Probing the Activator and Hydrophobic Substrate Binding Sites of PTEN by 31P NMR

Abstract

PTEN (Phosphatase and Tensin Homolog) antagonizes the PI3K signaling pathway by dephosphorylating PI(3,4,5)P3 at the inositol C-3, thus suppressing cell proliferation. PI(4,5)P2, the product of dephosphorylation, activates this enzyme, possibly by binding to the N terminal region of PTEN (not visible in the crystal structure). A distinct hydrophobic pocket near the active site has also been identified (by MD simulations). We have used 31P NMR as a probe of the spatial location and functional role of these potential phospholipid binding sites. At 242.7 MHz, the linewidths of phosphodiester resonances of phosphoinositides that bind exclusively to the active site (PI, PI(4)P) are broadened consistent with intermediate exchange. Estimated linewidths for the bound activator PI(4,5)P2 are smaller. Other amphiphiles can also be affected by PTEN in a specific fashion, for example, diC7PC which is used as a matrix in assays. This ligand is likely to bind in the hydrophobic pocket identified by simulations. 31P high resolution field cycling NMR, with PTEN spin-labeled on the active site cysteine, was used to determine the distance between the unpaired electron and the phosphorus nuclei of the different bound lipids. Results of these relaxation studies indicate there are discrete sites for both substrate and activator lipids that are also separate from the region occupied by nonspecific lipids such as diC7PC (or a nonionic detergent such as Triton X-100). The distances derived are used to propose a model of PTEN regulation by the activator and matrix lipids.