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Identification of Glycogen Synthase Kinase-3 substrates in Dictyostelium discoideum

Abstract

Glycogen synthase kinase-3 (GSK3) is a highly conserved multifunctional kinase that regulates many cellular processes in eukaryotes, including cell motility, microtubule function, and cell differentiation (Frame et al., 2001). Dictyostelium GSK3, GskA, has been shown to be essential for chemotaxis (Teo R et al., 2010), although only one GskA substrate, Daydreamer, has been identified so far (Kölsch et al., 2013). Interestingly, the chemotaxis phenotype of Daydreamer null cells was not as severe as the one observed for gskA_ (GskA null) cells, suggesting that there may be more proteins downstream GskA that are regulating chemotaxis. To identify direct downstream effectors of GSK3, a phosphoprotemic assay was performed before and after stimulation with the chemoattractant cAMP in wild-type and gskA- cells (Kölsch et al., 2013). This investigation provided potential direct GSK3 substrate candidates, GxcGG and SogA, based on the phosphorylation of the GSK3 consensus sequence (s/tXXXs/t) in wild-type cells but not in gskA- cells. Our results showed that GxcGG and SogA were phosphorylated in in vitro kinase assays by GSK3. Overall, both sogA- and gxcGG- cells displayed chemotaxis and developmental defects that resulted in incomplete fruiting body development. However they operate at different areas of the GSK3 signaling cascade as visualized by the cell membrane translocation of GFP tagged GxcGG and translocation to the cytosol of GFP tagged SogA during chemotaxis. My investigations suggest that SogA and GxcGG are substrates of GSK3 involved in the cAMP chemotaxis signaling pathway.

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