Four-jointed modulates growth and planar polarity by reducing the affinity of dachsous for fat.
Bottom Line: We have used both cell and in vitro assays to measure binding between Ft and Ds.We find that phosphorylation of Ds reduces its affinity for Ft in both of these assays.By expressing forms of Ds that lack the defined phosphorylation sites or have phosphomimetic amino acids at these positions, we demonstrate that effects of Fj on wing size and planar polarity can be explained by Fj phosphorylating these sites.
Affiliation: Medical Research Council (MRC) Centre for Developmental and Biomedical Genetics and Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, UK.Show MeSH
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Mentions: Ishikawa et al.  identified three conserved serines (S236, S561, and S881) in Ds cadherin domains that can be phosphorylated by Fj in vitro or in cell-based assays (Figure 3A). We mutated these three serines in ds-EGFP to alanine to obviate phosphorylation at these sites (dsS>Ax3-EGFP). Mutation of these serines did not disrupt the levels of expression or cell-surface localization (Figures 3C and 3D). Nor did it significantly impact on the behavior of the protein: in the absence of fj expression, dsS>Ax3-EGFP-expressing cells can still bind to ft-expressing cells at a similar level as do wild-type ds-EGFP-expressing cells (Figure 3B). But, in the cell aggregation assay, dsS>Ax3-EGFP-expressing cells did not respond to coexpression of GNT-Fj, unlike control ds-EGFP-expressing cells (Figure 3B). These results argue that these three phosphorylation sites are instrumental in the modulation of Ft-Ds binding affinity by Fj.
Affiliation: Medical Research Council (MRC) Centre for Developmental and Biomedical Genetics and Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, UK.