Generating P-Rex1 Chimera Proteins to Investigate the Mechanism of P-Rex2 and PTEN Binding

Abstract ID 95256 Poster Board 155 Mutations in phosphatidylinositol-3,4,5 trisphosphate-dependent Rac exchange factor 2 (P-Rex2) are often associated with diseases like cancer. P-Rex2 is regulated by phosphatase and tensin homologue (PTEN), a tumor suppressor that binds and inhibits P-Rex2 through an unclear mechanism. P-Rex1, a close family member of P-Rex2, shares a similar domain layout, but is not inhibited by PTEN. Previous studies show inconsistent results regarding the domains involved in P-Rex2-PTEN binding. While cross-linking mass spectrometry studies suggest that PTEN binds through the inositol polyphosphate 4-phosphatase-like (IP4P) subdomain and second PDZ (PDZ2) domain, truncation studies suggest that the pleckstrin homology (PH) domain and a portion of the IP4P subdomain of P-Rex2 are involved. We hypothesize that the PH domain and the IP4P subdomain are responsible for PTEN binding. In order to test our hypothesis, we have generated P-Rex1 chimeras containing either the PH or PDZ2 domain replaced with P-Rex2 sequence. GEF activity assay and phosphatase assay will be used to assess the relative activity of the chimeras compared to P-Rex1 wildtype, in order to characterize them for inhibition by PTEN and ability to inhibit PTEN. We expect P-Rex1 chimeras containing the domains responsible for P-Rex2–PTEN binding will have lower activity compared to P-Rex1 wild-type. Through this process, we will determine which domains facilitate the interaction between P-Rex2 and PTEN. This understanding of the mechanism for PTEN inhibition of P-Rex2 is an important step towards rational drug design against P-Rex2.