Stearic acid blunts growth-factor signaling via oleoylation of GNAI proteins

Abstract

Covalent attachment of C16:0 to proteins (palmitoylation) regulates protein function. Proteins are also S-acylated by other fatty acids including C18:0. Whether protein acylation with different fatty acids has different functional outcomes is not well studied. We show here that C18:0 (stearate) and C18:1 (oleate) compete with C16:0 to S-acylate Cys3 of GNAI proteins. C18:0 becomes desaturated so that C18:0 and C18:1 both cause S-oleoylation of GNAI. Exposure of cells to C16:0 or C18:0 shifts GNAI acylation towards palmitoylation or oleoylation, respectively. Oleoylation causes GNAI proteins to shift out of cell membrane detergent-resistant fractions where they potentiate EGFR signaling. Consequently, exposure of cells to C18:0 reduces recruitment of Gab1 to EGFR and reduces AKT activation. This provides a molecular mechanism for the anti-tumor effects of C18:0, uncovers a mechanistic link how metabolites affect cell signaling, and provides evidence that the identity of the fatty acid acylating a protein can have functional consequences.

Competition for cysteine acylation by C16:0 and C18:0 derived lipids is a global phenomenon in the proteome

“…The fact that two fatty acids can acylate a protein equally well does not mean that the stoichiometry of acylation in the cell with those two fatty acids is 1:1, since the stoichiometry also reflects the abundance of the two fatty acids in vivo. For instance, we find that GNAI3 can be comparably acylated by C15-az and C17-az yet in vivo we find by MS that GNAI3 is more abundantly palmitoylated than oleoylated (Nuskova et al, 2021). If we expose cells to C18:0, however, this ratio shifts towards oleoylation (Nuskova et al, 2021).…”

“…For instance, we find that GNAI3 can be comparably acylated by C15-az and C17-az yet in vivo we find by MS that GNAI3 is more abundantly palmitoylated than oleoylated (Nuskova et al, 2021). If we expose cells to C18:0, however, this ratio shifts towards oleoylation (Nuskova et al, 2021).…”

“…and additional examples of proteins with rapid acylation turnover have been reported (Degtyarev et al, 1993;El-Husseini Ael et al, 2002;Zhang et al, 2010). We previously found that within this 3-hour timeframe, C17:0-azide or C18:0 can be unsaturated in the cell, yielding proteins carrying acylation with C17:1-azide or C18:1, respectively (Nuskova et al, 2021). In contrast, C15:0-azide and C16:0 remain predominantly saturated.…”

“…This leads to labeling of both de novo synthesized proteins as well as proteins that undergo deacylation/reacylation cycles. For instance, RAS has a protein half-life of 24 h but an acylation half-life <30 min (Ahearn et al, 2018;Lin and Conibear, 2015), GNAI proteins have a half-life >12 h but an acylation half-life of 3 h (Nuskova et al, 2021),…”

“…If differential acylation takes place, does this lead to different functional consequences depending on the identity of the fatty acid? We recently showed that GNAI proteins can be differentially acylated by either palmitic acid or oleic acid on one amino acid, Cys3 (Nuskova et al, 2021). The identity of the fatty acid acylating GNAI proteins has functional consequences, since acylation with palmitic acid, but not with oleic acid, shifts GNAI proteins into detergent-resistant fractions of the cell membrane where they potentiate EGFR signaling.…”