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.
Highlights
Covalent attachment of C16:0 to proteins regulates protein function
We previously showed that C17:0-azide is functionally equivalent to C18:014. Using this assay followed by mass spectrometry, we previously screened for stearoylated proteins in HeLa cells[14]
Representative of two biological replicates. e Endogenous GNAI2 and GNAI3 are S-acylated on only one site, assayed using the acyl-PEG exchange (APE)
Summary
Covalent attachment of C16:0 to proteins (palmitoylation) regulates protein function. Exposure of cells to C18:0 reduces recruitment of Gab[1] 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. Metabolites can be enzymatically attached to proteins, thereby forming covalent post-translational modifications (PTMs) that directly regulate protein function. The cellular levels of these metabolites can directly convert into the stoichiometry of protein post-translational modification, thereby linking cell metabolism to cell signaling. Whether differential S-acylation of one residue leads to differential effects on protein function in animals is not well studied Fatty acids such as C16:0 or C18:0 are similar enough biophysically that one could imagine the resulting
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