Alkynyl-farnesol reporters for detection of protein S-prenylation in cells

Abstract

Protein S-prenylation is a lipid modification that regulates membrane-protein and protein-protein interactions in cell signaling. Though sites of protein S-prenylation can be predicted based upon conserved C-terminal CaaX or CC/CXC motifs, biochemical detection of protein S-prenylation in cells is still challenging. Herein, we report an alkynyl-isoprenol chemical reporter (alk-FOH) as an efficient substrate for prenyltransferases in mammalian cells that enables sensitive detection of S-farnesylated and S-geranylgeranylated proteins using bioorthogonal ligation methods. Fluorescent detection alleviates the need to deplete cellular isoprenoids for biochemical analysis of S-prenylated proteins and enables robust characterization of S-prenylated proteins, such as effectors that are injected into host cells by bacterial pathogens. This alkynyl-prenylation reporter provides a sensitive tool for biochemical analysis and rapid profiling of prenylated proteins in cells.

Figure 1

Bioorthogonal reporters of protein prenylation. A) CaaX protein S-farnesylation and S-geranylgeranylation (top). Rab protein dual S-geranylgeranylation (bottom). B) Scheme for metabolic labeling with prenylation reporters. C) Prenylation reporters. D) Detection tags.

Figure 2

Synthesis of prenylation reporters.

Figure 3

Comparative analysis of prenylation reporters. Jurkat cells were treated or not with 20 μM lovastatin for 24 hours before supplementing the media with prenylation reporters (50 μM, 4 hours). Cell lysates labeled with alkynyl-isoprenols (alk-FOH, alk-FOH-2 and alk-FOH-3) or azido-isoprenol (az-FOH) were conjugated via CuAAC to azido-rhodamine (az-rho) or alkynyl-rhodamine (alk-rho), respectively. Lysates (20 μg) were separated by SDS-PAGE and scanned for fluorescence (top panel) or stained with Coomassie blue as a loading control (lower panel).

Figure 4

Fluorescent visualization of protein S-prenylation on known prenylated proteins. A) Jurkat cells were treated or not with 20 μM lovastatin for 24 hours before supplementing the media with prenylation reporters (50 μM, 4 hours). Immunopurified Ras labeled with alkynyl-isoprenols (alk-FOH, alk-FOH-2 and alk-FOH-3) was conjugated via CuAAC to azido-rhodamine (az-rho), followed by separation by SDS-PAGE and fluorescence detection (top panels) or immunoblotting as a loading control (lower panel). B) HeLa cells transiently expressing HA-Ras^G12V, GFP-RhoA or GFP-Rab7 were treated or not with 10 μM FTI-277 or GGTI-2133 for 1 hour before supplementing the media with prenylation reporter alk-FOH (50 μM, 4 hours). Immunopurified proteins labeled with alk-FOH were conjugated via CuAAC to azido-rhodamine (az-rho), followed by separation by SDS-PAGE and fluorescence detection (top panels) or immunoblotting as a loading control (lower panel).

Figure 5

Alk-FOH analysis of SifA prenylation. A) HeLa cells transiently expressing HA-SifA were treated or not with either 10 μM FTI-277, GGTI-298, GGTI-2133 or 10 μM of both FTI-277 and GGTI-2133 for 1 hour before supplementing the media with prenylation reporter alk-FOH (50 μM, 4 hours). Immunopurified HA-SifA labeled with alk-FOH was conjugated via CuAAC to azido-rhodamine (az-rho), followed by separation by SDS-PAGE and fluorescence detection (top panels) or immunoblotting as a loading control (lower panel). B) HeLa cells transiently expressing HA-SifA, HA-SifA^C333S, HA-SifA^C334S, or HA-SifA^{C331,333,334S} were treated with prenylation reporter alk-FOH (50 μM, 4 hours). Immunopurified HA-SifA labeled with alk-FOH was conjugated via CuAAC to azido-rhodamine (az-rho), followed by separation by SDS-PAGE and fluorescence detection (top panels) or immunoblotting as a loading control (lower panel). Fluorescence was quantified by mean fluorescence intensity adjusted for loading and normalized for background (0) and strongest signal (1).