New Modes of GPCR Signalling

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Cyclin D1 Promotes Anchorage-independent Cell Survival by Inhibiting FOXO-Mediated Anoikis Lu Gan 1, Xiangliang Yang 1, Huibi Xu 1, Haojie Huang 2 1. College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China 2. Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA Aim: Adhesion to an appropriate extracellular matrix (ECM) is important for normal cells to survive, and detachment from such supportive matrices usually triggers a specific type of apoptosis termed anoikis. However, most cancer cells are commonly resistant to anoikis and can survive in the presence of an inappropriate ECM or in the absence of anchorage to the ECM. O-class Forkhead box (FOXO) transcription factors are critical regulators of diverse cellular processes, including apoptosis, cell-cycle arrest, DNA damage repair and oxidative stress resistance. In this study we determined whether FOXOs are involved in anoikis and its mechanism. Methods: Annexin V-PE apoptosis detection kit and soft agar colony formation assay were used to measure the anoikis in prostate cancer cells. FOXOs activities were determined using a generic FOXO luciferase reporter gene that contains three copies of forkhead response elements. Immunoprecipitation was used to determine the endogenous and exogenous interaction between cyclin D1 and FOXO. In vitro protein binding assay was used to confirm the interaction between the two proteins. In vitro kinase activity determined whether FOXO1 was phosphorylated by CDK4/CDK6. Results: FOXO1 and FOXO3a play an essential role in promoting cell detachment-induced anoikis. In contrast, elevated cyclin D1 inhibits anoikis. We further show that cyclin D1 interacts with FOXO proteins and impedes their transcriptional regulatory and anoikis-promoting functions. This effect of cyclin D1 requires its transcription repression domain but is independent of cyclin-dependent kinases CDK4 and CDK6. Moreover, we demonstrate that cancer-derived mutants of cyclin D1 are much more stable than wild-type cyclin D1 under anchorage-independent conditions and possess a greater antagonistic effect on FOXO-regulated anoikis and anchorage-independent growth of cancer cells. Conculsion: These data suggest that cyclin D1 may play a critical role in tumorigenesis and cancer metastasis by inhibiting the anoikis-promoting function of FOXO proteins. Keywords: Foxo; cyclin D1; Anoikis; protein-protein interaction Author: Lu Gan,PhD, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China, Tel: 86-27-87792147 E-mail: lugan@mail.hust.edu.cn
Investigate the Role of Rab27 in the Dcvs Docking and Priming Process in C. elegans Wan-juan Feng Key Laboratory of Molecular Biophysics, Ministry of Education, College of Life Science & Technology, Huazhong University of Science & Technology Objective Rab27 is a member of a large family of Ras-related small GTPases. Recent studies have suggested that Rab27 play an important role in dense-core vesicle exocytosis. However, which step does Rab27 take part in and what is the precise mechanisms by which the GTPases regulate dense-core vesicle exocytosis are not clear. Methods We have succeeded in monitoring the docking and fusion of single dense core vesicles (DCVs) employing total internal reflection fluorescence microscopy (TIRFM) in primary cultured C. elegans neurons. Results Using these approaches, we provide direct evidence that Rab27 regulates the docking step of DCVs at the plasma membrane in C. elegans. Keywords: Rab27, total internal reflection fluorescence microscopy(TIRFM), dense core vesicle(DCV) Author: E-mail: wanjuanfeng@gmail.com
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