Tools to analyze protein farnesylation in cells.
Farnesylation of proteins is catalyzed by protein farnesyl transferase (FTase) and is obligatory for the function of the oncoprotein Ras and a variety of other physiologically important proteins. The rapid and selective detection of cellular protein farnesylation status is crucial to understanding both the function of farnesylated proteins and FTase inhibitors. The unnatural FPP analogue 8-anilinogeranyl diphosphate (AGPP, 3b) is an effective alternative substrate for mammalian FTase. Using antibodies specific for the anilinogeranyl moiety, we show that the alcohol precursor (AGOH, 5b) of AGPP is incorporated into cellular proteins in an FTase dependent manner competitive with endogenous pools of FPP. Continuous treatment of HEK-293 cells with 100 microM AGOH for up to 3 days is neither cytotoxic or cytostatic. Antibodies to detect the unnatural anilinogeranyl group were raised against bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) bioconjugates of the activated hapten N-hydroxyphthalimido-succinyl-(S-anilinogeranyl)-L-cysteine methyl ester 9a. Polyclonal antisera containing anti-anilinogeranyl antibodies were generated by immunization of rabbits and a monoclonal anti-anilinogeranyl antibody was raised in mice. ELISA and western blotting of anilinogeranyl modified proteins were used to show the selectivity and measure the titer of the antibodies. The unnatural FPP analogue and corresponding antibodies provide a simple and rapid method for monitoring FTase activity in cells and detection of cellular proteins modified by AGOH 5a.
Troutman
Jerry M
JM
Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky 40536-0084, USA.
Roberts
Michael J
MJ
Andres
Douglas A
DA
Spielmann
H Peter
HP
D000818:Animals; D000906:Antibodies / Q000276:immunology; D002460:Cell Line; D006241:Haptens / Q000737:chemistry; D006801:Humans; D051379:Mice; D015394:Molecular Structure; D011106:Polyisoprenyl Phosphates / Q000737:chemistry; D017368:Protein Prenylation; D011817:Rabbits; D012717:Sesquiterpenes; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism
D000906:Antibodies; D006241:Haptens; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; C004808:farnesyl pyrophosphate; D018631:ras Proteins