Article Figures & Data
Figures
- FIG. 1.
Fer and Fyn kinases phosphorylate β-catenin Tyr-142, and this phosphorylation decreases α-catenin-β-catenin interaction. (A) GST-β-catenin fusion proteins (6.7 pmol, 0.8 μg) were phosphorylated with Fer kinase purified from transfected RWP1 cells as described in Materials and Methods. Phosphorylation was analyzed by Western blotting with anti-PTyr MAb. The membrane was stripped and reanalyzed against β-catenin to check that similar levels of GST-β-catenin were phosphorylated in all cases. (B) Wild-type GST-β-catenin or Tyr-142→Phe mutant (Y142F) was phosphorylated under the conditions indicated, either with Fer purified from transfected RWP1 cells, with Fyn or Yes kinases immunoprecipitated from transfected RWP1 cells, or with recombinant pp60c-src. Samples were analyzed by Western blotting with anti-PTyr MAb and reblotted with anti-β-catenin. (C) GST-β-catenin fusion proteins were phosphorylated with Fer as above. Pull down assays were then performed incubating the GST proteins with 40 μg of total cell extracts from RWP1. The amount of associated α-catenin was determined using specific MAb. WT, wild type β-catenin; Y142F and Y142E correspond to β-catenin mutants Tyr-142→Phe and Tyr-142→Glu, respectively. The estimated molecular masses of the bands detected with each antibody are indicated.
- FIG. 2.
Overexpression of Fer or Fyn kinases in RWP1 induces β-catenin phosphorylation and reduces α-catenin-β-catenin interaction. RWP1 cells were transfected with 5 μg of either pcDNA3-His-Fer (A), pEF-Bos-Fyn (B), or empty vector as control. After 48 h, cell extracts were prepared. (A) As shown in the upper panel, to verify Fer- kinase transfection, 5 μg of untransfected or transfected RWP1 total cell extracts was analyzed by SDS-PAGE and Western blot with anti-Xpress antibody corresponding to a tag that labels the transgene. As shown in the lower panel, 300 μg of the above-mentioned transfected-cell extracts was immunoprecipitated with anti-β-catenin followed by immunoblotting with antibodies against α-catenin, β-catenin, E-cadherin, and PTyr. The estimated molecular masses of the bands detected with each antibody are indicated. (B) Same as in (A) except that cells were transfected with pEF-Bos-Fyn and analyzed with an anti-Fyn MAb.
- FIG. 3.
IEC18 K-ras: an in vivo cell system with reduced α-catenin-β-catenin and E-cadherin-β-catenin interaction due to increased tyrosine phosphorylation. (A) Morphology of IEC18 and IEC18 K-ras cell lines. (B) IEC18 or IEC18 K-ras total cell extracts (5 μg) were analyzed by SDS-PAGE and immunoblotting with antibodies against α-catenin, E-cadherin, β-catenin, p120 catenin, or Pan ras to check the endogenous levels of each protein. (C) Extracts (300 μg) prepared from the indicated cells were immunoprecipitated with anti-β-catenin antibody, and this was followed by immunoblotting with the indicated MAbs. When indicated, genistein (100 μM) (GN) was added to the IEC-K-ras cell medium for 20 h before preparing the extracts.
- FIG. 4.
In IEC18 K-ras β-catenin, Tyr-142 and Tyr-654 are phosphorylated. Phosphorylation of Tyr-142 modulates the α-catenin-β-catenin interaction in vivo. (A) IEC18 K-ras cells were transfected with 5 μg of pcDNA3.1His-β-catenin (wild type or the indicated mutant forms) or empty vector as control. After 48 h, cell extracts were prepared, His-tagged β-catenin was purified by chromatography on nickel-agarose, and the level of phosphorylation was analyzed by Western blotting with PTyr MAb. Membrane was reanalyzed against β-catenin to check that similar levels of expression were obtained in all the cases. Y86F, Y142F, and Y654F correspond to β-catenin mutants Tyr-86→Phe, Tyr-142→Phe, and Tyr-654→Phe, respectively. (B) IEC18 K-ras cells were transfected with 5 μg of pcDNA3.1His-β-catenin (wild type or Tyr-142→Phe). His-tagged β-catenin complexes were analyzed with the indicated MAbs.
- FIG. 5.
Fer and Fyn kinases are activated in IEC18 K-ras. (A) IEC18 or IEC18 K-ras cells were transfected with 5 μg of pcDNA3-His-Fer or empty vector as control. After 48 h, cell extracts were prepared and His-tagged Fer kinase was purified. Proteins bound to the nickel-agarose were subjected to SDS-PAGE and Western blotting with anti-Xpress antibody to verify that similar levels of the transgene were expressed. Membrane was stripped and reblotted to analyze the PTyr content of Fer. (B) The activity of purified Fer was determined by adding 0.4 μg of GST-β-catenin under the phosphorylation conditions indicated in Materials and Methods for 1 h at 30°C. Samples were subjected to SDS-PAGE and Western blotting with antibodies against PTyr, β-catenin, and Xpress (to check that similar amounts of β-catenin and Fer were present in the reactions). (C and D) Assays were carried out as in panels A and B except that IEC18 or IEC18 K-ras cells were transfected with 5 μg of pEF-Bos-Fyn and extracts immunoprecipitated with anti-Fyn antibody. Determination of the β-catenin kinase activity present in the immunoprecipitate was performed as in panel B. The position of immunoglobulin G (IgG) heavy chains is indicated.
- FIG. 6.
p120 catenin recruits Fer and Fyn kinases to the junctional complex. IEC18 or IEC18 K-ras total cell extracts (300 μg) were immunoprecipitated with anti-p120 catenin (A) or anti-Fyn (B) antibodies, and this was followed by immunoblotting with the indicated MAbs. Lane NI corresponds to the result of an immunoprecipitation performed with an irrelevant antibody. (C) GST-cytoE-cadherin (15 pmol) was incubated or not with full-length p120 catenin (prephosphorylated with pp60c-src when indicated). Forty micrograms of total extract prepared from IEC18 K-ras transfected with 5 μg of pcDNA3-His-Fer was added. Protein complexes were pelleted down by affinity on glutathione-Sepharose beads, and proteins bound to the complex were analyzed by SDS-PAGE and Western blotting with MAbs against Fyn, the Xpress epitope, Yes, and p120 catenin. The estimated molecular masses of the bands detected with each antibody are indicated as well as the position of immunoglobulin G (IgG) heavy chains.
- FIG. 7.
Yes is associated with p120 catenin in IEC-K-ras cells. (A) IEC18 or IEC18 K-ras total cell extracts (300 μg) were immunoprecipitated with anti-Yes antibody, and the immune complexes were analyzed by sequential immunoblotting with antibodies against p120 catenin, Fyn, and Yes. Position of immunoglobulin G (IgG) heavy chains is indicated. (B) Eleven picomoles of GST or GST-p120 catenin fusion proteins was phosphorylated by pp60c-src under the conditions indicated in Materials and Methods. Pull down assays were performed with incubation of the GST proteins with 50 μg of total cell extracts prepared from IEC or IEC-K-ras. Presence of Fyn, Yes, or p120 catenin in the complex was analyzed by sequential analysis with specific MAbs. In the lane Input, a sample corresponding to 10% of the total cell extract used for the assay was loaded. The estimated molecular masses of the bands detected with each antibody are shown.
- FIG. 8.
Yes kinase is activated in IEC18 K-ras cells. IEC18 or IEC18 K-ras total cell extracts (300 μg) was immunoprecipitated with anti-Yes antibody. The immune complexes were analyzed with the anti-PTyr MAb (A) or subjected to an in vitro kinase assay with 0.5 μg of enolase (B) or GST-p120 catenin (C) as exogenous substrates. Samples were analyzed by SDS-PAGE and Western blotting with antibody against PTyr. Membranes were reblotted with MAb against Yes. To verify that similar amounts of substrates were used in both cases, Ponceau S staining (B) (central panel) and the anti-p120 blot (C) (central panel) are shown.
- FIG. 9.
Yes kinase activates Fer and Fyn. (A) IEC18 cells were transfected with 5 μg of pMIK-Neo-Yes kinase (wild type or the constitutively active form Tyr-535→Phe) or empty vector. After 48 h, cell extracts were prepared and 200 μg of total cell extracts were immunoprecipitated with anti-Fyn antibody. The immune complexes were analyzed with an anti-PTyr MAb or subjected to an in vitro kinase assay with 0.5 μg of GST-β-catenin as exogenous substrate and analyzed by sequential immunoblotting with antibodies against PTyr and Fyn. The position of immunoglobulin G (IgG) heavy chains is indicated. Very little phosphorylation was obtained when a similar assay was performed using GST-β-catenin (Y142F) as substrate (not shown). (B) IEC18 cells were transfected with 5 μg of pcDNA3-His-Fer kinase. After 48 h, cell extracts were prepared and His-tagged Fer kinase was purified by chromatography on Ni2+-agarose and eluted with 60 μl of lysis buffer plus 200 mM imidazole. In parallel, 300 μg of IEC18 or IEC18 K-ras total cell extracts was immunoprecipitated with anti-Yes antibody. The immune complexes were mixed with 20 μl of eluted-Fer kinase and incubated 1 h at 30°C in conditions indicated for kinase assays. Supernatants containing Fer were separated from the immunocomplexes spinning 30 s in a microcentrifuge and subjected to a GST-β-catenin kinase assay as mentioned above. The extent of tyrosine phosphorylation was analyzed blotting with an anti-kinase PTyr MAb. As control to verify that similar amounts of Yes were immunoprecipitated, immunocomplexes were also analyzed by Western blotting with anti-Yes antibody. Lane NI corresponds to the β-catenin phosphorylation obtained when the 20 μl of eluted-Fer was incubated with an IEC18 K-ras cell extract immunoprecipitated with an irrelevant antibody. No phosphorylation of β-catenin was observed when Fer kinase was omitted from the reaction or when GST-β-catenin (Y142F) was used as substrate. The molecular masses of 120 and 95 kDa correspond to GST-β-catenin and Fer, respectively.
- FIG. 10.
Proposed model of regulation of α-catenin-β-catenin interaction. In canonical adherens junctions, E-cadherin contacts with the actin cytoskeleton through β-catenin and α-catenin. p120 catenin is associated to Fer or/and Fyn kinases and normally is not bound to the E-cadherin complex. Following activation, Yes binds to the p120-Fer/Fyn complex and activates Fer/Fyn kinases. Either Yes, Fer/Fyn, or both phosphorylate Tyr residues in p120 catenin and enhance its binding to E-cadherin. Alternatively, the Fer/Fyn/Yes-p120 catenin complex binds to α-catenin through the association to Rho. The presence of Fer/Fyn kinases in the adhesion complex promotes the phosphorylation of β-catenin Tyr-142 and subsequent disruption of α-catenin-β-catenin interaction. For the sake of clarity, Fer, Fyn, Yes, and Rho proteins have not been depicted bound to the membrane, where they are probably located. For the same reason, p120 catenin is not presented simultaneously bound to Rho and E-cadherin, a fact that cannot be discarded.
