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Four-jointed modulates growth and planar polarity by reducing the affinity of dachsous for fat.

Brittle AL, Repiso A, Casal J, Lawrence PA, Strutt D - Curr. Biol. (2010)

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.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council (MRC) Centre for Developmental and Biomedical Genetics and Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, UK.

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Ds Phosphorylation Sites Mediate the Effects of Fj on Ft-Ds Binding Affinity(A) Diagram representing the structure of Ds protein and localization of phosphorylation sites. Ovals represent the cadherin domains of Ds. Dark gray marks the cadherin domains containing serines that were shown in S2 cells to be phosphorylated by Fj (CAD3 [S236], CAD6 [S561], CAD9 [S881]), and light gray represents cadherin domains containing conserved S/T residues that have not been shown to be phosphorylated by Fj [13].(B) Cell aggregation assay with Ds phosphorylation mutants. pAct-ds-EGFP, dsS>Ax3-EGFP (S236A, S561A, S881A), or dsS>Dx3-EGFP (S236D, S561D, S881D) was cotransfected with pMK33B empty vector or pMK33B-GNT-fj. The percentage of Ft-expressing cells binding to Ds-EGFP-expressing cells was determined. Binding of cells in the absence of CuSO4 (light gray) was compared to binding in the presence of 0.14 mM CuSO4 (dark gray) to determine whether Fj expression made a significant difference to binding (n = 3, error bars show standard deviation). Student's t tests were applied (∗∗∗p < 0.005) and are indicated for each pair of columns (±Cu). In addition, of the first eight columns (four column pairs), only column 4 (Ds + GNT-Fj, +Cu) shows a significant difference from column 1 (Ds, -Cu). The level of binding of Ft to DsS>Dx3-EGFP cells (columns 9 and 11) was significantly lower than to Ds-EGFP (column 1) and DsS>Ax3-EGFP (column 5) (p < 0.005). Note also that the level of binding of DsS>Dx3-EGFP cells (column 9) is not as low as Ds-EGFP when GNT-Fj (column 4) is coexpressed (p < 0.05), suggesting that phosphorylation of the serine residues has a stronger effect on the affinity of binding of Ds to Ft than mutation of these serine residues to aspartate.(C) Western blot comparing levels of Ds-EGFP, DsS>Ax3-EGFP, and DsS>Dx3-EGFP expression in transfected S2 cells.(D) Ratio of cell surface expression versus total levels of Ds-EGFP, DsS>Ax3-EGFP, and DsS>Dx3-EGFP in transfected S2 cells. The levels are not significantly different.
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fig3: Ds Phosphorylation Sites Mediate the Effects of Fj on Ft-Ds Binding Affinity(A) Diagram representing the structure of Ds protein and localization of phosphorylation sites. Ovals represent the cadherin domains of Ds. Dark gray marks the cadherin domains containing serines that were shown in S2 cells to be phosphorylated by Fj (CAD3 [S236], CAD6 [S561], CAD9 [S881]), and light gray represents cadherin domains containing conserved S/T residues that have not been shown to be phosphorylated by Fj [13].(B) Cell aggregation assay with Ds phosphorylation mutants. pAct-ds-EGFP, dsS>Ax3-EGFP (S236A, S561A, S881A), or dsS>Dx3-EGFP (S236D, S561D, S881D) was cotransfected with pMK33B empty vector or pMK33B-GNT-fj. The percentage of Ft-expressing cells binding to Ds-EGFP-expressing cells was determined. Binding of cells in the absence of CuSO4 (light gray) was compared to binding in the presence of 0.14 mM CuSO4 (dark gray) to determine whether Fj expression made a significant difference to binding (n = 3, error bars show standard deviation). Student's t tests were applied (∗∗∗p < 0.005) and are indicated for each pair of columns (±Cu). In addition, of the first eight columns (four column pairs), only column 4 (Ds + GNT-Fj, +Cu) shows a significant difference from column 1 (Ds, -Cu). The level of binding of Ft to DsS>Dx3-EGFP cells (columns 9 and 11) was significantly lower than to Ds-EGFP (column 1) and DsS>Ax3-EGFP (column 5) (p < 0.005). Note also that the level of binding of DsS>Dx3-EGFP cells (column 9) is not as low as Ds-EGFP when GNT-Fj (column 4) is coexpressed (p < 0.05), suggesting that phosphorylation of the serine residues has a stronger effect on the affinity of binding of Ds to Ft than mutation of these serine residues to aspartate.(C) Western blot comparing levels of Ds-EGFP, DsS>Ax3-EGFP, and DsS>Dx3-EGFP expression in transfected S2 cells.(D) Ratio of cell surface expression versus total levels of Ds-EGFP, DsS>Ax3-EGFP, and DsS>Dx3-EGFP in transfected S2 cells. The levels are not significantly different.

Mentions: Ishikawa et al. [13] 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.


Four-jointed modulates growth and planar polarity by reducing the affinity of dachsous for fat.

Brittle AL, Repiso A, Casal J, Lawrence PA, Strutt D - Curr. Biol. (2010)

Ds Phosphorylation Sites Mediate the Effects of Fj on Ft-Ds Binding Affinity(A) Diagram representing the structure of Ds protein and localization of phosphorylation sites. Ovals represent the cadherin domains of Ds. Dark gray marks the cadherin domains containing serines that were shown in S2 cells to be phosphorylated by Fj (CAD3 [S236], CAD6 [S561], CAD9 [S881]), and light gray represents cadherin domains containing conserved S/T residues that have not been shown to be phosphorylated by Fj [13].(B) Cell aggregation assay with Ds phosphorylation mutants. pAct-ds-EGFP, dsS>Ax3-EGFP (S236A, S561A, S881A), or dsS>Dx3-EGFP (S236D, S561D, S881D) was cotransfected with pMK33B empty vector or pMK33B-GNT-fj. The percentage of Ft-expressing cells binding to Ds-EGFP-expressing cells was determined. Binding of cells in the absence of CuSO4 (light gray) was compared to binding in the presence of 0.14 mM CuSO4 (dark gray) to determine whether Fj expression made a significant difference to binding (n = 3, error bars show standard deviation). Student's t tests were applied (∗∗∗p < 0.005) and are indicated for each pair of columns (±Cu). In addition, of the first eight columns (four column pairs), only column 4 (Ds + GNT-Fj, +Cu) shows a significant difference from column 1 (Ds, -Cu). The level of binding of Ft to DsS>Dx3-EGFP cells (columns 9 and 11) was significantly lower than to Ds-EGFP (column 1) and DsS>Ax3-EGFP (column 5) (p < 0.005). Note also that the level of binding of DsS>Dx3-EGFP cells (column 9) is not as low as Ds-EGFP when GNT-Fj (column 4) is coexpressed (p < 0.05), suggesting that phosphorylation of the serine residues has a stronger effect on the affinity of binding of Ds to Ft than mutation of these serine residues to aspartate.(C) Western blot comparing levels of Ds-EGFP, DsS>Ax3-EGFP, and DsS>Dx3-EGFP expression in transfected S2 cells.(D) Ratio of cell surface expression versus total levels of Ds-EGFP, DsS>Ax3-EGFP, and DsS>Dx3-EGFP in transfected S2 cells. The levels are not significantly different.
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fig3: Ds Phosphorylation Sites Mediate the Effects of Fj on Ft-Ds Binding Affinity(A) Diagram representing the structure of Ds protein and localization of phosphorylation sites. Ovals represent the cadherin domains of Ds. Dark gray marks the cadherin domains containing serines that were shown in S2 cells to be phosphorylated by Fj (CAD3 [S236], CAD6 [S561], CAD9 [S881]), and light gray represents cadherin domains containing conserved S/T residues that have not been shown to be phosphorylated by Fj [13].(B) Cell aggregation assay with Ds phosphorylation mutants. pAct-ds-EGFP, dsS>Ax3-EGFP (S236A, S561A, S881A), or dsS>Dx3-EGFP (S236D, S561D, S881D) was cotransfected with pMK33B empty vector or pMK33B-GNT-fj. The percentage of Ft-expressing cells binding to Ds-EGFP-expressing cells was determined. Binding of cells in the absence of CuSO4 (light gray) was compared to binding in the presence of 0.14 mM CuSO4 (dark gray) to determine whether Fj expression made a significant difference to binding (n = 3, error bars show standard deviation). Student's t tests were applied (∗∗∗p < 0.005) and are indicated for each pair of columns (±Cu). In addition, of the first eight columns (four column pairs), only column 4 (Ds + GNT-Fj, +Cu) shows a significant difference from column 1 (Ds, -Cu). The level of binding of Ft to DsS>Dx3-EGFP cells (columns 9 and 11) was significantly lower than to Ds-EGFP (column 1) and DsS>Ax3-EGFP (column 5) (p < 0.005). Note also that the level of binding of DsS>Dx3-EGFP cells (column 9) is not as low as Ds-EGFP when GNT-Fj (column 4) is coexpressed (p < 0.05), suggesting that phosphorylation of the serine residues has a stronger effect on the affinity of binding of Ds to Ft than mutation of these serine residues to aspartate.(C) Western blot comparing levels of Ds-EGFP, DsS>Ax3-EGFP, and DsS>Dx3-EGFP expression in transfected S2 cells.(D) Ratio of cell surface expression versus total levels of Ds-EGFP, DsS>Ax3-EGFP, and DsS>Dx3-EGFP in transfected S2 cells. The levels are not significantly different.
Mentions: Ishikawa et al. [13] 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.

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.

View Article: PubMed Central - PubMed

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
Related in: MedlinePlus