Regulation of the PTEN Tumor Suppressor: Identification of the Active Protein Complex
Annual rept. 1 Apr 2003-31 Mar 2004
JOHNS HOPKINS UNIV BALTIMORE MD SCHOOLOF MEDICINE
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The PTEN tumor suppressor gene is among the most commonly mutated genes in prostate cancer. PTEN antagonizes the PI3KAKT pathway by dephosphorylating the 3, position of phosphoinositides. We had previously found that phosphorylation of the PTEN Tail regulates its membrane recruitment by regulating its interaction with a protein complex. The main aim of this proposal is to identify the components of this protein membrane complex and test their functional role in the PI3K pathway apoptosis or cell cycle in prostate cancer cell lines. Importantly, in the first year of the project we have found two alleles of PTEN that either enhance or decrease membrane recruitment. We have also generated expression plasmids containing these mutations with the Flag-His tag system to be used in the immunoaffinity purification experiments. Notably, we have determined the optimal conditions to use in the PTEN purification protein complex. We have found that the use of a specific crosslinker prior to the lysis of the cells is essential for the stabilization of the PTEN protein complex. Finally, we have performed a large-scale purification using these conditions and found several specific bands interacting with PTEN that are currently being sequenced.
- Medicine and Medical Research