Measurement of protein farnesylation and geranylgeranylation in vitro, in cultured cells and in biopsies, and the effects of prenyl transferase inhibitors.
The importance of the post-translational lipid modifications farnesylation and geranylgeranylation in protein localization and function coupled with the critical role of prenylated proteins in malignant transformation has prompted interest in their biology and the development of farnesyl transferase and geranylgeranyl transferase inhibitors (FTIs and GGTIs) as chemical probes and anticancer agents. The ability to measure protein prenylation before and after FTI and GGTI treatment is important to understanding and interpreting the effects of these agents on signal transduction pathways and cellular phenotypes, as well as to the use of prenylation as a biomarker. Here we describe protocols to measure the degree of protein prenylation by farnesyl transferase or geranylgeranyl transferase in vitro, in cultured cells and in tumors from animals and humans. The assays use [(3)H]farnesyl diphosphate and [(3)H]geranylgeranyl diphosphate, electrophoretic mobility shift, membrane association using subcellular fractionation or immunofluorescence of intact cells, [(3)H]mevalonic acid labeling, followed by immunoprecipitation and SDS-PAGE, and in vitro transcription, translation and prenylation in reticulocyte lysates. These protocols require from 1 d (enzyme assays) to up to 3 months (autoradiography of [(3)H]-labeled proteins).
Department of Drug Discovery, Moffitt Cancer Center, Tampa, Florida, USA.
Norbert
N
Berndt
Saïd M
SM
Sebti
D000818:Animals; D001706:Biopsy; D002478:Cells, Cultured; D051231:Farnesyltranstransferase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D051232:Geranylgeranyl-Diphosphate Geranylgeranyltransferase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D050356:Lipid Metabolism; D008322:Mammals; D017368:Protein Prenylation / Q000502:physiology*; D021381:Protein Transport
D051231:Farnesyltranstransferase; D051232:Geranylgeranyl-Diphosphate Geranylgeranyltransferase