The PTEN/MMAC1 tumor suppressor gene is inactivated by homozygous deletion or loss of heterozygosity in several types of human cancers. Its encoded protein is a lipid phosphatase that interrupts signaling initiated by activated phosphoinositol-3-kinase (PI3K). Termination of PI3K signaling interferes with the activation of its downstream effectors, including protein kinase B (PKB/Akt). As Akt is known to influence a variety of important molecules involved in crucial homeostatic mechanisms, PTEN is optimally situated to precisely direct cellular behavior.

As surprisingly little is known regarding PTEN regulation and interaction with other cellular mediators, we have explored PTEN function with three technical approaches: somatic cell gene targeted interruption of PTEN function, phosphorylation analysis of purified PTEN protein, and mass spectrometric identification of proteins complexed with PTEN. PTEN targeting was performed in the immortalized, non-transformed BJ-hTERT cell line and ultimately, interruption of only one allele was successful. We believe this resulted from the induction of p53-mediated oncogenic senescence as PTEN deletion allowed PI3K deregulation. Phosphorylation analysis was completely successful with 100% amino acid coverage obtained with purified PTEN protein. However, the modified residues we identified have already been reported; no novel sites were found with our method. An exhaustive search for PTEN interacting proteins was carried out using cell lines stably expressing FLAG-tagged PTEN. We utilized anti-FLAG protein purification followed by electrophoresis, gel staining, and LC/MS/MS and narrowed putative novel interactors to those which were most interesting. Unfortunately, we were only able to determine that many proteins were non-specifically bound to our immunoprecipitation beads or antibody. We have hope that newly developed cell lines will be a more productive reagent and will enable the discovery of novel post-translational modifications and interacting protein networks.