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Identification of direct substrates of GSK-3 and analysis of substrate functions in Dictyostelium discoideum


Recently studies have shown that glycogen synthase kinase- 3 (GSK-3) is essential for chemotaxis in Dictyostelium discoideum (Teo et al., 2010). To investigate the substrates of which GSK-3 may be acting through to have such dramatic effects, a phosphoproteomic assay was performed to detect proteins that change in phosphorylation levels when GSK-3 is absent from the cell. These data provided us with many potential direct GSK-3 substrate candidates. Some of those potential substrates include the proteins PdeD, DDB0231558, GnrA, DcT, Phg2, and Rapgap1. Through our investigations Phosphodiesterase D (PdeD) and Dynacortin (DcT) were found to be phosphorylated by GSK-3 in vitro. Additionally, these proteins were not phosphorylated in vitro when alanine substitution point mutations were present in the GSK-3 recognition and phosphorylation site. pdeD null cells have subtle chemotaxis defects and when PdeD is over-expressed, developmental defects are observed. dcT null cells also show chemotaxis defects in speed and developmental defects. GSK-3 is known to regulate Daydreamer, a protein that has been found to be necessary for chemotaxis. However, it has been unknown until now if GSK-3 phosphorylates other substrates involved in the known chemotaxis pathways. My investigations have been able to demonstrate that GSK-3 fits into the known chemotaxis signal transduction pathways through the phosphorylation of PdeD and DcT. Furthermore, these studies demonstrate that phosphoproteomics can be used to identify direct substrates and are highly sensitive in the detection of small changes in the phosphorylation levels of proteins

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