Microtubules Inhibit E-Cadherin Adhesive Activity by Maintaining Phosphorylated p120-Catenin in a Colon Carcinoma Cell Model

Abstract

Tight regulation of cadherin-mediated intercellular adhesions is critical to both tissue morphogenesis during development and tissue homeostasis in adults. Cell surface expression of the cadherin-catenin complex is often directly correlated with the level of adhesion, however, examples exist where cadherin appears to be inactive and cells are completely non-adhesive. The state of p120-catenin phosphorylation has been implicated in regulating the adhesive activity of E-cadherin but the mechanism is currently unclear. We have found that destabilization of the microtubule cytoskeleton, independent of microtubule plus-end dynamics, dephosphorylates p120-catenin and activates E-cadherin adhesion in Colo 205 cells. Through chemical screening, we have also identified several kinases as potential regulators of E-cadherin adhesive activity. Analysis of several p120-catenin phosphomutants suggests that gross dephosphorylation of p120-catenin rather than that of specific amino acids may trigger E-cadherin adhesion. Uncoupling p120-catenin binding to E-cadherin at the membrane causes constitutive adhesion in Colo 205 cells, further supporting an inhibitory role of phosphorylated p120-catenin on E-cadherin activity.

Fig 1. Microtubule disruption dephosphorylates p120 and stimulates cadherin-based adhesion in Colo 205 cells.

(A-F) Brightfield microscopy of non-adhesive and adhesion-activated Colo 205 cells. Scale bar = 50 μm. (A-C) Parental or p120 knockdown cells were treated with either 10 μM nocodazole or an equal volume of DMSO for 1.5 hours. (D-F) Parental cells were pre-treated with 10 μM taxol for 2 hours then treated with 2 μg/mL 19A11 activating E-cadherin mAb for 4 hours. (G) Colo 205 cells expressing EB1-EGFP were treated with various concentrations of nocodazole and imaged at 10 minutes via fluorescent confocal microscopy. Scale bar = 5 μm. The same cells were then imaged by brightfield microscopy (scale bar = 50 μM) after 2 hours of treatment to examine the extent of adhesion activation, then fixed in methanol and immunostained for α-tubulin to examine the extent of microtubule disruption (scale bar = 5 μm). (H) Cells were treated for 1.5 hours with various concentrations of nocodazole then lysed in 1% triton x-100. Samples were resolved on 6% SDS-PAGE with 20 μM Phos-tag reagent then visualized via Western blotting. Red stars = higher molecular weight in non-activated samples, cyan stars = lower molecular weight in activated samples.

Fig 2. Chemical perturbation of certain serine/threonine kinases activates cadherin adhesion in Colo 205 cells.

(A-P) Cells were treated with various drugs known to inhibit ser/thr kinases at a range of concentrations for 1.5–6 hours (S1 Table). If cells remained non-adhesive, the brightfield image shown corresponds to the highest dose tested. If cells became adhesive, the lowest dose tested with an effect is shown. Scale bar = 50 μm. (Q) Cells that became adhesive were lysed in 1% triton x-100 and resolved by 6% SDS-PAGE with 20 μM Phos-tag reagent. The extent of p120 dephosphorylation was then visualized via Western blotting. (R) Brightfield images of cells treated with either 55 mM LiCl or 50 μM D4476 after knockdown of p120.

Fig 3. The overall state of p120 phosphorylation regulates cadherin adhesive activity.

(A-J) Brightfield images of Colo 205 cells expressing mouse p120 non-phosphorylatable mutants with knockdown of endogenous human p120 by siRNA. Scale bar = 50 μm. (K) Cells in A-J were lysed in 1% triton x-100, resolved on 6% SDS-PAGE, and visualized via Western blotting.

Fig 4. Additional phosphorylated residues of p120 may contribute to the regulation of cadherin-based adhesion.

Colo 205 cells expressing various mouse p120 phosphomimetic mutants with endogenous human p120 knockdown by siRNA before treatment. Untreated (-), 10 μM nocodazole (N), and 55 mM LiCl (L) treated cells were lysed in 1% triton x-100 and resolved by 6% SDS-PAGE with 20 μM Phos-tag reagent. The extent of p120 dephosphorylation was then visualized via Western blotting.

Fig 5. A p120-binding deficient E-cadherin mutant exhibits constitutive adhesion in Colo 205 cells.

Colo 205 cells expressing human E-cadherin constructs behind stable knockdown of endogenous human E-cadherin by shRNA specific to an untranslated region. (A) Brightfield images of cells treated with either 10 μM nocodazole or an equal volume of DMSO. Scale bar = 50 μm. (B) Cells were lysed in 1% triton x-100 and resolved by 6% SDS-PAGE with 20 μM Phos-tag reagent. The extent of p120 dephosphorylation and E-cadherin expression was then visualized via Western blotting. (C) Subfractionation of cells in hypotonic buffer resolved by 7.5% SDS-PAGE and visualized by Western blotting. M = membrane, C = cytosol.