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  current news   Press   selected story    
     
  30th March 2011  
 

Protein interactions of PTEN and its cancer-associated G20E mutant compared by stable isotope labeling by amino acids in cell culture-based parallel affinity purification.

 
 




Authors
Jayantha GUNARATNE1, Mei Xian GOH2, Lee Foon SWA1, Fen Yee LEE2, Emma SANFORD2, Loke Meng WONG2, Kelly A. HOGUE1, Walter P. BLACKSTOCK1, and Koichi OKUMURA2.

1 - Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore 138673.

2 - Cancer Science Institute of Singapore, National University of Singapore, 28 Medical Drive, Singapore 117456.

Published in J Biol Chem. 17 March 2011.

Abstract
The tumor suppressor PTEN (Phosphatase and Tensin homologue) negatively regulates the phosphatidyl-inositol-3-kinase (PI3K) pathway through its lipid phosphatase activity and is one of the most commonly lost tumor suppressors in human cancers. Though the tumor suppressive function involves PTENís lipid phosphatase-dependent and Ėindependent activities, the mechanism leading to the phosphatase-independent function of PTEN is poorly understood. Some PTEN mutants have lipid phosphatase activity but fail to suppress cell growth. Here we use a cancer associated mutant, G20E, to gain insight into the phosphatase-independent function of PTEN by investigating protein-protein interactions using mass spectrometry (MS)-based stable isotope labeling by amino acids in cell culture (SILAC). A strategy named parallel affinity purification (PAP) and SILAC has been developed to prioritize interactors and to compare the interactions between wild-type and G20E PTEN. Clustering of the prioritized interactors acquired by the PAP-SILAC approach shows three distinct clusters: 1) wild-type specific interactors, 2) interactors unique to the G20E mutant and 3) proteins common to wild-type and mutant. These interactors are mainly involved in cell migration and apoptosis pathways. We further demonstrate that the wild-type specific interactor, NUDTL16L1, is required for the regulatory function of wild-type PTEN in cell migration. These finding contribute to a better understanding of the mechanisms of PTENís phosphatase-dependent and -independent functions.

 
 

 
 


Figure Legend : (A) PAP-SILAC allows reliable identification and prioritization of PTEN interactors. Parallel affinity purification in combination with SILAC (PAP-SILAC) for wild-type was carried-out using GFP trap and anti-PTEN antibody, whereas GFP-trap and anti-FLAG were used for G20E. Only common proteins with higher SILAC ratios from PAP for each experiment were extracted for clustering to identify their specificities. The heatmap of PAP-SILAC results allows categorizing of PTEN wild type and G20E interactors. (B) PTENís new interactors are involved in the migration and apoptosis signaling pathways. Network maps of cell migration and apoptosis for PTEN interactors from SILAC based PAP-SILAC were created by using information gathered from published literature and MetaCoreTM version 6.0. The double-headed arrow indicates interaction of proteins. Facilitation is a single-headed green arrow and inhibition is a red stroke. The known partner proteins of PTEN are implicated in the control of migration and apoptosis.

For more information on Walter BLACKSTOCKís laboratory, please click here.