title: In vitro reconstitution of activation of PLCepsilon by Ras and Rho GTPases.
abstract: Phosphatidylinositol-specific phospholipase C (PLC) enzymes catalyze the hydrolysis of phophatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] to diacylglycerol (DAG) and inositol 1,4,5-triphosphate [Ins(1,4,5)P3]. PLCepsilon is a recently discovered isoform that has been shown to be activated by members of the Ras and Rho families of guanosine trisphosphatases (GTPases) as well as subunits of heterotrimeric G-proteins. We describe a method for expressing a truncated PLCepsilon variant as an MBP fusion protein in E. coli. Subsequently, we describe the methodology necessary to reconstitute this protein with K-Ras-4B and RhoA GTPases and measure its activation.
authors.affiliation: Cancer Research UK Centre for Cell and Molecular Biology, Chester Beatty Laboratories, The Institute of Cancer Research, London, UK.
authors.forename: Natalia Lamuño
authors.initials: NL
authors.lastname: Gandarillas
authors.forename: Tom D
authors.initials: TD
authors.lastname: Bunney
authors.forename: Michelle B
authors.initials: MB
authors.lastname: Josephs
authors.forename: Peter
authors.initials: P
authors.lastname: Gierschik
authors.forename: Matilda
authors.initials: M
authors.lastname: Katan
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000201:enzymology*; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D002474:Cell-Free System; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000166:cytology; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D054801:Phosphoinositide Phospholipase C / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D051381:Rats; D017384:Sequence Deletion; D012995:Solubility; D018631:ras Proteins / Q000302:isolation & purification* / Q000494:pharmacology*; D020741:rho GTP-Binding Proteins / Q000302:isolation & purification* / Q000494:pharmacology*
chemical_list: D007527:Isoenzymes; D054801:Phosphoinositide Phospholipase C; D018631:ras Proteins; D020741:rho GTP-Binding Proteins