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oai:repisalud.isciii.es:20.500.12105/70002024-09-27T20:40:06Zcom_20.500.12105_2052com_20.500.12105_2051col_20.500.12105_19609

00925njm 22002777a 4500 dc Oeste, Clara L author Díez-Dacal, Beatriz author Bray, Francesca author García de Lacoba, Mario author de la Torre, Beatriz G author Andreu, David author Ruiz-Sánchez, Antonio J author Pérez-Inestrosa, Ezequiel author Garcia-Dominguez, Carlota A author Rojas-Cabañeros, Jose Maria author Pérez-Sala, Dolores author 2011-01-06 Ras proteins are crucial players in differentiation and oncogenesis and constitute important drug targets. The localization and activity of Ras proteins are highly dependent on posttranslational modifications at their C-termini. In addition to an isoprenylated cysteine, H-Ras, but not other Ras proteins, possesses two cysteine residues (C181 and C184) in the C-terminal hypervariable domain that act as palmitoylation sites in cells. Cyclopentenone prostaglandins (cyPG) are reactive lipidic mediators that covalently bind to H-Ras and activate H-Ras dependent pathways. Dienone cyPG, such as 15-deoxy-Δ(12,14)-PGJ(2) (15d-PGJ(2)) and Δ(12)-PGJ(2) selectively bind to the H-Ras hypervariable domain. Here we show that these cyPG bind simultaneously C181 and C184 of H-Ras, thus potentially altering the conformational tendencies of the hypervariable domain. Based on these results, we have explored the capacity of several bifunctional cysteine reactive small molecules to bind to the hypervariable domain of H-Ras proteins. Interestingly, phenylarsine oxide (PAO), a widely used tyrosine phosphatase inhibitor, and dibromobimane, a cross-linking agent used for cysteine mapping, effectively bind H-Ras hypervariable domain. The interaction of PAO with H-Ras takes place in vitro and in cells and blocks modification of H-Ras by 15d-PGJ(2). Moreover, PAO treatment selectively alters H-Ras membrane partition and the pattern of H-Ras activation in cells, from the plasma membrane to endomembranes. These results identify H-Ras as a novel target for PAO. More importantly, these observations reveal that small molecules or reactive intermediates interacting with spatially vicinal cysteines induce intramolecular cross-linking of H-Ras C-terminus potentially contributing to the modulation of Ras-dependent pathways. PLoS One. 2011;6;6(1):e15866 10.1371/journal.pone.0015866 1932-6203 1932-6203 PloS one 21253588 http://hdl.handle.net/20.500.12105/7000 The C-terminus of H-Ras as a target for the covalent binding of reactive compounds modulating Ras-dependent pathways