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Cooperation of distinct Rac-dependent pathways to stabilise E-cadherin adhesion.

Erasmus JC, Welsh NJ, Braga VM - Cell. Signal. (2015)

Bottom Line: In contrast, depletion of another EGFR family member, ErbB3, did not interfere with either process.However, in a strong divergence from EGFR RNAi phenotype, DOCK180 depletion did not perturb actin recruitment or cadherin localisation at junctions.Rather, reduced DOCK180 levels impaired the resistance to mechanical stress of pre-formed cell aggregates.

View Article: PubMed Central - PubMed

Affiliation: Molecular Medicine, National Heart and Lung Institute, Faculty of Medicine, Imperial College London, Sir Alexander Fleming Building, SW7 2AZ London, UK.

No MeSH data available.


EGFR, but not ErbB3, is required for cadherin-dependent Rac activation.A, Following induction of cell–cell contacts, keratinocyte lysates were immunoprecipitated with anti-E-cadherin antibodies, anti-α3-integrin (positive control) or no antibody (control) and probed with antibodies against proteins shown on the left of panels.B–C, Keratinocytes were treated with EGFR (oligo #1, oligo #2), ErbB3 (E3) or control (scr) siRNA oligos for 48 h. Equal amount of protein was separated on SDS–PAGE and probed with antibodies against proteins shown on the left of each panel.D–I, Keratinocytes were transfected with different siRNA oligos and junctions were initiated for 5 min by the addition of calcium ions and active Rac levels measured.D and G, Proteins were precipitated with GST–PAK-Crib beads (Rac∙GTP) and lysates (Total Rac) were probed with anti-Rac antibodies. The amount of GST fusion protein in each sample was evaluated by Amido Black staining (PAK-Crib).E and H, Depletion of EGFR or ErbB3 are shown. Beta-tubulin is shown as a loading control.F and I, Cell–cell-adhesion-dependent Rac activation was quantified and normalised to Rac·GTP levels at time 0 (no cell–cell contacts) for each siRNA group. Data is representative of 3 independent experiments (thereafter N = 3). *, p < 0.05; **, p < 0.005; n.s., non-significant.
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f0005: EGFR, but not ErbB3, is required for cadherin-dependent Rac activation.A, Following induction of cell–cell contacts, keratinocyte lysates were immunoprecipitated with anti-E-cadherin antibodies, anti-α3-integrin (positive control) or no antibody (control) and probed with antibodies against proteins shown on the left of panels.B–C, Keratinocytes were treated with EGFR (oligo #1, oligo #2), ErbB3 (E3) or control (scr) siRNA oligos for 48 h. Equal amount of protein was separated on SDS–PAGE and probed with antibodies against proteins shown on the left of each panel.D–I, Keratinocytes were transfected with different siRNA oligos and junctions were initiated for 5 min by the addition of calcium ions and active Rac levels measured.D and G, Proteins were precipitated with GST–PAK-Crib beads (Rac∙GTP) and lysates (Total Rac) were probed with anti-Rac antibodies. The amount of GST fusion protein in each sample was evaluated by Amido Black staining (PAK-Crib).E and H, Depletion of EGFR or ErbB3 are shown. Beta-tubulin is shown as a loading control.F and I, Cell–cell-adhesion-dependent Rac activation was quantified and normalised to Rac·GTP levels at time 0 (no cell–cell contacts) for each siRNA group. Data is representative of 3 independent experiments (thereafter N = 3). *, p < 0.05; **, p < 0.005; n.s., non-significant.

Mentions: We and others have previously shown that EGFR co-precipitates with E-cadherin receptors [13] and that the EGFR kinase activity is required for Rac activation following E-cadherin contact formation [8]. However, EGFR family members can form homo- and heterodimers, and it is not clear whether all family members are able to interact with E-cadherin. Out of the four different EGFR family members, EGFR, ErbB2 and ErbB3 are expressed in normal keratinocytes [25] as confirmed in this study (Figs. 1A, S1). From these, only EGFR was able to co-precipitate with E-cadherin when cell–cell junctions are initiated for 30 min (Fig. 1A). As a positive control, α3β1 integrin also precipitated with EGFR, but not ErbB2 or ErbB3. These results indicate that, when cell–cell junctions are formed, EGFR monomers or homodimers, but not other EGFR family members, may co-cluster with E-cadherin.


Cooperation of distinct Rac-dependent pathways to stabilise E-cadherin adhesion.

Erasmus JC, Welsh NJ, Braga VM - Cell. Signal. (2015)

EGFR, but not ErbB3, is required for cadherin-dependent Rac activation.A, Following induction of cell–cell contacts, keratinocyte lysates were immunoprecipitated with anti-E-cadherin antibodies, anti-α3-integrin (positive control) or no antibody (control) and probed with antibodies against proteins shown on the left of panels.B–C, Keratinocytes were treated with EGFR (oligo #1, oligo #2), ErbB3 (E3) or control (scr) siRNA oligos for 48 h. Equal amount of protein was separated on SDS–PAGE and probed with antibodies against proteins shown on the left of each panel.D–I, Keratinocytes were transfected with different siRNA oligos and junctions were initiated for 5 min by the addition of calcium ions and active Rac levels measured.D and G, Proteins were precipitated with GST–PAK-Crib beads (Rac∙GTP) and lysates (Total Rac) were probed with anti-Rac antibodies. The amount of GST fusion protein in each sample was evaluated by Amido Black staining (PAK-Crib).E and H, Depletion of EGFR or ErbB3 are shown. Beta-tubulin is shown as a loading control.F and I, Cell–cell-adhesion-dependent Rac activation was quantified and normalised to Rac·GTP levels at time 0 (no cell–cell contacts) for each siRNA group. Data is representative of 3 independent experiments (thereafter N = 3). *, p < 0.05; **, p < 0.005; n.s., non-significant.
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f0005: EGFR, but not ErbB3, is required for cadherin-dependent Rac activation.A, Following induction of cell–cell contacts, keratinocyte lysates were immunoprecipitated with anti-E-cadherin antibodies, anti-α3-integrin (positive control) or no antibody (control) and probed with antibodies against proteins shown on the left of panels.B–C, Keratinocytes were treated with EGFR (oligo #1, oligo #2), ErbB3 (E3) or control (scr) siRNA oligos for 48 h. Equal amount of protein was separated on SDS–PAGE and probed with antibodies against proteins shown on the left of each panel.D–I, Keratinocytes were transfected with different siRNA oligos and junctions were initiated for 5 min by the addition of calcium ions and active Rac levels measured.D and G, Proteins were precipitated with GST–PAK-Crib beads (Rac∙GTP) and lysates (Total Rac) were probed with anti-Rac antibodies. The amount of GST fusion protein in each sample was evaluated by Amido Black staining (PAK-Crib).E and H, Depletion of EGFR or ErbB3 are shown. Beta-tubulin is shown as a loading control.F and I, Cell–cell-adhesion-dependent Rac activation was quantified and normalised to Rac·GTP levels at time 0 (no cell–cell contacts) for each siRNA group. Data is representative of 3 independent experiments (thereafter N = 3). *, p < 0.05; **, p < 0.005; n.s., non-significant.
Mentions: We and others have previously shown that EGFR co-precipitates with E-cadherin receptors [13] and that the EGFR kinase activity is required for Rac activation following E-cadherin contact formation [8]. However, EGFR family members can form homo- and heterodimers, and it is not clear whether all family members are able to interact with E-cadherin. Out of the four different EGFR family members, EGFR, ErbB2 and ErbB3 are expressed in normal keratinocytes [25] as confirmed in this study (Figs. 1A, S1). From these, only EGFR was able to co-precipitate with E-cadherin when cell–cell junctions are initiated for 30 min (Fig. 1A). As a positive control, α3β1 integrin also precipitated with EGFR, but not ErbB2 or ErbB3. These results indicate that, when cell–cell junctions are formed, EGFR monomers or homodimers, but not other EGFR family members, may co-cluster with E-cadherin.

Bottom Line: In contrast, depletion of another EGFR family member, ErbB3, did not interfere with either process.However, in a strong divergence from EGFR RNAi phenotype, DOCK180 depletion did not perturb actin recruitment or cadherin localisation at junctions.Rather, reduced DOCK180 levels impaired the resistance to mechanical stress of pre-formed cell aggregates.

View Article: PubMed Central - PubMed

Affiliation: Molecular Medicine, National Heart and Lung Institute, Faculty of Medicine, Imperial College London, Sir Alexander Fleming Building, SW7 2AZ London, UK.

No MeSH data available.