Dynamic fatty acylation of p21N-ras.

Abstract

To study the acylation of p21N‐ras with palmitic acid we have used cells which express the human N‐ras gene to high levels under control of the steroid‐inducible MMTV–LTR promoter. Addition of [3H]palmitate to these cells resulted in detectable incorporation of label into p21N‐ras within 5 min, which continued linearly for 30‐60 min. Inhibition of protein synthesis for up to 24 h before addition of [3H]palmitate had no effect on acylation of p21N‐ras, suggesting that this can occur as a late post‐translational event. Acylated p21N‐ras with a high SDS–PAGE mobility is found only in the membrane fraction, whereas approximately 50% of the [35S]methionine‐labelled p21N‐ras is cytoplasmic and has a lower mobility. Conversion of the acylated high mobility form to a deacylated form of slightly lower mobility can be achieved with neutral hydroxylamine, which is known to cleave thioesters. This treatment also results in partial removal of p21N‐ras from the membranes. A remarkably high rate of turnover of the palmitate moiety can be demonstrated by pulse–chase studies (t1/2 approximately 20 min in serum‐containing medium) which cannot be attributed to protein degradation. The data suggest an active acylation–deacylation cycle for p21N‐ras, which may be involved in its proposed function as a signal transducing protein.

Association of Drosophila cysteine string proteins with membranes

“…Unlike myristoylation, palmitoylation of cysteine residues is a reversible modification. Pulse-chase experiments have demonstrated a fast turnover of palmitate group of several proteins, including H-ras and N-Ras [13], the transferrin receptor [14] and neuronal growth cone protein GAP-43 [15]. It is possible that this dynamic modification, like reversible phosphorylation, can regulate a variety of cellular functions.…”

In situ selectivity profiling and crystal structure of SML-8-73-1, an active site inhibitor of oncogenic K-Ras G12C

“…However, despite the slower rate, these data unambiguously demonstrate that SML is able to compete irreversibly, preventing GN from exchanging back in. This suggests that a covalent inhibitor may be able to overcome the endogenous pool of GDP/GTP within the cell due to the relatively long 14-to 24-h t 1/2 of Ras if appropriate dosing regimens can be developed that maintain exposure of G12C K-Ras to covalent inhibitors (32,33). We note that this assay method will be useful in developing inhibitors of K-Ras G12C with more "drug-like" characteristics, and it also may be applied to other protein targets for which cysteine-targeted irreversible therapeutics are being developed.…”

Ras proteins: paradigms for compartmentalised and isoform-specific signalling

“…Mutagenesis of the H-Ras palmitoylated cysteines 181 and 184 revealed that H-Ras mono-palmitoylated on cysteine 184 is trapped within the Golgi complex; in contrast, mono-palmitoylation of cysteine 181 enables cell surface localisation [61]. Importantly, palmitoylation unlike farnesylation is labile with half-lives estimated to be a few minutes to a few hours depending on the methods used for analysis [62][63][64]. These are much shorter than the 21 hour half life of H-Ras meaning that palmitoylated Ras isoforms must go through many cycles of acylation and deacylation.…”