Abstract
The tumor suppressor PTEN (phosphatase and tensin homologue) negatively regulates the 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 the lipid phosphatase-dependent and -independent activities of PTEN, the mechanism leading to the phosphatase-independent function of PTEN is understood poorly. 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 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 involved mainly 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 findings contribute to a better understanding of the mechanisms of the phosphatase-dependent and -independent functions of PTEN.
Highlights
Opment of tumors in a variety of tissues [1, 2]
Mutant by a parallel affinity purification with quantitative mass spectrometry proving it to be a useful strategy for reducing false positives, capturing true interactors, and prioritizing them
Quantitative mass spectrometry-based proteomic methods such as SILAC remove much of the “noise” of nonspecific interactors found in a standard immunoprecipitation experiments and allow a first round of in silico analysis to provide information on biological function
Summary
Cell Culture for SILAC—The U87MG human malignant glioma cell line (obtained from ATCC; HTB14) was used to make stable cell lines that expressed FLAP, FLAP-WT PTEN, and -G20E PTEN. Protein lysates (50 mg) were incubated with either FLAG beads (Sigma) or GFP-trap-agarose (ChromoTek). The cell lysates were prepared by Nonidet P-40 lysis buffer, and each lysate (50 mg) was incubated with GFP-trap-agarose. Immunoblotting for Co-precipitation—The precipitated samples of anti-PTEN, anti-FLAG beads, and GFP-trap were immunoblotted and detected by specific antibodies: PTEN A2B1, IQGAP1 H-109 (Santa Cruz Biotechnology), ubiquitinspecific peptidase 7 (USP7) (Bethyl Laboratories), CALU, FLAG M2 (Sigma), and V5 (Invitrogen) with ECL reagent (GE Healthcare). After serum starvation for 16 h, 2.5 ϫ 105 cells in DMEM 0.5% FBS were seeded into the upper chamber with medium containing 10% FBS in the lower chamber using 24-well plates and incubated for 8 h under normal cell culture condition.
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