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Gankyrin plays an essential role in Ras-induced tumorigenesis through regulation of the RhoA/ROCK pathway in mammalian cells
Jiang-Hong Man, … , Hui-Yan Li, Xue-Min Zhang
Jiang-Hong Man, … , Hui-Yan Li, Xue-Min Zhang
Published July 12, 2010
Citation Information: J Clin Invest. 2010;120(8):2829-2841. https://doi.org/10.1172/JCI42542.
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Research Article Oncology

Gankyrin plays an essential role in Ras-induced tumorigenesis through regulation of the RhoA/ROCK pathway in mammalian cells

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Abstract

Activating mutations in Ras proteins are present in about 30% of human cancers. Despite tremendous progress in the study of Ras oncogenes, many aspects of the molecular mechanisms underlying Ras-induced tumorigenesis remain unknown. Through proteomics analysis, we previously found that the protein Gankyrin, a known oncoprotein in hepatocellular carcinoma, was upregulated during Ras-mediated transformation, although the functional consequences of this were not clear. Here we present evidence that Gankyrin plays an essential role in Ras-initiated tumorigenesis in mouse and human cells. We found that the increased Gankyrin present following Ras activation increased the interaction between the RhoA GTPase and its GDP dissociation inhibitor RhoGDI, which resulted in inhibition of the RhoA effector kinase Rho-associated coiled coil–containing protein kinase (ROCK). Importantly, Gankyrin-mediated ROCK inhibition led to prolonged Akt activation, a critical step in activated Ras–induced transformation and tumorigenesis. In addition, we found that Gankyrin is highly expressed in human lung cancers that have Ras mutations and that increased Gankyrin expression is required for the constitutive activation of Akt and tumorigenesis in these lung cancers. Our findings suggest that Gankyrin is a key regulator of Ras-mediated activation of Akt through inhibition of the downstream RhoA/ROCK pathway and thus plays an essential role in Ras-induced tumorigenesis.

Authors

Jiang-Hong Man, Bing Liang, Yue-Xi Gu, Tao Zhou, Ai-Ling Li, Tao Li, Bao-Feng Jin, Bing Bai, Hai-Ying Zhang, Wei-Na Zhang, Wei-Hua Li, Wei-Li Gong, Hui-Yan Li, Xue-Min Zhang

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Figure 4

Gankyrin mediates the interaction of RhoA and RhoGDI1 and regulates Akt phosphorylation through the RhoA/ROCK/PTEN signaling pathway.

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Gankyrin mediates the interaction of RhoA and RhoGDI1 and regulates Akt ...
(A) Serum-starved HEK293 cells, transfected with control or Gankyrin siRNA, were treated with EGF for the indicated times. The lysates of these cells were subjected to immunoprecipitation with indicated antibodies. The relative amount of the coimmunoprecipitated RhoA normalized to the immunoprecipitated RhoGDI, in 0 minutes, is designated as 1.0, and the numbers under the first row indicate the relative amount of RhoA for other time points. (B) As described in Figure 2A, NIH3T3 cells were induced with 4-OHT for the indicated times. The lysates of these cells were subjected to immunoprecipitation with anti-RhoGDI1 antibody and immunoblotting with indicated antibodies. (C) SDS-PAGE and autoradiography of NIH3T3-ER:RasG12V-control shRNA and Gankyrin shRNA cells, which were serum starved overnight and treated for 0–48 hours with 4-OHT. Extracts immunoprecipitated with anti-ROCK2 were incubated with a substrate of a fusion protein of GST-MYPT1 (amino acids 654–880). Phosphorylation of MYPT1 was then detected by autoradiography. (D) RhoA WT or RhoA Q63L mutant were overexpressed with Flag-Gankyrin or vector through infection with retroviral vectors in NIH3T3 cells. Extracts immunoprecipitated with anti-ROCK2 were incubated with a substrate of a fusion protein of GST-MYPT1. We then detected the phosphorylation of MYPT1 with the indicated phospho-specific antibodies. (E) MEF-Pten WT and MEF-Pten knockout cells stably expressed Gankyrin through infection with a retroviral vector encoding human Gankyrin. After that, the cells were serum starved overnight and then treated with EGF for the indicated times. The lysates of these cells were analyzed by immunoblot using indicated antibodies.
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