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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.


DOCK180 is required for E-cadherin-mediated Rac activation. Cells were treated with DOCK180 (oligo #1, oligo #2), SOS1 (oligo #1) or control (scr) siRNA oligos.A–D, Lysates were prepared and used to determine efficiency of knockdown (A, B) and its specificity for DOCK180 or SOS1 depletion (C, D). Equal amounts of protein were separated on SDS–PAGE and probed with antibodies against proteins as shown on the left of each panel.E–H, Activation levels of Rac after 5 min of cell–cell contact formation. Pull down using GST–PAK-Crib was performed to precipitate active Rac (Rac∙GTP).E and G, Samples were probed with antibodies listed on the left of panels. Fusion proteins in each sample were evaluated by Amido black staining (PAK-Crib). Depletion of DOCK180 and SOS1 is shown and β-tubulin used as a loading control.F and H, Cell–cell adhesion-dependent Rac activation. Rac·GTP was quantified at different time points and normalised to Rac·GTP levels at time 0 (no cell–cell contacts) for each siRNA group (scr, DOCK180 or SOS1). N = 3; *, p < 0.03; **, p < 0.003; n.s., non-significant.
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f0015: DOCK180 is required for E-cadherin-mediated Rac activation. Cells were treated with DOCK180 (oligo #1, oligo #2), SOS1 (oligo #1) or control (scr) siRNA oligos.A–D, Lysates were prepared and used to determine efficiency of knockdown (A, B) and its specificity for DOCK180 or SOS1 depletion (C, D). Equal amounts of protein were separated on SDS–PAGE and probed with antibodies against proteins as shown on the left of each panel.E–H, Activation levels of Rac after 5 min of cell–cell contact formation. Pull down using GST–PAK-Crib was performed to precipitate active Rac (Rac∙GTP).E and G, Samples were probed with antibodies listed on the left of panels. Fusion proteins in each sample were evaluated by Amido black staining (PAK-Crib). Depletion of DOCK180 and SOS1 is shown and β-tubulin used as a loading control.F and H, Cell–cell adhesion-dependent Rac activation. Rac·GTP was quantified at different time points and normalised to Rac·GTP levels at time 0 (no cell–cell contacts) for each siRNA group (scr, DOCK180 or SOS1). N = 3; *, p < 0.03; **, p < 0.003; n.s., non-significant.

Mentions: DOCK180 RNAi in keratinocytes did not reduce SOS1 protein levels or vice versa (Fig. 3A–B) nor interfered with the levels of proteins in the cadherin complex or Rho small GTPases (Fig. 3C–D). When keratinocyte cell–cell contacts were initiated for 30 min, Rac∙GTP levels increased significantly following treatment with control or SOS1 siRNA oligos (Fig. 3G–H). However, cells with reduced DOCK180 protein had no significant increase in active Rac levels (Fig. 3E–F). Thus, DOCK180, but not SOS1, is likely to be one of the GEFs responsible for Rac activation by new junctions in keratinocytes. Our results suggest a selectivity among distinct GEFs via which Rac is activated upon engagement of cadherin receptors at cell–cell contacts. Our data is also supported by a previous report on depletion of ELMO2 in MDCK and indicates that DOCK180 may be important for junction signalling in cell types other than keratinocytes. DOCK180 was not formally tested in MDCK cells because of toxicity caused by its depletion. However, in contrast to MDCK cells [7], DOCK180 RNAi did not reduce keratinocyte cell viability (data not shown) and could be tested herein.


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

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

DOCK180 is required for E-cadherin-mediated Rac activation. Cells were treated with DOCK180 (oligo #1, oligo #2), SOS1 (oligo #1) or control (scr) siRNA oligos.A–D, Lysates were prepared and used to determine efficiency of knockdown (A, B) and its specificity for DOCK180 or SOS1 depletion (C, D). Equal amounts of protein were separated on SDS–PAGE and probed with antibodies against proteins as shown on the left of each panel.E–H, Activation levels of Rac after 5 min of cell–cell contact formation. Pull down using GST–PAK-Crib was performed to precipitate active Rac (Rac∙GTP).E and G, Samples were probed with antibodies listed on the left of panels. Fusion proteins in each sample were evaluated by Amido black staining (PAK-Crib). Depletion of DOCK180 and SOS1 is shown and β-tubulin used as a loading control.F and H, Cell–cell adhesion-dependent Rac activation. Rac·GTP was quantified at different time points and normalised to Rac·GTP levels at time 0 (no cell–cell contacts) for each siRNA group (scr, DOCK180 or SOS1). N = 3; *, p < 0.03; **, p < 0.003; n.s., non-significant.
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f0015: DOCK180 is required for E-cadherin-mediated Rac activation. Cells were treated with DOCK180 (oligo #1, oligo #2), SOS1 (oligo #1) or control (scr) siRNA oligos.A–D, Lysates were prepared and used to determine efficiency of knockdown (A, B) and its specificity for DOCK180 or SOS1 depletion (C, D). Equal amounts of protein were separated on SDS–PAGE and probed with antibodies against proteins as shown on the left of each panel.E–H, Activation levels of Rac after 5 min of cell–cell contact formation. Pull down using GST–PAK-Crib was performed to precipitate active Rac (Rac∙GTP).E and G, Samples were probed with antibodies listed on the left of panels. Fusion proteins in each sample were evaluated by Amido black staining (PAK-Crib). Depletion of DOCK180 and SOS1 is shown and β-tubulin used as a loading control.F and H, Cell–cell adhesion-dependent Rac activation. Rac·GTP was quantified at different time points and normalised to Rac·GTP levels at time 0 (no cell–cell contacts) for each siRNA group (scr, DOCK180 or SOS1). N = 3; *, p < 0.03; **, p < 0.003; n.s., non-significant.
Mentions: DOCK180 RNAi in keratinocytes did not reduce SOS1 protein levels or vice versa (Fig. 3A–B) nor interfered with the levels of proteins in the cadherin complex or Rho small GTPases (Fig. 3C–D). When keratinocyte cell–cell contacts were initiated for 30 min, Rac∙GTP levels increased significantly following treatment with control or SOS1 siRNA oligos (Fig. 3G–H). However, cells with reduced DOCK180 protein had no significant increase in active Rac levels (Fig. 3E–F). Thus, DOCK180, but not SOS1, is likely to be one of the GEFs responsible for Rac activation by new junctions in keratinocytes. Our results suggest a selectivity among distinct GEFs via which Rac is activated upon engagement of cadherin receptors at cell–cell contacts. Our data is also supported by a previous report on depletion of ELMO2 in MDCK and indicates that DOCK180 may be important for junction signalling in cell types other than keratinocytes. DOCK180 was not formally tested in MDCK cells because of toxicity caused by its depletion. However, in contrast to MDCK cells [7], DOCK180 RNAi did not reduce keratinocyte cell viability (data not shown) and could be tested herein.

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.