Direct activation of purified phospholipase C epsilon by RhoA studied in reconstituted phospholipid vesicles.
Phospholipase C-epsilon (PLC-epsilon) was shown recently to be a downstream effector of Rho GTPases, and we have used an in vitro phospholipid vesicle reconstitution system with purified proteins to show this regulation to be direct. This chapter describes high-level expression of a hexahistidine-tagged fragment of PLC-epsilon encompassing the catalytic core of the enzyme through the tandem RA domains by use of a recombinant baculovirus and High Five insect cells. The recombinant protein is purified to homogeneity using metal chelate affinity and size exclusion chromatography. The small GTPase RhoA also is expressed to high levels in a lipidated form after baculovirus expression in High Five cells and is purified to near homogeneity after detergent extraction and metal chelate affinity chromatography. The capacity of GTPgammaS-bound RhoA to stimulate the phospholipase activity of PLC-epsilon is assessed by reconstitution of the RhoA in mixed-detergent phospholipid micelles containing PtdIns(4,5)P2 substrate.
Seifert
Jason P
JP
Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, USA.
Snyder
Jason T
JT
Sondek
John
J
Harden
T Kendall
TK
D000818:Animals; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004789:Enzyme Activation; D006801:Humans; D008081:Liposomes; D054801:Phosphoinositide Phospholipase C; D017368:Protein Prenylation; D010738:Type C Phospholipases / Q000302:isolation & purification* / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000502:physiology*
D008081:Liposomes; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; C423320:phospholipase C epsilon; D020742:rhoA GTP-Binding Protein