The role of GEF-H1 regulation in RhoA signaling pathway in myeloid leukemia cells

• Main Authors: Yuan-Chen Chang, 張元貞
• Other Authors: Zee-Fen Chang
• Format: Others
• Language: en_US
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/87331792688232513095

碩士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 92 === Phorbol-12-myristate-13-acetate (PMA) induces proliferation, differentiation, or apoptosis depending on the cell type. In the case of the erythroblastic D2 cell line, which is a cytokine-independent variant derived from TF-1 cells, PMA treatment induces a half of D2 cells adhesion and differentiates into monocyte/macrophage-like morphology, while the other population remains in suspension and undergoes apoptosis. In our laboratory, we have shown that RhoA acts as a switcher in determining whether D2 cells undergo differentiation or apoptosis in response to PMA. RhoA activity is downregulated in PMA-induced adherent cells, while in suspended cells RhoA activity remains elevated. In this study, I aimed to understand the mechanism responsible for differential regulation of RhoA in D2 cells upon PMA treatment. By using the strategy of GST-RhoA affinity capture, we identified protein GEF-H1 that was specifically interacting with RhoA in D2 cells. It has been reported that majority of GEF-H1 is colocalized with microtubules when expressed in cultured mammalian cells. However, in my study I found that subcellular distribution of GEF-H1 was mainly in the cytosolic fraction but not bound to microtubules in D2 cells. In PMA-induced attached cells, GEF-H1 became associated with microtubules, whereas in PMA-induced suspension cells GEF-H1 remained in the cytosol. Since GEF-H1 is a GEF specific for RhoA, I proposed that the elevated level of GEF-H1 in cytosolic fraction plays a role in modulating RhoA activity, contributing to PMA-induced apoptosis. In support of this hypothesis, I found that overexpression of active form of GEF-H1, which lacks microtubule binding domain, increased PMA-induced apoptosis in D2 cells, and that knockdown of GEF-H1 decreased RhoA activity and PMA-induced apoptosis concomitantly. In summary, I discovered that the subcellular localization of GEF-H1 is differently regulated in PMA-induced suspended and attached D2 cells. This difference takes a part in modulation of RhoA activity, thus affecting cell spreading and survival in response to PMA stimulation.