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Raf activation by Ras and promotion of cellular metastasis require phosphorylation of prohibitin in the raft domain of the plasma membrane

Oncogene volume 32, pages 777–787 (2013)Cite this article

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Abstract

Prohibitin (PHB) is indispensable for Ras-induced Raf-1 activation, cell migration and growth; however, the exact role of PHB in the molecular pathogenesis of cancer metastasis remains largely unexamined. Here, we found a positive correlation between plasma membrane-associated PHB and the clinical stages of cancer. The level of PHB phosphorylated at threonine 258 (T258) and tyrosine 259 (Y259) in human cancer-cell membranes correlated with the invasiveness of cancer cells. Overexpression of phosphorylated PHB (phospho-PHB) in the lipid-raft domain of the cell membrane enhanced cell migration/invasion through PI3K/Akt and Raf-1/ERK activation. It also enhanced epithelial–mesenchymal transition, matrix metalloproteinase-2 activity and invasiveness of cancer cells in vitro. Immunoprecipitation analysis demonstrated that phospho-PHB associated with Raf-1, Akt and Ras in the membrane and was essential for the activation of Raf-1 signaling by Ras. Mice implanted with cancer cells stably overexpressing PHB in the plasma membrane showed enlarged cervical tumors, enhanced metastasis and shorter survival time compared with mice implanted with cancer cells without PHB overexpression. Dephosphorylation of PHB at T258 by site-directed mutagenesis diminished the in vitro and in vivo effects of PHB. These results suggest that increase in phospho-PHB T258 in the raft domain of the plasma membrane has a role in the Ras-driven activation of PI3K/Akt and Raf-1/ERK-signaling cascades and results in the promotion of cancer metastasis.

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Acknowledgements

This work was supported by the National Science Council, Taiwan (grants NSC 96-2313-B-001-005-MY3 and NSC 99-2313-B-001-004-MY3 to Shu-Mei Liang), and Academia Sinica (to Shu-Mei Liang and Chi-Ming Liang). We thank Chi-Ying Huang of National Yang Ming University, Taiwan for designing phosphorylated peptides to make antibodies against phospho-PHB. We also thank Shu-Chen Shen and Tzu-Wen Tai of the Scientific Instrument Center, Academia Sinica for their assistance with confocal microscopy analyses and flow cytometric analysis.

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Authors and Affiliations

  1. Institute of Biotechnology, College of Bioscience and Biotechnology, National Cheng Kung University, Tainan, Taiwan

    C-F Chiu & S-M Liang

  2. Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan

    C-F Chiu, S-W Hung & S-M Liang

  3. Genomics Research Center, Academia Sinica, Taipei, Taiwan

    M-Y Ho, J-M Peng, C-M Liang & S-M Liang

  4. Department of Pathology, Shuang Ho Hospital, Taipei Medical University, Taipei, Taiwan

    W-H Lee

  5. Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan

    C-M Liang

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  1. C-F Chiu
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Correspondence to C-M Liang or S-M Liang.

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Chiu, CF., Ho, MY., Peng, JM. et al. Raf activation by Ras and promotion of cellular metastasis require phosphorylation of prohibitin in the raft domain of the plasma membrane. Oncogene 32, 777–787 (2013). https://doi.org/10.1038/onc.2012.86

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