High Sensitivity SELDI Analysis of NF1 Interactors in Mammals Drosophila and Yeast

摘要

The role of neurofibromin in cellular growth control is complex with available data suggesting that neurofibromin is a bifunctional modulator that may regulate proliferation by coordinating the activities of numerous molecular pathways. One important step in clarifying neurofibromin's function is identification of critical interactors whose detection and characterization may define downstream targets of neurofibromin and regulators of its activity. The purpose of our research was identification of neurofibormin-associated proteins in multiple experimental systems using high sensitivity surface enhanced laser desorption/ionization (SELDI) mass spectrometry. Our primary efforts were aimed at overcoming the longstanding difficulties of manipulating normal and mutant forms of neurofibromin in mammalian cells and our progress has been severely limited by technical difficulties that prevented controlled exogenous neurofibromin expression in target cells. We have focused on using tetracycline- inducible HSV amplicon systems to re-introduce wild type neurofibromin into neurofibromin-deficient primary mouse embryo fibroblasts and human neuroblastoma cells. Our preliminary analyses in NIH3T3 fibroblasts, neurobibromin-deficient MEFs, and human Schwann cells derived from NFl- associated tumors show tight control of tetracycline-inducible gene expression and suggest that the amplicon system will be a valuable tool in the identification of neurofibromin- associated proteins in a broad spectrum of cell types, including those that are pathologically relevant to the NFl phenotype.