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Berlin Brandenburg

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  • 1
    In: Traffic, June 2010, Vol.11(6), pp.813-826
    Description: Fibroblast growth factor 2 (FGF2) is a potent mitogen that is exported from cells by an endoplasmic reticulum (ER)/Golgi‐independent mechanism. Unconventional secretion of FGF2 occurs by direct translocation across plasma membranes, a process that depends on the phosphoinositide phosphatidylinositol 4,5‐biphosphate (PI(4,5)P2) at the inner leaflet as well as heparan sulfate proteoglycans at the outer leaflet of plasma membranes; however, additional core and regulatory components of the FGF2 export machinery have remained elusive. Here, using a highly effective RNAi screening approach, we discovered Tec kinase as a novel factor involved in unconventional secretion of FGF2. Tec kinase does not affect FGF2 secretion by an indirect mechanism, but rather forms a heterodimeric complex with FGF2 resulting in phosphorylation of FGF2 at tyrosine 82, a post‐translational modification shown to be essential for FGF2 membrane translocation to cell surfaces. Our findings suggest a crucial role for Tec kinase in regulating FGF2 secretion under various physiological conditions and, therefore, provide a new perspective for the development of a novel class of antiangiogenic drugs targeting the formation of the FGF2/Tec complex.
    Keywords: Fgf2 ; Membrane Translocation ; Non‐Classical Export ; Phosphorylation ; Rnai Screening ; Tec Kinase ; Unconventional Protein Secretion
    ISSN: 1398-9219
    E-ISSN: 1600-0854
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  • 2
    Language: English
    In: Journal of cell science, 01 June 2014, Vol.127(Pt 11), pp.2433-47
    Description: α2β1 integrin is one of the most important collagen-binding receptors, and it has been implicated in numerous thrombotic and immune diseases. α2β1 integrin is a potent tumour suppressor, and its downregulation is associated with increased metastasis and poor prognosis in breast cancer. Currently, very little is known about the mechanism that regulates the cell-surface expression and trafficking of α2β1 integrin. Here, using a quantitative fluorescence-microscopy-based RNAi assay, we investigated the impact of 386 cytoskeleton-associated or -regulatory genes on α2 integrin endocytosis and found that 122 of these affected the intracellular accumulation of α2 integrin. Of these, 83 were found to be putative regulators of α2 integrin trafficking and/or expression, with no observed effect on the internalization of epidermal growth factor (EGF) or transferrin. Further interrogation and validation of the siRNA screen revealed a role for KIF15, a microtubule-based molecular motor, as a significant inhibitor of the endocytic trafficking of α2 integrin. Our data suggest a novel role for KIF15 in mediating plasma membrane localization of the alternative clathrin adaptor Dab2, thus impinging on pathways that regulate α2 integrin internalization.
    Keywords: Dab2 ; Endocytic Trafficking ; Integrin ; Kif15 ; Adaptor Proteins, Signal Transducing -- Metabolism ; Breast Neoplasms -- Genetics ; Cell Membrane -- Metabolism ; Integrin Alpha2beta1 -- Metabolism ; Kinesin -- Metabolism ; Tumor Suppressor Proteins -- Metabolism
    ISSN: 00219533
    E-ISSN: 1477-9137
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  • 3
    Language: English
    In: Cell Host & Microbe, 2011, Vol.9(1), pp.32-45
    Description: Hepatitis C virus (HCV) is a major causative agent of chronic liver disease in humans. To gain insight into host factor requirements for HCV replication, we performed a siRNA screen of the human kinome and identified 13 different kinases, including phosphatidylinositol-4 kinase III alpha (PI4KIIIα), as being required for HCV replication. Consistent with elevated levels of the PI4KIIIα product phosphatidylinositol-4-phosphate (PI4P) detected in HCV-infected cultured hepatocytes and liver tissue from chronic hepatitis C patients, the enzymatic activity of PI4KIIIα was critical for HCV replication. Viral nonstructural protein 5A (NS5A) was found to interact with PI4KIIIα and stimulate its kinase activity. The absence of PI4KIIIα activity induced a dramatic change in the ultrastructural morphology of the membranous HCV replication complex. Our analysis suggests that the direct activation of a lipid kinase by HCV NS5A contributes critically to the integrity of the membranous viral replication complex. ► 13 human kinases, including PI4KIIIα, are required for HCV replication ► HCV NS5A recruits PI4KIIIα to replication sites & stimulates its kinase activity ► PI4P levels are elevated in HCV-containing cells in vitro and in vivo ► Activity of PI4KIIIα is critical for the integrity of viral replication sites
    Keywords: Biology
    ISSN: 1931-3128
    E-ISSN: 1934-6069
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  • 4
    Language: English
    In: Biotechnology Journal, January 2010, Vol.5(1), pp.39-49
    Description: RNA interference (RNAi) has emerged as a powerful technique for studying loss‐of‐function phenotypes by specific down‐regulation of gene expression, allowing the investigation of virus‐host interactions by large‐scale high‐throughput RNAi screens. Here we present a robust and sensitive small interfering RNA screening platform consisting of an experimental setup, single‐cell image and statistical analysis as well as bioinformatics. The workflow has been established to elucidate host gene functions exploited by viruses, monitoring both suppression and enhancement of viral replication simultaneously by fluorescence microscopy. The platform comprises a two‐stage procedure in which potential host factors are first identified in a primary screen and afterwards re‐tested in a validation screen to confirm true positive hits. Subsequent bioinformatics allows the identification of cellular genes participating in metabolic pathways and cellular networks utilised by viruses for efficient infection. Our workflow has been used to investigate host factor usage by the human immunodeficiency virus‐1 (HIV‐1), but can also be adapted to other viruses. Importantly, we expect that the description of the platform will guide further screening approaches for virus‐host interactions. The ViroQuant‐CellNetworks RNAi Screening core facility is an integral part of the recently founded BioQuant centre for systems biology at the University of Heidelberg and will provide service to external users in the near future.
    Keywords: High‐Throughput Screening ; Hiv ; Rna Interference ; Small Interfering Rna
    ISSN: 1860-6768
    E-ISSN: 1860-7314
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