Sensitive and Rapid Analysis of Protein Palmitoylation with a Synthetic Cell-Permeable Mimic of Src Oncoproteins

Abstract

The localization of oncogenic Src and Ras proteins to cellular plasma membranes is critical for the proliferation of specific cancers. In addition to other lipid modifications, these proteins require posttranslational palmitoylation of specific cysteine residues by the enzyme palmitoyl acyltransferase (PAT) in order to be stably anchored at plasma membranes. Hence, the identification of inhibitors of protein palmitoylation has significant potential to define a new class of antitumor agents. However, studies of protein palmitoylation have been hindered by the dynamic and reversible nature of cysteine acylation and the lack of sensitive and convenient assays of PAT activity. To facilitate the rapid identification of compounds that affect protein palmitoylation, we report the solid-phase synthesis of a fluorescent cell-permeable palmitoyl acyltransferase substrate that mimics the N-terminus of Src family proteins. Metabolic radiolabeling and epifluorescence microscopy of Jurkat lymphocytes treated with this Src-mimetic lipopeptide revealed that this compound is palmitoylated intracellularly, which confers localization at cellular plasma membranes. Addition of the palmitoylation inhibitor 2-bromopalmitic acid to substrate-treated cells blocked palmitoylation and diminished substrate-mediated plasma membrane fluorescence. Analysis of inhibition of palmitoylation by flow cytometry revealed that this fluorescent lipopeptide substrate represents a highly sensitive molecular probe of palmitoyl acyltransferase activity that enables unprecedented high-throughput assays of protein palmitoylation.

A fluorescent probe for cysteine depalmitoylation reveals dynamic APT signaling

“…21 These approaches reveal the balance between palmitoylation and depalmitoylation, but cannot reveal potential upstream regulatory mechanisms specifically of the depalmitoylases. Therefore, to study the regulation and dynamics of S -palmitoylation, there is a need for methods to monitor the activity levels of depalmitoylases in physiological contexts.…”

Use of micellar electrokinetic chromatography to measure palmitoylation of a peptide

“…Previous work utilizing fluorescently-labeled peptide substrates for monitoring palmitoylation observed increases in fluorescence intensity following palmitoylation of a peptide[43]. To determine whether the fluorescence properties of GAP-peptide were altered by palmitoylation, the fluorescence spectra of the doubly palmitoylated and non-palmitoylated peptide were measured in a neutral aqueous buffer.…”

Cellular palmitoylation and trafficking of lipidated peptides

“…The conserved DHHC domain of these proteins was hypothesized to represent a signature catalytic motif for PATs and has provided a starting point for the characterization of human PATs. For example, we recently demonstrated that human HIP-14 (DHHC17) is a farnesyl-directed PAT that causes oncogenic transformation of fibroblasts when over- Lipidated peptides that mimic palmitoylation motifs have been used by us to quantify palmitoylation in vitro (24)(25)(26)(27) and by others to study trafficking, localization, and membrane interactions in live cells (46)(47)(48)(49)(50). In particular, studies by Schroeder et al (46,47) were instrumental in demonstrating that lipopeptides that mimic protein palmitoylation sites are useful tools for the analyses of PAT activity and protein trafficking.…”