Abstract
BackgroundAvailable in vitro and in vivo methods for verifying protein substrates for posttranslational modifications via farnesylation or geranylgeranylation (for example, autoradiography with 3H-labeled anchor precursors) are time consuming (weeks/months), laborious and suffer from low sensitivity.ResultsWe describe a new technique for detecting prenyl anchors in N-terminally glutathione S-transferase (GST)-labeled constructs of target proteins expressed in vitro in rabbit reticulocyte lysate and incubated with 3H-labeled anchor precursors. Alternatively, hemagglutinin (HA)-labeled constructs expressed in vivo (in cell culture) can be used. For registration of the radioactive marker, we propose to use a thin layer chromatography (TLC) analyzer. As a control, the protein yield is tested by Western blotting with anti-GST- (or anti-HA-) antibodies on the same membrane that has been previously used for TLC-scanning. These protocols have been tested with Rap2A, v-Ki-Ras2 and RhoA (variant RhoA63L) including the necessary controls. We show directly that RasD2 is a farnesylation target.ConclusionSavings in time for experimentation and the higher sensitivity for detecting 3H-labeled lipid anchors recommend the TLC-scanning method with purified GST- (or HA-) tagged target proteins as the method of choice for analyzing their prenylation capabilities in vitro and in vivo and, possibly, also for studying the myristoyl and palmitoyl posttranslational modifications.
Highlights
Available in vitro and in vivo methods for verifying protein substrates for posttranslational modifications via farnesylation or geranylgeranylation are time consuming, laborious and suffer from low sensitivity
Optimization of experimental parameters and analysis of the prenylation behaviour of the protein Rap2A The proposed new procedure starts with a polymerase chain reaction (PCR)-amplification of the glutathione Stransferase (GST)-Rap2A open reading frame (Genbank accession of Rap2A BC070031) followed by in vitro transcription and translation using rabbit reticulocyte lysate in the presence of a 3H-labeled isoprenoid donor
Detection of incorporated radioactive label is performed by scanning with the thin layer chromatography (TLC) analyzer
Summary
Available in vitro and in vivo methods for verifying protein substrates for posttranslational modifications via farnesylation or geranylgeranylation (for example, autoradiography with 3H-labeled anchor precursors) are time consuming (weeks/months), laborious and suffer from low sensitivity. Several other CaaX proteins from the Rho family of GTPases [25,26] and Rap1A [27] are involved in tumorigenesis as well Since their lipid modifications are essential for their biological function [10,28,29,30,31], inhibitors of prenyltransferases (PTases), especially of FTase [32,33,34] attracted the interest of pharmaceutical research as anticancer drugs. Two such compounds made it to phase III trials [35,36]. There is evidence that inhibitors of prenylation may be useful in the treatment of other diseases such as infestation with protozoa [6,37]
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