Abstract 4868: In vitro and in cellulo mass spectrometry study of TEAD1 palmitoylation
Immunology(2020)
Abstract
The HIPPO pathway plays an important role in cancer progression and one of its essential components are the transcription factor proteins from the TEAD family. The TEAD family consists of 4 different family members (TEAD1-TEAD4) who modulate gene expression through the interaction with co-activator proteins such as YAP1 and TAZ. Recent studies have shown that TEADs are partially palmitoylated. Activity and stability of TEAD proteins are regulated by this modification. Here, we attempt to better understand the palmitoylation status of TEAD proteins in cells. In a first step, we have developed LC/MS/MS-based assays for the detection of palmitoylated TEAD peptides using recombinant TEAD1 protein. Subsequently, we have set out to analyze endogenous TEAD proteins using YAP1 activated malignant mesothelioma H2052 tumor cells. H2052 tumor cells express preferentially TEAD1 protein. TEAD1 was immunoprecipitated from H2052 tumor cells and subjected to peptidic digest and LC/MS/MS analysis. Peptide mapping permitted to confirm the expected site of palmitoylation (cysteine 359 of TEAD1). In addition, novel sites of both palmitoylation and myristoylation were detected on the lysine residue 336 of TEAD1. While these hydrophobic peptides could not be recovered after in-gel digestion of immunoprecipitation enriched TEAD1 of H2052 cells, digestion performed directly on immunoprecipitation beads allowed us to recover those peptides and hence to confirm the existence of TEAD lysine-myristoylation and lysine-palmitoylation in cells. In this study, we show the development of an MS-based strategy to characterize post translational modifications directly on endogenous TEAD1 proteins present in H2052 tumor cells. Citation Format: Armelle Buzy, Olivier Guichard, Jean-Luc Zacchayus, Olivier Venier, Olivier Courtin, Iris Valtingojer, Laurent Debussche, Vincent Mikol, Jean-Claude Guillemot. In vitro and in cellulo mass spectrometry study of TEAD1 palmitoylation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4868.
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Key words
vitroandin cellulomass spectrometry study,tead1 palmitoylation
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