Involvement of the tyrosine kinase fer in cell adhesion - PubMed
Involvement of the tyrosine kinase fer in cell adhesion
R Rosato et al. Mol Cell Biol. 1998 Oct.
Abstract
The Fer protein belongs to the fes/fps family of nontransmembrane receptor tyrosine kinases. Lack of success in attempts to establish a permanent cell line overexpressing it at significant levels suggested a strong negative selection against too much Fer protein and pointed to a critical cellular function for Fer. Using a tetracycline-regulatable expression system, overexpression of Fer in embryonic fibroblasts was shown to evoke a massive rounding up, and the subsequent detachment of the cells from the substratum, which eventually led to cell death. Induction of Fer expression coincided with increased complex formation between Fer and the cadherin/src-associated substrate p120(cas) and elevated tyrosine phosphorylation of p120(cas). beta-Catenin also exhibited clearly increased phosphotyrosine levels, and Fer and beta-catenin were found to be in complex. Significantly, although the levels of alpha-catenin, beta-catenin, and E-cadherin were unaffected by Fer overexpression, decreased amounts of alpha-catenin and beta-catenin were coimmunoprecipitated with E-cadherin, demonstrating a dissolution of adherens junction complexes. A concomitant decrease in levels of phosphotyrosine in the focal adhesion-associated protein p130 was also observed. Together, these results provide a mechanism for explaining the phenotype of cells overexpressing Fer and indicate that the Fer tyrosine kinase has a function in the regulation of cell-cell adhesion.
Figures

Time course of Fer overexpression. Lysates were prepared from cells cultured in the absence of tetracycline (Fer-overexpressing cells) for the indicated times. Each lane contains 20 μg of total cell protein. The filters were blotted with the RC-20 anti-pTyr monoclonal antibody (A) or anti-Fer antisera (B). The location of the 94-kDa Fer kinase is indicated in panel B. WB, Western blot.

Time course of Fer overexpression-induced detachment and reattachment. (A) tet-Fer cells were cultured in the presence (a, Fer expression repressed) or absence (b and c, 15 and 34 h, respectively, after tetracycline withdrawal) of tetracycline, and cell morphology and detachment were evaluated microscopically. (B) Floating cells were collected at different time points as indicated and counted. The bars represent the percentages of floating cells in the total number of cells (attached plus floating cells) at each time point. The control at t = 51 h represents cells grown in the continuous presence of tetracycline. (C) Reattachment of floating cells. Cells which had been floating for the indicated amount of time after tetracycline withdrawal were reincubated with tetracycline-containing medium to repress Fer expression. The percentage of cells from each time point which either re-attached or remained floating is indicated after 24 h in medium with tetracycline. The data shown are representative of two independently performed experiments.

Cell cycle analysis of cells after Fer induction. (A) Flow cytometry. DNA content of control cells lacking Fer induction was compared to DNA content of floating cells after 1.5 days of Fer overexpression. Black areas denote G0/G1 (left peak, 2C DNA) or G2/M (right peak, 4C DNA) populations. The hatched area in between represents the population of cells in S phase (2C-4C DNA). The percentage of cells in each phase was calculated by including only live cells (population of cells with less than 2C DNA was excluded). PI, propidium iodide. (B) DNA laddering. DNAs were isolated from the same cultures depicted in panel A, 1.5 days after tetracycline withdrawal, and run on a 1.5% agarose gel. Each lane contains 10 μg of DNA. Lane 1, cells grown in the presence of tetracycline; lane 2, Fer-overexpressing attached cells; lane 3, Fer-overexpressing floating cells.

Decreased tyrosine phosphorylation of the Crk-associated substrate p130 but not of the focal adhesion kinase Fak in Fer-overexpressing cells. This figure is representative of three independently performed experiments. Total lysates or immunoprecipitates were blotted with anti-p130 monoclonal antibodies (A) or anti-FAK monoclonal antibodies (B). as indicated below each panel. The locations of p130 and p125FAK are indicated. IgL, immunoglobulin light chain. WB, Western blot.

Association of p120cas with Fer. Lysates were prepared at 0, 12, and 24 h after withdrawal of tetracycline, which induces Fer expression. Total lysates or immunoprecipitates were blotted with anti-Cas monoclonal (A), anti-pTyr monoclonal (B), or anti-Fer polyclonal (C) antibodies as indicated below the panels. The locations of p120cas and p94Fer (FER) are indicated. WB, Western blot.

Induction of Fer expression modifies the composition of the E-cadherin–catenin complex. Cells were at confluency. Antibodies used for immunoprecipitation (IP) or Western blotting (WB) of whole-cell lysates (A) and E-cadherin immunoprecipitates (B) are indicated. IgH, immunoglobulin heavy chain; IgL, immunoglobulin light chain.

α-Catenin is unaffected by Fer overexpression. Lysates (A) and immunoprecipitates (B) were prepared as described in the legend to Fig. 4 and immunoprecipitated with anti-α-catenin monoclonal antibodies. Antibodies used for Western blotting (WB.) are indicated below the panels. The location of the 102-kDa α-catenin protein is indicated. IgH, immunoglobulin heavy chain; IgL, immunoglobulin light chain.

β-catenin coimmunoprecipitates with Fer and becomes tyrosine phosphorylated upon induction of Fer expression. Lysates (A) and β-catenin (B), Fer (C), and p120cas (D) immunoprecipitates were prepared at 0, 12, and 24 h after withdrawal of tetracycline. Antibodies used for immunoprecipitation (IP) and Western blotting (WB) are indicated above and below each panel, respectively. The locations of β-catenin, α-catenin, and Fer are indicated. Note that the extra lower-molecular-weight species which reacts with the β-catenin antibodies and is seen in some of the extracts (but not lysates, which are processed more rapidly) most likely represents partial proteolytic degradation products of β-catenin as indicated in reference . IgH, immunoglobulin heavy chain.
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