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First published online June 28, 2004
doi: 10.1242/10.1242/jcs.01174

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Continuous association of cadherin with ß-catenin requires the non-receptor tyrosine-kinase Fer

¹ Department of Biological Sciences, The University of Iowa, Iowa City, IA 52242, USA
² Department of Biochemistry, Room 641 Botterell Hall, Queen's University, Kingston, Ontario, K7L 3N6, Canada
³ Department of Pathology, Division of Cancer Biology and Genetics, Room A309 Botterell Hall, Queen's University Cancer Research Institute, Kingston, Ontario, K7L 3N6, Canada
⁴ Laboratory of Cell Adhesion and Metastasis, Mayo Clinic, Griffin Cancer Research Building, 4500 San Pablo Road, Jacksonville, FL 32224, USA

View larger version (30K): [in a new window]   Fig. 1. Binding of Fer to p120ctn. (A) Full-length and GST fusion constructs of Fer used in the in vitro binding assay. CC1, CC2 and CC3 correspond to coiled-coil domains. (B) Recombinant GST-Fer peptides were purified by affinity chromatography on glutathione-agarose and incubated with biotin-conjugated CBP-p120ctn immobilized on streptavidin-coated wells. Bound protein was determined by ELISA using anti-GST antibody and the appropriate HRP-conjugated secondary antibody. (C) Dose response of binding of the GST-Fer constructs to immobilized p120ctn. Immobilized p120ctn was incubated with increasing concentrations of GST-Fer constructs 1-7 and bound protein determined as described in B.

View larger version (28K): [in a new window]   Fig. 2. Binding of p120ctn to Fer. (A) Full-length and GST constructs of p120ctn used in the in vitro binding assay. Filled rectangles represent the Armadillo domains. (B) Recombinant GST-p120ctn peptides conjugated to biotin were immobilized on streptavidin-coated wells and incubated with His-tagged Fer-3. Bound protein was determined by ELISA using anti-His-tag antibody and HRP-conjugated second antibody. (C) Dose response of binding of the biotinylated GST-p120ctn peptides to His-Fer-3. His-Fer-3 was incubated with increasing concentrations of immobilized GST-p120ctn constructs 1 to 10, and bound protein was determined as described in B.

View larger version (23K): [in a new window]   Fig. 3. Effect of cell-permeable Antennapedia fusion peptides that mimic the Fer-p120ctn binding site on in vitro binding between Fer and p120ctn, and on the composition of the cadherin complex of proteins in E8 chick retina cells. (A) Biotin-labeled CBP-p120ctn was immobilized on streptavidin wells and incubated with 3 µg GST-Fer per well in the presence of increasing concentrations of competing peptides. Bound Fer was estimated using anti-GST antibody. Abbreviations: FerP and FerCo, Antennapedia cell-permeable peptides covalently attached to the Fer-7 or the reverse Fer-7 sequence, respectively; p120P and p120Co, Antennapedia peptides covalently attached to the P10 or the reverse P10 sequence. (B) Intact E8 retina cells were incubated in the presence of the indicated peptide for 45 minutes, 60 minutes or 120 minutes at room temperature. The cells were homogenized in buffer containing 1% NP-40 and immunoprecipitated with NCD-2. The resulting immuno-complexes were analysed by western blot with the indicated antibody. (C) Intact E8 retina cells were incubated in the presence of the indicated peptide for 60 minutes at room temperature, lysed in RIPA buffer, immunoprecipitated with anti-ß-catenin antibody and analysed by western blot using anti-phosphotyrosine antibody. The blots were then stripped and blotted with anti-ß-catenin antibody. The relative intensity of the ß-catenin western blot bands was measured by densitometry and is presented as the ratio between phosphotyrosine ß-catenin and total ß-catenin. (D) Retina cells were incubated with the indicated peptides and labeled with a cell-impermeable biotinylation reagent for the indicated time. The cells were lysed as above, cell-surface N-cadherin pulled down with avidin-coated beads, fractionated by SDS-PAGE and visualized by western blot with anti-N-cadherin antibody. An aliquot of the total lysate was also analyzed by western blot, for comparison.

View larger version (69K): [in a new window]   Fig. 4. Effect of cell permeable Antennapedia fusion peptides that mimic the Fer-p120ctn binding site on cell adhesion and neurite outgrowth. (A,B) Adhesion of E8 chick retina cells to cadherin (A) and laminin (B). Dissociated E8 chick neural retina cells were incubated for 1 hour at room temperature in the presence of the indicated peptide. Equal numbers of cells were then aliquoted into wells precoated with the extracellular domain of N-cadherin, Fc-N-cad (A) or with laminin (B) and incubated for 1 hour in the presence of the indicated peptides. Adherent cells were stained with crystal violet and quantified by absorbance at 560 nm. No adhesion is observed onto uncoated, blocked wells (No Ncad). Adhesion is expressed as the percentage of adhesion in the absence of added peptides (A) or in the presence of control peptide FerCo (B). (C,D) Neurite outgrowth. E7 chick retina cells were plated on slides coated with anti-N-cadherin antibody NCD-2 or laminin. Single cells bearing neurites longer than two cell diameters were counted and expressed as a percentage of the total adherent cells (C). Cells and neurites were visualized under phase contrast (D).

View larger version (25K): [in a new window]   Fig. 5. Effect of cell permeable Antennapedia fusion peptides that mimic the p120ctn binding site on N-cadherin on the composition and function of the cadherin complex of proteins in E9 chick retina cells. (A) Intact E9 retina cells were incubated in the presence of the indicated peptide for 60 minutes or 120 minutes at room temperature. The cells were homogenized in buffer containing 1% NP-40 and immunoprecipitated with NCD-2. The resulting immuno-complexes were analyzed by western blot with the indicated antibody. (B) Intact E9 retina cells were incubated in the presence of the indicated peptide for 60 minutes at room temperature lysed in RIPA buffer, immunoprecipitated with anti-ß-catenin antibody and analysed by western blot using anti-phosphotyrosine antibody. The blots were then stripped and blotted with anti-ß-catenin antibody. (C) Retina cells were incubated with the indicated peptides and labeled with a cell-impermeable biotinylation reagent. The cells were lysed as above, cell-surface N-cadherin `pulled down' with avidin-coated beads, fractionated by SDS-PAGE and visualized by western blot with anti-N-cadherin antibody. An aliquot of the total lysate was also analyzed by western blot, for comparison. (D) Dissociated E8 chick neural retina cells were incubated for 1 hour at room temperature in the presence of the indicated peptide. Adhesion to immobilized Fc-N-cad is expressed as the percentage of adhesion in the absence of added peptide.

View larger version (21K): [in a new window]   Fig. 6. In vitro phosphorylation of PTP1B by Fer. Purified, phosphatase-dead (C215S) GST-PTP1B was immobilized on glutathione-coated wells and incubated with recombinant wild-type Fer (FerWT) or kinase-dead Fer (FerD743R), and the amount of phosphotyrosine incorporated into PTP1B was measured using anti-phosphotyrosine antibodies. (A) PTP1B carrying the C215S mutation and additional mutations on tyrosine 66 (C215S/Y66F), tyrosine 152 (C215S/Y152F) or both tyrosine 66 and 152 (C215S/Y66/152F) was used as substrate and phosphorylation evaluated by ELISA using HRP-conjugated anti-phosphotyrosine antibody. (B) Immobilized kinase dead GST-PTP1B was incubated with Fer, eluted with SDS sample buffer and the amount of phosphotyrosine evaluated by western blot with anti-phosphotyrosine antibody (top). The same membrane was stripped and reprobed with anti-PTP1B antibody (bottom).

View larger version (28K): [in a new window]   Fig. 7. Effect of FerP on cells expressing mutant ß-catenin. GST, GST/wild-type-ß-catenin, GST/Y142F-ß-catenin or GST/Y654F-ß-catenin were introduced into E8 chick neural retina cells using BioPorter and the cells treated with FerP or FerCo peptides for 1 hour. (A) Cells were lysed in nonionic detergent, immunoprecipitated with anti-cadherin antibody and immunoblotted with the indicated antibody. (B) Intact cells were aliquoted into wells precoated with Fc-N-cad and assayed for adhesion. Adhesion is expressed as the percentage of adhesion among cells treated with GST and CoP.

View larger version (51K): [in a new window]   Fig. 8. Expression and localization of N-cadherin and associated proteins among immortalized fibroblasts derived from wild-type and ferD743R mice. Fibroblasts derived from wild-type (WT) or ferD743R (D743R) embryos were cultured to confluence, washed free of serum and lysed in mild detergent buffer. (A) Equal amounts of protein from cell lysates were analysed by western blot using the indicated antibodies. (B) Equal amounts of protein were immunoprecipitated with anti-pan-cadherin, anti-ß-catenin or anti-PTP1B antibodies and the immunoprecipitates analysed by western blot using the indicated antibodies. (C) WT or D743R cells were stained with Alexa-568/phalloidin and anti-pan-cadherin or anti-ß-catenin antibodies, as indicated, followed by the appropriate Alexa-488-conjugated second antibody, and visualized by confocal microscopy. Among WT cells, cadherin `zippers' are clearly seen at cell boundaries, where actin and cadherin or actin and ß-catenin overlap (arrowhead). In D743R cells, there is no detectable cadherin or ß-catenin at cell boundaries (arrowhead). Scale bar, 10 µm. (D) Aggregation of WT and D743R cells in suspension cultures. Single cells from WT or D743R cultures were prepared by trypsin dissociation and allowed to aggregate in the presence of 1 mM Ca2+ or 5 mM EGTA for 3 hours at 37°C and with shaking at 80 rpm, in 30 mm dishes. Cells were visualized under phase-contrast and photographed using an Axiovert25 microscope and Axiovision system (Zeiss).

View larger version (34K): [in a new window]   Fig. 9. Rescue of D743R cells by expression of wild-type Fer or Y654F ß-catenin. (A) D743R cells were transfected with wild-type Fer cDNA and stable cell lines were selected. Equivalent amounts of protein from cell lysates were analysed by immunoblotting with anti-Fer antibody. `D743R+WTFer' indicates lysates of D743R cells transfected with wild-type Fer cDNA. (B) WT, D743R and D743R cells transfected with wild-type Fer cDNA (D743R + WT) were lysed in neutral detergent, immunoprecipitated with anti-pan-cadherin antibody and analysed by western blot with anti-ß-catenin and anti-PTP1B antibodies. (C) Cells grown on multiple-well slides were fixed, permeabilized and stained with Alexa-568/phalloidin and anti-cadherin or anti-ß-catenin antibody followed by Alexa-488-labeled secondary antibody. Notice that cadherin and ß-catenin are again localized at cell boundaries in D743R cells expressing wild-type Fer. Scale bar, 10 µm. (D) D743R cells were incubated with GST/Y654F-ß-catenin, GST/wild-type-ß-catenin, or GST/Y142F-ß-catenin in the presence of BioPorter, the cells were lysed, immunoprecipitated with anti-GST antibody and immunoblotted with anti-pan-cadherin antibody. The immunoblots were stripped and reprobed with anti-GST antibody. (E) D743R cells expressing GST/Y654F-ß-catenin were visualized under phase contrast and photographed using an Axiovert 25 microscope and Axiovision (Zeiss) after 3 hours at 37°C in 30 mm dishes rotated at 80 rpm.

View larger version (54K): [in a new window]   Fig. 10. Association of cadherin with ß-catenin and PTP1B in mouse tissue and effect of EGF on D743R cells. (A) Brain lysates from wild-type or ferD743R mice were immunoprecipitated with anti-pan-cadherin antibody and analysed by western blot with the indicated antibodies. The relative intensities of the cadherin, ß-catenin and PTP1B bands were determined by densitometry and are presented as ratios. (B) WT or D743R cells grown to semiconfluence were washed free of serum and incubated with or without 100 ng ml–1 EGF for 2 hours. The cells were then lysed in neutral detergent, immunoprecipitated with anti-pan-cadherin antibody and analysed by western blot with anti-ß-catenin and anti-PTP1B antibodies. (C) Alternatively, cells were lysed in RIPA buffer, immunoprecipitated with anti-ß-catenin antibody and analysed by western blot with anti-phosphotyrosine antibody.