Novel phosphoinositide-protein interactions and inhibition of Akt activation

The PI3K/Akt signaling pathway is critical for normal growth and development of a cell. The activation of Akt, while playing an important role in cell survival, has also been linked to cancer and diabetes. A specific Akt inhibitor is predicted to be of immense value in cancer treatment. Akt activation requires recruitment to the plasma membrane through interaction of its N-terminal pleckstrin homology (PH) domain with the phosphoinositide products of phosphatidylinositol-3-kinase (PI3K); phosphatidylinositol-3,4-bisphosphate (PI(3,4)P₂) and (PI(3,4,5)P ₃). Two synthetic peptide libraries were screened for binding to the Akt PH domain using competitive displacement of PI(3,4)P₂. One library consisted of random octamers and the second library was biased with alternate racemic glutamate or aspartate amino acids. Twenty-seven sequences were obtained and analyzed for Akt PH binding and the three sequences with highest affinity were chosen for further study. Each peptide demonstrated low micromolar in vitro inhibition of Akt binding to PI(3,4)P ₂ and demonstrated Akt selectivity over other PH domains. However, the affinity was determined to be nonspecific and inhibition of phosphoinositide-binding was determined to be due to masking of the phosphoinositides, not binding the Akt PH domain. When attached to a highly basic membrane permeable sequence, two peptides were able to enter cells and delay membrane localization of an expressed EGFP-Akt PH construct. The basic peptides demonstrated minimal cellular toxicity and inhibition of Akt activation presumably through masking newly formed phosphoinositides at the cell membrane to prevent Akt activation.; Analysis of protein-phosphoinositide interactions can indicate possible pathways in which a protein may be involved in cell signaling. Peroxiredoxin 1 (Prx1) was identified in a screen for high affinity phosphatidylinositol-3,4,5 trisphosphate (PI(3,4,5)P₃) binding proteins with PI(3,4,5)P ₃ PIP Beads™. The murine Prx1 gene was clone and characterized for phosphoinositide binding. Photoaffinity labeling was employed to examine phosphoinositide interactions with Rab5 effector proteins, Dnm1p, caspase 3, caspase 8, caspase 9, Unc104, EVH1, and Dbl. Novel tandem PH domain constructs were created in an attempt to create a more sensitive reporter protein of phosphoinositides with in vitro assays, though none exhibited any advantage over wild type PH domain.