The reversible phosphorylation of proteins is an important regulatory mechanism in eukaryotes, being essential during cell cycle progression. Cell cycle alterations may result in uncontrolled proliferation, leading to tumor development. Fibroblast growth factor (FGF2) acts as a proliferative agent in majority of cells. However, in some cellular contexts it shows antiproliferative functions. Therefore, FGF2 could be an important mechanism of tumor suppression. In adrenocortical carcinoma cells (Y1 cell line), FGF2 transiently inhibits G0/G1 transition, and irreversibly blocks cells in G2/M. To better understand the signaling pathways activated or inhibited by FGF2, it is essential to identify phosphoproteins in large scale. Therefore, in this project we intend to standardize the phosphopeptides enrichment technique and to analyze the phosphoproteome of Y1 cells after FGF2 treatment. First, the peptides will be fractionated by cation exchange chromatography followed by phosphopeptide enrichment using TiO2 or Fe3+ columns. Samples will be analyzed by mass spectrometry and the phosphorylation sites will be identified. Using bioinformatics, the signaling pathways and Gene Ontology categories that are preferentially phosphorylated by FGF2 are going to be identified in order to elucidate the molecular mechanisms that may play a role in the cell cycle blockage induced by FGF2.
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