ABSTRACT
Transcription factors (TFs) are essential for the expression of all proteins, including those involved in human health and disease. However, TFs are resistant to proteomic characterization because they are frequently masked by more abundant proteins due to the limited dynamic range of capillary liquid chromatography-tandem mass spectrometry and protein database searching. Purification methods, particularly strategies that exploit the high affinity of TFs for DNA response elements (REs) on gene promoters, can enrich TFs prior to proteomic analysis to improve dynamic range and penetrance of the TF proteome. For example, trapping of TF complexes specific for particular REs has been achieved by recovering the element DNA-protein complex on solid supports. Additional methods for improving dynamic range include two- and three-dimensional gel electrophoresis incorporating electrophoretic mobility shift assays and Southwestern blotting for detection. Here we review methods for TF purification and characterization. We fully expect that future investigations will apply these and other methods to illuminate this important but challenging proteome.
Subject(s)
Proteomics/methods , Tandem Mass Spectrometry/methods , Transcription Factors/chemistry , Animals , Chromatography, Liquid/methods , Databases, Protein , Electrophoresis/methods , Humans , Response Elements , Transcription Factors/genetics , Transcription Factors/isolation & purification , Transcription Factors/metabolism , Transcriptional ActivationABSTRACT
Fibroblast growth factors (FGFs) and fibroblast growth factor receptors (FGFRs) are major regulators of skeletal growth and development. Signal transduction via FGFRs is complex and mediates proliferation, differentiation, or migration depending upon the cellular context. Members of the Spry gene family antagonize the FGFR signal transduction pathway and inhibit lung morphogenesis, angiogenesis, and chondrogenesis. We examined the expression of Spry2 in the osteoblastic MC3T3-E1 cell line. MC3T3-E1 cells express Spry2 in response to FGF1 stimulation. Treatment of MC3T3-E1 cells with FGF1 results in the expression of Spry2 in a manner consistent with an early response gene. Pharmacological inhibitors of mitogen-activated protein kinase activation inhibit FGF1-induced expression of Spry2 mRNA. Transient overexpression of Spry2 in MC3T3-E1 resulted in decreased FGF1-mediated extracellular signal-regulated kinase phosphorylation and FGF1-stimulated osteopontin promoter activity. Furthermore, we show that Spry2 interacts with Raf-1 in a glutathione-S-transferase pulldown assay and that this interaction may involve multiple sites. Finally, Spry2 expression precedes the onset of the expression of osteoblast differentiation markers in an in vitro assay of primary osteoblast differentiation. Taken together, these results indicate that Spry2 expression is an early response to stimulation by FGF1 in MC3T3-E1 cells and acts as a feedback inhibitor of FGF1-induced osteoblast responses, possibly through interaction with Raf1.