B). Flow cytometry was used to detect the fluorescence intensity after mitochondrial damage (C). Mitochondrial ROS production was assessed after MitoSOX Red staining using microscopy and flow cytometry (D). E, F Representative confocal images of Caki/OTUB1 knockout (KO) cells were transiently co-transfected with plasmids harboring Flag-OTUB1 WT and each single mutant of Flag-OTUB1 (Y26A and Y26E) in the presence of pDsRed2-mito (F). Protein expression was detected using western blotting (E). The nuclei were stained with DAPI, and the length of the mitochondria was measured using ZEN3.4 (F). The band intensity of all proteins was analyzed using ImageJ. The values in graph A–F represent the mean ± SEM (A, F) or mean ± SD (B–E) of three independent experiments. ^*P < 0.001 compared to the vector-transfected cells. ^#P < 0.001 compared to the Flag-OTUB1 Y26A-transfected cells. ^†P < 0.05 compared to the compared to the vector-transfected cells. ^&P < 0.05 compared to the Flag-OTUB1 Y26A-transfected cells. ^**P < 0.01 compared to the vector-transfected OTUB1 KO cells. ^##P < 0.001 compared to the vector-transfected OTUB1 KO cells.

Fig. 4. Src and SRMS kinase phosphorylated OTUB1 at Y26 residue, which determined substrate specificity.

A Caki cells were transiently transfected with vector, Flag-OTUB1 WT, and Flag-OTUB1 Y26A plasmids. B Phosphorylation of OTUB1 was detected using immunoprecipitation (IP) with anti-phospho-tyrosine antibody (4G10). C Caki cells were transiently transfected with vector, Flag-OTUB1 WT, Flag-OTUB1 Y26A, and Flag-OTUB1 Y26F plasmids. Phosphorylation of OTUB1 at Y26 was detected using western blotting with productive phospho-tyrosine 26 OTUB1 antibody. D Caki cells were transiently transfected with GFP siRNA or 85 types of tyrosine kinases siRNA. The band intensity of Raptor was analyzed using ImageJ. E Caki cells were treated with Src inhibitors or transiently transfected with Cont siRNA and Src siRNA in the presence of HA-Ub. The ubiquitination of endogenous Raptor was detected using the ubiquitination assay. F Caki cell were transiently transfected with Cont siRNA, Src siRNA, or SRMS siRNA. G Caki cells were treated with 2 µM Src inhibitor (LCB03-0100 and KB SRC 4) for the indicated time points. H Caki cells were treated with Src inhibitors or transiently transfected with Cont siRNA and Src siRNA. The interaction was demonstrated using immunoprecipitation. I Caki cells were transiently transfected with vector, Flag-OTUB1 WT, Flag-OTUB1 Y26A and Flag-OTUB1 Y26F plasmids. Protein expression was detected using western blotting. The band intensity of all proteins was analyzed using ImageJ. The values in graphs represent the mean ± SD of three independent experiments. ^*P < 0.01 compared to control. ^#P < 0.01 compared to the Flag-OTUB1 WT-transfected cells.

Fig. 5. Phosphorylation of OTUB1 at tyrosine 26 by Src kinase played a critical role in stabilizing Raptor.

A Caki cells were co-transfected with plasmids harboring Flag-OTUB1 WT and each single mutant of Flag-OTUB1 (Y26A and Y26E) with Cont or Src siRNA in the presence of HA-Ub. The ubiquitination of endogenous Raptor was detected using the ubiquitination assay. B Representative confocal images of Caki/pDsRed2-mito cells transfected with Flag-OTUB1 Y26E plasmid and then treated with 2 µM Src inhibitor (LCB03-0100 and KB SRC 4) for 24 h. The nuclei were stained with DAPI, and the length of the mitochondria was measured using ZEN3.4. C–E Caki cells were transiently transfected with Flag-OTUB1 Y26E plasmid and then treated with 2 µM Src inhibitor (LCB03-0100 and KB SRC 4) for 24 h. Flow cytometry was used to detect the fluorescence intensity after mitochondrial damage (C). Mitochondrial ROS production was assessed after MitoSOX Red staining using microscopy and flow cytometry (D). Protein expression was detected using western blotting (E). The band intensity of all proteins was analyzed using ImageJ. The values in graphs A–E represent the mean ± SEM (B) or mean ± SD (A, C–E) of three independent experiments. ^*P < 0.05 compared to control. ^#P < 0.05 compared to the Flage-OTUB1 WT-transfected cells. ^**P < 0.001 compared to vector-transfected cells. ^&P < 0.001 compared to the Src inhibitors treatment in vector-transfected cells.

Fig. 6. UBC13 was important for OTUB1-mediated stabilization of Raptor.

A Caki cells were transiently transfected with vector, Flag-OTUB1 WT, and Flag-OTUB1 Y26A plasmids. The interaction was demonstrated using immunoprecipitation. B, C Caki cells were transiently co-transfected with OTUB1 siRNA (B) or Flag-OTUB1 plasmid (C) and six types of E2 siRNA. D Caki cells were transiently transfected with Raptor and Raptor mutants (K579R, K736R, K774R, and K789R). The ubiquitination of endogenous Raptor was detected using the ubiquitination assay. E Caki cells were transiently co-transfected with OTUB1 siRNA and Raptor WT or Raptor mutants (K774R and K789R). The ubiquitination of endogenous Raptor was detected using the ubiquitination assay. F Caki cells were transiently transfected with Cont siRNA and Raptor siRNA. After 1 day, the cells were transfected with OTUB1 siRNA and Raptor or Raptor mutants (K579R, K736R, K774R, and K789R). Protein expression was detected using western blotting. The band intensity of all proteins was analyzed using ImageJ. The values in graphs represent the mean ± SD of three independent experiments. ^*P < 0.001 compared to control. ^#P < 0.05 compared to the Flag-OTUB1 Y26A-transfected cells. ^†P < 0.001 compared to the compared to the OTUB1 siRNA-transfected cells. ^&P < 0.001 compared to the Flag-OTUB1 WT plus Cont siRNA-transfected cells. ^**P < 0.01 compared to the Flag-OTUB1 WT-transfected cells. ^##P < 0.001 compared to the Flag-OTUB1 WT plus OTUB1 siRNA-transfected cells.

Fig. 7. OTUB1 Y26 residue increased sensitivity to anti-cancer drug due to degradation of Raptor.

A Caki cells were transduced with lentivirus containing either a non-target (NT) or Raptor shRNA sequence. Colony formation was measured using crystal violet staining. Cell growth was determined using cell counting. B, C Caki cells were transiently co-transfected with Raptor and Cont siRNA (B), OTUB1 siRNA (B), Flag-OTUB1WT (C), or Flag-OTUB1 Y26A (C). Colony formation was measured using crystal violet staining. Cell growth was determined using cell counting. D, E Caki (D) and A549 (E) cells were transiently transfected with vector, Flag-OTUB1 WT, and Flag-OTUB1 Y26A plasmids, and then treated with 1 µM doxorubicin and 25 µM oxaliplatin for 24 h. Apoptosis and protein expression were measured using flow cytometry and western blotting. The values in graphs A–E represent the mean ± SD of three independent experiments. ^*P < 0.01 compared to the shNT-infected cells. ^#P < 0.01 compared to the vector and Cont siRNA-transfected cells. ^†P < 0.01 compared to the vector and OTUB1 siRNA-transfected cells. ^&P < 0.01 compared to the vector^-transfected cells. ^$P < 0.01 compared to the Flag-OTUB1 Y26A-transfected cells. ^**P < 0.001 compared to the doxorubicin or oxaliplatin treatment in vector-transfected cells. ^##P < 0.01 compared to doxorubicin or oxaliplatin treatment in Flag-OTUB1 Y26A-transfected cells.

Fig. 8. Raptor expression contributed to poor prognosis of patients with RCC.

A Raptor mRNA expression in RCC cancer tissue and normal kidney using the Yusenko et al. renal cancer dataset from the Oncomine database (https://www.onco-mine.org/). B Survival analysis of the TCGA patient cohort in Oncolnc. n = 208. Patients from the TCGA cohort were divided into high and low Raptor-expressing groups based on the average Raptor expression level. C, D Investigation of protein expression in 40 paired primary renal tumor tissues and the corresponding normal adjacent tissues. E Correlation analysis of p-OTUB1/Raptor, p-Drp1/Raptor and p-OTUB1/p-Drp1 protein levels. F Scheme showing the mechanisms of OTUB1-mediated Raptor stabilization. The values in graphs A, B, D, E represent the mean ± SD of three independent experiments.