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    Probing adenylation: using a fluorescently labelled ATP probe to directly label and immunoprecipitate VopS substrates.

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    Authors
    Lewallen, Daniel M.
    Steckler, Caitlin J.
    Knuckley, Bryan
    Chalmers, Michael J.
    Thompson, Paul R
    UMass Chan Affiliations
    Department of Biochemistry and Molecular Pharmacology
    Document Type
    Journal Article
    Publication Date
    2012-06-01
    Keywords
    Adenosine Triphosphate
    Bacterial Proteins
    Cell Line, Tumor
    Fluorescent Dyes
    Humans
    Immunoprecipitation
    Kinetics
    Limit of Detection
    Signal Transduction
    Vibrio parahaemolyticus
    cdc42 GTP-Binding Protein
    Biochemistry
    Enzymes and Coenzymes
    Medicinal-Pharmaceutical Chemistry
    Therapeutics
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    Link to Full Text
    http://dx.doi.org/10.1039/c2mb25053e
    Abstract
    The bacterial effector VopS from Vibrio parahaemolyticus modifies host Rho GTPases to prevent downstream signalling, which leads to cell rounding and eventually apoptosis. While previous studies have used [alpha-(32)P] ATP for studying this enzyme, we sought to develop a non-radioactive chemical probe of VopS function. To guide these studies, the kinetic parameters were determined for a variety of nucleotides and the results indicated that the C6 position of adenosine was amenable to modification. Since Fl-ATP is a commercially available ATP analogue that is fluorescently tagged at the C6 position, we tested it as a VopS substrate, and the results show that VopS uses Fl-ATP to label Cdc42 in vitro and in MCF7 whole cell extracts. The utility of this probe was further demonstrated by immunoprecipitating Fl-ATP labeled Cdc42 as well as several novel substrate proteins. The proteins, which were identified by LC-MS/MS, include the small GTPases Rac1 and Cdc42 as well as several proteins that are potential VopS substrates and may be important for V. parahaemolyticus pathology. In total, these studies identify Fl-ATP as a valuable chemical probe of protein AMPylation.
    Source
    Mol Biosyst. 2012 Jun;8(6):1701-6. doi: 10.1039/c2mb25053e. Epub 2012 Mar 28. Link to article on publisher's site
    DOI
    10.1039/c2mb25053e
    Permanent Link to this Item
    http://hdl.handle.net/20.500.14038/50026
    Notes

    At the time of publication, Paul Thompson was not yet affiliated with UMass Medical School.

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    Link to Article in PubMed
    ae974a485f413a2113503eed53cd6c53
    10.1039/c2mb25053e
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