A tagging-via-substrate approach to detect the farnesylated proteome using two-dimensional electrophoresis coupled with Western blotting.
Prenylation is a post-translational modification critical for the proper function of multiple physiologically important proteins, including small G-proteins, such as Ras. Methods allowing rapid and selective detection of protein farnesylation and geranylgeranylation are fundamental for the understanding of prenylated protein function and for monitoring efficacy of drugs such as farnesyltransferase inhibitors (FTIs). Although the natural substrates for prenyltransferases are farnesyl pyrophosphate and geranylgeranyl pyrophosphate, farnesyltransferase has been shown to incorporate isoprenoid analogues into protein substrates. In this study, protein prenyltransferase targets were labeled using anilinogeraniol, the alcohol precursor to the unnatural farnesyl pyrophosphate analogue 8-anilinogeranyl diphosphate in a tagging-via-substrate approach. Antibodies specific for the anilinogeranyl moiety were used to detect the anilinogeranyl-modified proteins. Coupling this highly effective labeling/detection method with two-dimensional electrophoresis and subsequent Western blotting allowed simple, rapid analysis of the complex farnesylated proteome. For example, this method elucidated the differential effects induced by two chemically distinct FTIs, BMS-214,662 and L-778,123. Although both FTIs strongly inhibited farnesylation of many proteins such as Lamins, NAP1L1, N-Ras, and H-Ras, only the dual prenylation inhibitor L-778,123 blocked prenylation of Pex19, RhoB, K-Ras, Cdc42, and Rap1. This snapshot approach has significant advantages over traditional techniques, including radiolabeling, anti-farnesyl antibodies, or mass spectroscopy, and enables dynamic analysis of the farnesylated proteome.
Onono
Fredrick O
FO
Department of Hematology, Hemostasis, Oncology, and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany.
Morgan
Michael A
MA
Spielmann
H Peter
HP
Andres
Douglas A
DA
Subramanian
Thangaiah
T
Ganser
Arnold
A
Reuter
Christoph W M
CW
D000814:Aniline Compounds / Q000494:pharmacology; D015153:Blotting, Western / Q000379:methods; D045744:Cell Line, Tumor; D015180:Electrophoresis, Gel, Two-Dimensional; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051231:Farnesyltranstransferase / Q000037:antagonists & inhibitors / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D020014:K562 Cells; D008954:Models, Biological; D017368:Protein Prenylation; D020543:Proteome / Q000032:analysis* / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods*; D013379:Substrate Specificity
D000814:Aniline Compounds; D004791:Enzyme Inhibitors; D007093:Imidazoles; D020543:Proteome; C425231:L 778,123; D051231:Farnesyltranstransferase