In vitro reconstitution of activation of PLCepsilon by Ras and Rho GTPases

Methods Mol Biol. 2009:462:379-89. doi: 10.1007/978-1-60327-115-8_24.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Baculoviridae / enzymology*
  • Cell Line
  • Cell Membrane / chemistry
  • Cell-Free System
  • Chromatography, Affinity
  • Chromatography, Gel
  • Enzyme Activation / drug effects
  • Escherichia coli / cytology
  • Isoenzymes / genetics
  • Isoenzymes / isolation & purification
  • Isoenzymes / metabolism
  • Phosphoinositide Phospholipase C / genetics
  • Phosphoinositide Phospholipase C / isolation & purification*
  • Phosphoinositide Phospholipase C / metabolism*
  • Rats
  • Sequence Deletion
  • Solubility
  • ras Proteins / isolation & purification*
  • ras Proteins / pharmacology*
  • rho GTP-Binding Proteins / isolation & purification*
  • rho GTP-Binding Proteins / pharmacology*

Substances

  • Isoenzymes
  • Phosphoinositide Phospholipase C
  • ras Proteins
  • rho GTP-Binding Proteins