Probing adenylation: using a fluorescently labelled ATP probe to directly label and immunoprecipitate VopS substrates.
Department of Biochemistry and Molecular Pharmacology
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
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.
DOI of Published Version
Mol Biosyst. 2012 Jun;8(6):1701-6. doi: 10.1039/c2mb25053e. Epub 2012 Mar 28. Link to article on publisher's site
Lewallen, Daniel M.; Steckler, Caitlin J.; Knuckley, Bryan; Chalmers, Michael J.; and Thompson, Paul R., "Probing adenylation: using a fluorescently labelled ATP probe to directly label and immunoprecipitate VopS substrates." (2012). Thompson Lab Publications. 36.