In vitro reconstitution of activation of PLCepsilon by Ras and Rho GTPases.
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.
Gandarillas
Natalia Lamuño
NL
Cancer Research UK Centre for Cell and Molecular Biology, Chester Beatty Laboratories, The Institute of Cancer Research, London, UK.
Bunney
Tom D
TD
Josephs
Michelle B
MB
Gierschik
Peter
P
Katan
Matilda
M
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*
D007527:Isoenzymes; D054801:Phosphoinositide Phospholipase C; D018631:ras Proteins; D020741:rho GTP-Binding Proteins