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First published online 2 March 2004
doi: 10.1242/jcs.01001

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EGFR signaling to p120-catenin through phosphorylation at Y228

Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, TN 37232-6840, USA

View larger version (37K): [in a new window]   Fig. 1. Characterization of p120 Y228 phosphospecific monoclonal antibody. (A) Detection of phosphorylated p120 on whole cell lysate western blots. 3T3 cells with or without expression of transforming Src 527F were washed for 2 minutes with PBS containing pervanadate, then lysed with RIPA containing pervanadate. Whole cell lysates were separated by SDS-PAGE on a 7% gel and western blotted with mAb pY228 to phosphorylated p120, mAb pp120 to total p120, and mAb PY20, a general phosphotyrosine antibody. The entire blot is shown. (B) mAb pY228 detects p120 by immunoprecipitation. p120, Y228-phosphorylated p120, or various tyrosine phosphorylated proteins were immunoprecipitated from Src-transformed 3T3 cell lysate with mAb 15D2, mAb pY228, or PY20, respectively. Immunoprecipitation with HA-tag antibody 12CA5 served as a negative control. Immunoprecipitates were then western blotted with either mAb pY228, pp120, or PY20. The entire blots are shown for the pY228 and PY20 panels. (C) mAb pY228 is specific for phosphorylated p120. p120 was immunoprecipitated from A431 cells with p120 mAb 15D2. Immunoprecipitates were treated with or without lambda protein phosphatase (New England Biolabs) for 30 minutes at 30°C. Samples were western blotted with mAb pY228 or pp120. (D) mAb pY228 is specific for p120 phosphorylated at Y228. Cos-7 cells were transiently co-transfected with transforming Src (RcRSV c-Src 527F) together with either empty RcRSV vector (lane 1), or RcRSV vector containing mp120-1A (lane 2), mp120-1A/8F (lane 3), or mp120-1A/228F (lane 4), using Superfect reagent (Qiagen). Transfected p120 was specifically immunoprecipitated with murine-specific p120 mAb 8D11, and western blotted with mAb pY228, pp120 or PY20.

View larger version (75K): [in a new window]   Fig. 2. EGFR activation induces p120 phosphorylation at Y228. A431 cells were serum starved for 16-18 hours, then stimulated for 5 minutes with (+) or without (–) EGF (100 ng/ml). (A) Biochemical characterization of p120 mAB pY228. After EGF treatment, cells were lysed in RIPA buffer containing pervanadate. Lysates were probed by western blotting with a p120 phosphospecific mAB to Y228 (pY 228) or a general p120 mAB (pp120). EGFR immunoprecipitates were blotted with PY20 (EGFR, PY20), a general phosphotyrosine antibody, or with an EGFR monoclonal antibody (EGFR). (B) Immunofluorescent localization of Y228-phospho-p120 before (–) and after (+) EGF treatment. After EGF treatment, cells were washed for 2 minutes with PBS (containing pervanadate), then fixed with methanol. Y228-phosphorylated p120 was stained with mAb pY228 (green). Total p120 was stained with pAb F1SH (red). The individual images were merged to facilitate comparison (merge). 40x magnification. (C) Coimmunoprecipitation of Y228-phospho-p120 with E-cadherin. E-cadherin was immunoprecipitated with mAb HECD-1 before (–) and after (+) EGF treatment. Immunoprecipitates were probed by western blotting for E-cadherin (E-cad), p120 and Y228-phosphorylated p120 (pY228). Total p120 and Y228-phosphorylated p120 levels in the starting whole cell lysate (WCL) were also analyzed by western blotting.

View larger version (32K): [in a new window]   Fig. 3. Constitutive high level phosphorylation of p120 at Y228 in carcinoma cell lines. (A) Variable levels of p120 phosphorylation at Y228. p120 was immunoprecipitated from the indicated human carcinoma cell lines and western blotted with mAb pY228 (pY228) or mAb pp120 (p120). E-cadherin levels were monitored by western blotting of whole cell lysates with mAb HECD-1. (B) Cadherin-dependent p120 phosphorylation. SkBr3 and MDA-MB-453 cells, which lack E-cadherin expression, were infected with control or E-cadherin containing retrovirus. Polyclonal cell lines were generated by selection in G418. Immunoprecipitated p120 was detected by western blotting with mAb pY228 (pY228) or mAb pp120 (p120). E-cadherin was detected in whole cell lysates by western blotting with mAb HECD-1 (E-cadherin). All cells were washed with PBS containing pervanadate for 2 minutes prior to lysis with pervanadate-containing RIPA buffer.

View larger version (70K): [in a new window]   Fig. 4. p120 phosphorylation at Y228 during adherens junction formation. (A) Characterization of a p120 knockdown and add-back model system. Endogenous p120 levels were stably knocked down with retrovirally transduced siRNA specific for human p120 (pRS.h) in parental A431 cells, and clonally derived pRS.h-expressing A431 cell lines were subsequently infected with retroviral vector alone or murine p120 constructs WT-mp120-3A or mp120-3A/7F (which contains phenylalanine mutations at the seven known p120 Y phosphorylation sites). Murine p120 is unaffected by the human p120 siRNA because of mismatches at the nucleotide level. RIPA lysates were probed by western blotting. Human and mouse p120 together were detected by western blotting with mAb pp120 (Total p120). Tubulin was simultaneously detected as a loading control. Murine p120 (mp120-3A) was selectively detected with the murine-specific mAb 8D11. E-cadherin expression was detected with HECD-1. (B) Morphologic effects of WT- and 7F-p120 expression in p120 knockdown cells. The cells lines in A were trypsinized, plated overnight and photographed on a phase contrast microscope (10x magnification). No differences were detected between cells rescued with wild type (mp120-3A) and mutant (mp120-3A/7F) p120. (C) Effects of junction formation on p120 Y228 phosphorylation. p120 Y228 phosphorylation was monitored by western blotting of cell lysates after calcium switch (see Materials and Methods). Total p120 (p120) and Y228 phosphorylated p120 (pY228) were quantitated in the presence of calcium (NS=no switch), or at the indicated times after calcium add-back. (D) Effects of EGFR inhibition. The experiment in C was repeated in the presence of the EGFR inhibitor AG1478 (300 nM). (E) A431 pRS.h (p120 knockdown) cells expressing mp120-3A or mp120-3A/7F were subjected to calcium switch assay as in C, and then examined by immunofluorescence with p120-specific antibody pAb F1SH at the indicated times after add-back of calcium (63x magnification).

View larger version (41K): [in a new window]   Fig. 5. The major p120 Y phosphorylation sites are not required for E-cadherin-mediated strong cell adhesion. Hanging drop aggregation assays were conducted with p120-deficient cells expressing human p120 siRNA alone (vector), or the same cells reconstituted with murine p120 constructs expressing WT-p120 (mp120-3A) or mutant p120 (mp120-3A/7F). The latter construct contained phenylalanine (F) mutations at the seven major sites of p120 tyrosine phosphorylation. Cells were photographed on a phase-contrast light microscope (10x magnification).

View larger version (31K): [in a new window]   Fig. 6. Effects of EGFR and Src inhibitors on p120 Y228 phosphorylation. (A) A431 cells were treated for 1 hour with DMSO alone or DMSO containing the indicated inhibitors. Drugs concentrations were as follows: AG1478, 300 nM; EKI, 1 µM; PP1, 5 µM; SU6656, 10 µM. Cells were then washed for 2 minutes with PBS containing pervanadate, then lysed with RIPA containing pervanadate. Cell lysates were blotted with mAb pY228 (pY 228) or mAb pp120 (p120). EGFR was immunoprecipitated from whole cell lysates and western blotted with antibodies to phosphotyrosine (EGFR, PY20) or EGFR (EGFR). (B) A431 cells were prepared as described in A, except that cells were treated with increasing concentrations of SU6656 as indicated. Whole cell lysates were western blotted with mAb pp120 or mAb pY228.

View larger version (43K): [in a new window]   Fig. 7. EGF signaling to p120 Y228 in A431 cells is independent of Src. (A) Validation of SU6656 activity. Phosphorylation of p120 by Src was monitored by in vitro kinase assay in the presence of increasing concentrations of the Src-family inhibitor SU6656. 32P-labeled samples were separated by SDS-PAGE, transferred to nitrocellulose, and visualized by autoradiography (15-minute exposure). As a control for sample loading, p120 was subsequently quantitated by western blotting with mAb-pp120 (5-second exposure). (B) EGF-induced p120 phosphorylation at Y228 in A431 cells is not blocked by SU6656. A431 cells were serum starved for 18 hours, then treated for 1 hour with DMSO alone or DMSO containing the indicated concentrations of SU6656. Cells were then incubated for 5 minutes with or without EGF (100 ng/ml), and lysed in RIPA containing pervanadate. Total p120 (p120) and Y228-phosphorylated p120 (pY228) levels were quantitated by western blotting. EGFR phosphorylation was determined by western blotting EGFR immunoprecipitates with the phosphotyrosine-specific mAb PY20 (EGFR, PY20) or with an EGFR-specific mAb (EGFR). (C) Src-independent EGF-induced p120 phosphorylation in HER-14 cells. HER-14 cells are murine Swiss 3T3 fibroblasts stably transfected with a human EGFR construct. The cells were serum starved for 12.5 hours in the presence of 4 µg/ml each insulin and transferrin to improve viability, then incubated for one hour with DMSO alone or with DMSO containing 2 µM SU6656. Cells were then treated for 5 minutes with or without EGF (100 ng/ml) prior to lysis in RIPA including pervanadate. p120 immunoprecipitates generated with mAb 15D2 were divided, and then western blotted with mAb pY228 or mAb pp120. (D) EGF induces p120 Y228 phosphorylation in the absence of Src kinase activity. SYF cells are murine embryonic fibroblasts derived from mice that are null for Src, Yes and Fyn. Other Src-family kinases have not been detected in these cells. Polyclonal EGFR-expressing cell lines were generated from SYF cells expressing either c-Src (+c-Src) or kinase dead c-Src (kd-Src). Cells were serum starved for 24 hours, EGF stimulated (100 ng/ml) and analyzed as described in B for p120 phosphorylation at Y228.

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