Prohibitin regulates mTOR pathway via interaction with FKBP8

doi:10.1007/s11684-020-0805-6

Front. Med.

2021, Vol. 15

Issue (3) : 448-459 https://doi.org/10.1007/s11684-020-0805-6

RESEARCH ARTICLE

Prohibitin regulates mTOR pathway via interaction with FKBP8

Jiahui Zhang¹, Yanan Yin¹, Jiahui Wang¹, Jingjing Zhang², Hua Liu², Weiwei Feng², Wen Yang¹, Bruce Zetter³(

), Yingjie Xu¹(

)

¹. Department of Biochemistry and Molecular and Cell Biology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
². Department of Obstetrics and Gynecology, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
³. Vascular Biology Program, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA

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Abstract

The ability of tumor cells to sustain continuous proliferation is one of the major characteristics of cancer. The activation of oncogenes and the mutation or inactivation of tumor suppressor genes ensure the rapid proliferation of tumor cells. The PI3K--Akt--mTOR axis is one of the most frequently modified signaling pathways whose activation sustains cancer growth. Unsurprisingly, it is also one of the most commonly attempted targets for cancer therapy. FK506 binding protein 8 (FKBP8) is an intrinsic inhibitor of mTOR kinase that also exerts an anti-apoptotic function. We aimed to explain these contradictory aspects of FKBP8 in cancer by identifying a “switch” type regulator. We identified through immunoprecipitation--mass spectrometry-based proteomic analysis that the mitochondrial protein prohibitin 1 (PHB1) specifically interacts with FKBP8. Furthermore, the downregulation of PHB1 inhibited the proliferation of ovarian cancer cells and the mTOR signaling pathway, whereas the FKBP8 level in the mitochondria was substantially reduced. Moreover, concomitant with these changes, the interaction between FKBP8 and mTOR substantially increased in the absence of PHB1. Collectively, our finding highlights PHB1 as a potential regulator of FKBP8 because of its subcellular localization and mTOR regulating role.

Keywords prohibitin 1 FKBP8 mTOR cell proliferation cancer

Corresponding Author(s): Bruce Zetter,Yingjie Xu

Just Accepted Date: 27 October 2020 Online First Date: 11 January 2021 Issue Date: 18 June 2021

Cite this article:

Jiahui Zhang,Yanan Yin,Jiahui Wang, et al. Prohibitin regulates mTOR pathway via interaction with FKBP8[J]. Front. Med., 2021, 15(3): 448-459.

URL:

https://academic.hep.com.cn/fmd/EN/10.1007/s11684-020-0805-6
https://academic.hep.com.cn/fmd/EN/Y2021/V15/I3/448

Fig.1 FKBP8 is localized in the mitochondria through its C terminal and the overexpression of non-mitochondrial-localized FKBP8 inhibits the mTOR pathway. (A) HEK293 cells were stably transfected with C-terminal HA-tagged FKBP8, incubated with 200 nmol/L Mito-tracker green for 45 min, and subjected to immunofluorescence assay with anti-HA (red) antibody. Nuclei were stained with DAPI (blue). Scale bar, 10?mm. (B) Fluorescence analysis of HeLa cells stably transfected with N-terminal EGFP-tagged FKBP8 and stained by Mito-tracker red. Nuclei are stained with DAPI (blue). Scale bar, 10?mm. (C) SK-OV-3 cells were incubated with 200 nmol/L Mito-tracker green for 45 min and subjected to immunofluorescence assay with anti-FKBP8 (red) antibody. Nuclei were stained with DAPI (blue). Scale bar, 10?mm. (D) HEK293 cells were transfected with plasmid expressing C-terminal HA-tagged FKBP8 for 48 h followed by serum starvation overnight. Proteins were extracted and subjected to Western blot analysis. mTOR pathway signaling was assessed by p-P70S6K. AKT was used as loading control. (E) HEK293 cells were transfected with plasmid expressing N-terminal EGFP-tagged FKBP8 for 48 h and subjected to serum starvation overnight. Proteins were extracted and subjected to Western blot analysis. mTOR pathway signaling was assessed by p-mTOR and p-P70S6K. GAPDH was used as loading control.

Fig.2 FKBP8 interacts with PHB1. (A) IP–MS of FKBP8-HA in HEK293 cells. Lysates of PHB-HA-expressing cells were immunoprecipitated with HA resin. Bait complexes were analyzed by LC–MS/MS. High-confidence interacting proteins had normalized weighted D scores>1 and Z-scores>4. (B) HEK293 cells were transiently transfected with PHB1-HA and EGFP-FKBP8 plasmids. Proteins were subjected to IP using HA magnetic beads, and co-precipitation FKBP8 (EGFP) was detected by Western blot. Whole cell lysates (input) showed that all plasmids were expressed in the transfected cells. (C) HEK293 cells were transfected with FKBP8-HA plasmid, proteins were subjected to IP using HA magnetic beads, and co-precipitation endogenous PHB1 was detected by Western blot. (D) Immunofluorescent staining of endogenous FKBP8 (red) and PHB1 (green) in Mes-Sa cells. Nuclei were stained with DAPI. Scale bar, 10?mm. (E) In situ PLA between FKBP8 and PHB1 was performed with mouse anti-FKBP8 and rabbit anti-PHB1 antibodies using Duolink PLA technology. Each PLA signal (red fluorescent puncta) is indicative of one detected FKBP8–PHB1 interaction event in Mes-Sa cells. Red, PLA signal; blue, DAPI; green, Mito-tracker green. Scale bar, 10?mm.

Fig.3 Silencing of PHB1 decreases cell proliferation and colony formation abilities. (A) SK-OV-3 cells were transfected with siPHB1 for 72 and 96 h, and then proteins were extracted and subjected to Western blot analysis. (B) MTT assay showed that cell proliferation ability was inhibited in SK-OV-3 siPHB1 cells compared with siCTL cells. Data are shown as mean±SD. ***P<0.001. (C) Colony forming assay showed decreased colony formation and colony cell number in HeLa cells transfected with siPHB1, and PHB1 protein level was determined by Western blot. Data are shown as mean±SD. **P<0.01. (D) HeLa cells were transfected with siPHB1, incubated in the absence of serum for 20 h, and stimulated with 150 nmol/L insulin for 15 or 30 min, and then proteins were extracted and subjected to Western blot analysis. (E) Mes-Sa and Mes-Sa-Dx5 cells were transfected with siPHB1 and treated with 250 or 500 nmol/L paclitaxel for 24 h, and then proteins were extracted and subjected to Western blot analysis.

Fig.4 PHB1 knockdown results in the release of FKBP8 from the mitochondria and increased interaction with mTOR. (A) HeLa cells were transfected with siPHB1, incubated in the absence of serum for 20 h, and stimulated with 150 nmol/L insulin for 30 min, and then proteins were extracted and subjected to Western blot analysis. (B) OVCAR5 cells were transfected with siPHB1 and treated with 250 nmol/L paclitaxel overnight. Mitochondrial fraction and cytosol were isolated, and the amounts of PHB1 and FKBP8 were analyzed by Western blot. Transketolase was used as cytosol loading control. (C) HeLa cells were transfected with siPHB1 or siCTL and cultured under normal condition, and proteins were extracted 72 h post-transfection and subjected to Western blot analysis. (D) qPCR analysis of the mRNA level of FKBP8 in HeLa cells after siPHB1transfection. GAPDH was used as internal reference. Data represent the mean±SD of three independent experiments; two-tailed unpaired t-test, ****P<0.0001; N.S., no significance. (E) HeLa cells were transfected with siPHB1 for 72 h and incubated with 20 mmol/L MG132 for 8 h, and then proteins were extracted and subjected to Western blot analysis. (F) Mes-Sa cells were transfected with siPHB1, proteins were subjected to IP using an endogenous mTOR antibody, and co-precipitation FKBP8 was detected by Western blot.

Fig.5 PHB1 protein expression was detected in ovarian cancer (A) and normal tissues (B) by IHC staining. Boxed areas are enlarged below each image. Scale bars, 100 µm.

Tab.1 Correlation analysis of PHB1 expression with ovarian tumor tissues

Tab.2 Correlation analysis of PHB1 expression with pathology, stage, and treatment of patients with ovarian cancer

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