Protein Protocols Protein Protocols

Radiolabeled Prenyl Alcohols and Anaolgs 641
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Incorporation of Radiolabeled Prenyl Alcohols
and Their Analogs into Mammalian Cell Proteins
A Useful Tool for Studying Protein Prenylation
Alberto Corsini, Christopher C. Farnsworth, Paul McGeady,
Michael H. Gelb, and John A. Glomset
1. Introduction
Prenylated proteins comprise a diverse family of proteins that are posttranslationally
modified by either a farnesyl group or one or more geranylgeranyl groups (1–3). Recent
studies suggest that members of this family are involved in a number of cellular processes,
including cell signaling (4–6), differentiation (7–9), proliferation (10–12),
cytoskeletal dynamics (13–15), and endocytic and exocytic transport (4,16,17). The
authors’ studies have focused on the role of prenylated proteins in the cell cycle (18).
Exposure of cultured cells to competitive inhibitors (statins) of 3-hydroxy-3methylglutaryl
Coenzyme A (HMG-CoA) reductase not only blocks the biosynthesis
of mevalonic acid (MVA), the biosynthetic precursor of both farnesyl and geranylgeranyl
groups, but pleiotropically inhibits DNA replication and cell-cycle progression (10,18–20).
Both phenomena can be prevented by the addition of exogenous MVA (10,18,19). The
authors have observed that all-trans-geranylgeraniol (GGOH) and, in a few cases,
all-trans-farnesol (FOH) can prevent the statin-induced inhibition of DNA synthesis
(21). In an effort to understand the biochemical basis of these effects, the authors have
developed methods for the labeling and two-dimensional gel analysis of prenylated
proteins that should be widely applicable.
Because of the relative diversity of prenylated proteins, it is important to use analytical
methods that differentiate between them. A useful approach discussed here is to
selectively label farnesylated or geranylgeranylated proteins using [ 3H] labeled FOH
or GGOH (22–24), followed by one-dimensional SDS-PAGE or high-resolution twodimensional
gel electrophoresis (2DE) of the labeled proteins. Since the enzymes that
transfer prenyl groups to proteins utilize the corresponding prenyl alcohol pyrophosphate
(FPP or GGPP) as substrate, these prenols are thought to undergo two phosphorylation
steps prior to their subsequent utilization (2,3). The discovery of a GGOH
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
641