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-Δ12,14-PGJ2 (15d-PGJ2) and Δ12-PGJ2 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-PGJ2. 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.

This work was supported by European Union Cooperation in the field of Scientific and Technical research (COST) Action CM1001, grant SAF2009-11642 from Ministerio de Ciencia e Innovación and Red Temática de Investigación Cooperativa from Instituto de Salud Carlos II (Spain) RD07/0064/0007 to DP-S and RD07/0064/0000 to EP-I. Work at Universitat Pompeu Fabra was supported by grants BIO2005-07592-CO2-02 and BIO2008-04487-CO3-02 from Ministerio de Ciencia e Innovación (Spain) to DA, and by the regional government of Catalunya (SGR2005-00494). JMR’s work is supported by Fondo de Investigaciones Sanitarias-Intrasalud (PS09/00562) and Red Temática de Investigación Cooperativa RD06/0020/0003 from Instituto de Salud Carlos III, and the Spanish Association Against Cancer (AECC). CLO is the recipient of a predoctoral fellowship from the Formación de Personal Investigador (FPI) program (Ministerio de Ciencia e Innovación, Spain, BES-2010-033718). CAG-D is the recipient of a fellowship from Fondo de Investigaciones Sanitarias-Beca de Formación en Investigación. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscrip