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First published online 24 October 2006
doi: 10.1242/jcs.03236

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Tyrosine phosphorylation of p145^met mediated by EGFR and Src is required for serum-independent survival of human bladder carcinoma cells

¹ Graduate School of Science and Technology, Kobe University, Nada, Kobe 657-8501, Japan
² Department of Internal Medicine, University of Virginia School of Medicine, Charlottesville, Virginia 22903, USA
³ Laboratory of Molecular Biology, Research Center for Environmental Genomics, Kobe University, Nada, Kobe 657-8501, Japan

View larger version (50K): [in a new window]   Fig. 1. Serum starvation promotes tyrosine phosphorylation of a 145 kDa protein in a Src- and EGFR-dependent manner in bladder carcinoma 5637 cells. (A) Human 5637 cells (1x106 cells/dish) were cultured in normal conditions (10% FCS) for 48 hours. After the treatment, cells were cultured for an additional 48 hours in normal conditions (), 10% FCS plus 10 µM PP2 (), without serum () or without serum plus 10 µM PP2 (). Cell number was determined at 24, 48, 72 and 96 hours post initial treatment. (B) Whole cell extracts (30 µg/lane) were prepared from carcinoma 5637 cells that had been cultured in normal (10% FCS, denoted FCS) or serum-free medium (for 1 to 48 hours), and analyzed by immunoblotting (IB) with an anti-phosphotyrosine antibody (PY99), an antibody against the activated and tyrosine-phosphorylated Src family protein-tyrosine kinases (pSFK) (pY418) or an anti-ß tubulin antibody. In the top panel (IB: PY99), the positions of a 145 kDa tyrosine-phosphorylated protein (pp145) and molecular size markers (25-250 kDa) are indicated. (C) 5637 cells were cultured in normal (+ FCS) or serum-free (– FCS) medium for 24 hours. For the final 1 hour of the serum starvation treatment, cells were incubated in the absence or the presence of the following substances: 50 µM genistein (Gen), 50 µM daidzein (Ddz), 10 µM PP2, 10 µM PP3, 1 µM SU6656 or 5 µM AG99. Whole cell extracts (30 µg/lane) were analyzed by immunoblotting with anti-phosphotyrosine antibody. The position of pp145 is indicated. (D) Carcinoma 5637 cells were treated as in panel A. After the treatments (96 hours post treatment), cell death (black bars) and caspase 3/7 protease activity (grey bars) of the whole cell extracts (20 µg/assay) were determined by Trypan Blue exclusion and a synthetic substrate Ac-DEVD-AMC, respectively. Data shown are mean ± s.d. of three independent experiments. *P<0.01 compared with control.

View larger version (49K): [in a new window]   Fig. 2. Activation of SFKs and EGFR in serum-starved 5637 cells. Whole cell extracts (300 µg/lane) were prepared from carcinoma 5637 cells that had been cultured in normal (10% FCS, denoted `+') or serum-free conditions (for 24 hours, denoted `–') and subjected to immunoprecipitation (IP) with anti-Src antibody, anti-Yes antibody, anti-Fyn antibody or anti-EGFR antibody as described. The immunoprecipitates were analyzed by immunoblotting with the homologous antibody (Src, Yes, Fyn, or EGFR), anti-activated SFK antibody (pY418), an antibody that recognizes the inactive, carboxyl-terminal tyrosine-phosphorylated form of SFKs (pY529) and anti-phosphotyrosine antibody PY99. Asterisks indicate the positions of each protein.

View larger version (48K): [in a new window]   Fig. 3. Molecular identification of pp145 as ß-subunit of c-Met protein. (A) Immunopurification of pp145. Whole cell extracts were prepared from carcinoma 5637 cells cultured in serum-starved conditions for 24 hours. The extracts (–FCS, 300 µg/lane) were subjected to immunoprecipitation with anti-phosphotyrosine antibody and the immunoprecipitates were analyzed by silver staining. A control immunoprecipitate prepared from the normally grown cell extracts (+ FCS, 300 µg/lane) was also analyzed. The position of a 145 kDa protein (pp145) is indicated. (B) MS identification of four peptide fragments annotated to be part of c-Met. Mass values of four peptide fragments were obtained from the 145 kDa tyrosine-phosphorylated protein digested with trypsin (see Materials and Methods for detail) and that were annotated to be part of the human p145met using the MASCOT database search algorithm. Also shown are amino acid numbers (start-end) and expected (expt) as well as calculated mass values (calc) for each peptide fragment. Note that a peptide fragment annotated to be the amino acid residues 988-1004 contained mass value equivalent to one phosphate. (C) Schematic structure of p145met. The whole amino acid sequence (1390 amino acids) of the human p145met is shown. The amino acid sequences matching those of the known partial sequences by mass spectrometry analysis (see panel B) are shown in red. Also shown are the N-terminal signal sequence (residues 1-24, underlined), a C-terminal end of potential proteolytic cleavage site (residues 303-307, arrowhead), transmembrane region (residues 933-955, dashed line), catalytic domain (residues 1078-1345, squared), and tyrosine phosphorylation sites (residues 1003, 1230, 1234, 1235, 1313, 1349 and 1365, underlined in bold).

View larger version (56K): [in a new window]   Fig. 4. Identification and characterization of p145met, which is predominantly tyrosine phosphorylated in serum-starved 5637 cells. (A) Whole cell extracts were prepared from normally grown (control), HGF-treated (+ HGF, 125 ng/ml, 30 minutes), or serum-starved (– FCS, 24 hours) 5637 cells and immunoprecipitated (300 µg/lane) with either anti-Met antibody (IP: Met) or anti-phosphotyrosine antibody PY99 (IP: PY99). The immunoprecipitates were analyzed by immunoblotting with either anti-Met antibody (IB: Met) or anti-phosphotyrosine antibody PY99. The whole cell extracts (W, 30 µg/lane) were also analyzed by direct immunoblotting to serve as positive controls. Note that the position of p145met matches exactly the position of the tyrosine-phosphorylated p145. Asterisks indicate the positions of p180met, the precursor form of p145met. (B) Whole cell extracts prepared as in A (control, + HGF, and – FCS: each 600 µg/analysis) were immunoprecipitated with anti-Met antibody and analyzed by immunoblotting with anti-Met antibody or phospho-specific anti-Met antibodies: pY1003, pY1234/1235, pY1349 or pY1365. The positions of the unphosphorylated or phosphorylated p145 [p(p)145met] are indicated. (C) Carcinoma 5637 cells were cultured in normal (10% FCS, 1 hour) or serum-free condition (24 hours) in the absence or the presence of HGF (50 µg/ml), K252a (10 µM) and U0126 (10 µM). After treatment, cells were extracted and examined for phosphorylation of p145met (IB: PY99) and p42/p44 MAPK (IB: pMAPK) (30 µg/lane). (D) Carcinoma 5637 cells were treated with either HGF (250 ng/ml) or serum-free medium (–FCS) for the indicated times. Whole cell extracts (30 µg/lane) were prepared and analyzed by immunoblotting with anti-Met antibody. The positions of p180met (asterisk) and p145met are indicated.

View larger version (34K): [in a new window]   Fig. 5. EGFR ligands and EGFR act as an upstream regulator of tyrosine phosphorylation of p145met, activation of Src and survival in serum-starved 5637 cells. (A) Normally grown 5637 cells (+ FCS) were treated with 125 ng/ml HGF (+ FCS/HGF), conditioned RPMI1640 media from 24-hour normally grown 5637 cells (Cond./+ FCS med), conditioned RPMI1640 media from 24-hour serum-starved 5637 cells (Cond./– FCS med), or fresh RPMI1640 medium containing either 10% FCS (Fresh/+ FCS med) or no FCS (Fresh/– FCS med) for 1 hour. After treatment, whole cell extracts (20 µg/lane) were prepared and analyzed by immunoblotting with either anti-phosphotyrosine antibody PY99 or anti-phosphoMAPK antibody. The positions of pp145met, pp44, and pp42 are indicated. (B) Normally grown 5637 cells were treated with the indicated concentrations (0, 1 or 10 ng/ml) of EGF for 10 minutes in the absence or presence of 1 µg/ml anti-EGFR monoclonal antibody mAb528 or 1 µg/ml normal mouse IgG. After treatment, whole cell extracts were prepared and analyzed by immunoprecipitation (200 µg/lane) and/or immunoblotting (20 µg/lane for direct analysis) with the indicated antibodies. The positions of pp170, pp145, pp60src and p170/EGFR are indicated. (C) Whole cell extracts were prepared from 5637 cells that had been treated with or without 10 ng/ml EGF for 10 minutes as in panel B, and subjected to immunoprecipitation (200 µg/lane) with either anti-EGFR or anti-Met antibodies followed by immunoblotting with either anti-phosphotyrosine antibody PY99, anti-EGFR or anti-Met antibodies. The positions of pp170, pp145, p170/EGFR and p145met are indicated. (D) Carcinoma 5637 cells were treated with either normal medium (10% FCS) containing 125 ng/ml HGF for 1 hour or serum-free medium for 24 hours in the absence or the presence of 1.25 mM CuCl2, 1.25 mM MgCl2, 1 µg/ml anti-EGFR monoclonal antibody mAb528 or 1 µg/ml normal mouse IgG. After the treatments, whole cell extracts (20 µg/lane) were prepared and analyzed by immunoblotting with either anti-phosphotyrosine antibody PY99 or anti-Met antibody. The positions of pp145met, p145met and p180met (asterisk) are indicated. (E) Carcinoma 5637 cells (1x106 cells/dish) were cultured in normal conditions (10% FCS) for 48 hours, and then further cultured for an additional 48 hours in serum-free medium containing none (closed circles), 1 µg/ml anti-EGFR monoclonal antibody mAb528 (closed triangles) or 1 µg/ml normal mouse IgG (closed squares). Cell number was determined at 24, 48, 72 and 96 hour post initial treatment as in Fig. 1A. (F) Carcinoma 5637 cells were treated as in E. After treatment (96 hours post treatment), cell death (black bar) and caspase 3/7 protease activity (grey bar) of the whole cell extracts (20 µg/assay) were determined as in Fig. 1D. Data shown are mean ± s.d. of three independent experiments. *P<0.01 compared with levels in the control.

View larger version (37K): [in a new window]   Fig. 6. p145met is required for survival of serum-starved 5637 cells: effect of HGF-induced down-regulation of p145met. (A) Normally grown 5637 cells (C, control) were treated with 10% FCS plus 125 ng/ml HGF for 24 hours (H, HGF-treated). After treatment, the cells were further treated for the indicated times (1-24 hours) in new medium containing 10% FCS plus 125 ng/ml HGF (10% FCS) or 125 ng/ml HGF alone (no FCS). After treatment, whole cell extracts were prepared and analyzed by immunoprecipitation and/or immunoblotting as in Fig. 1B, Fig. 4C and Fig. 5B. The positions of pp145met, p145met, pp60src, p60src, pp44 and pp42 are indicated. (B) Carcinoma 5637 cells (1x106 cells/dish) were cultured in normal conditions (10% FCS) for 24 hours, and then cultured for 24 hours in 10% FCS plus 125 ng/ml HGF () or 10% FCS plus 125 ng/ml HGF plus 1.25 mM CuCl2 (). After treatment, cells were treated for an additional 48 hours in serum-free media. Cell number was determined at 24, 48, 72 and 96 hours post initial treatment as in Fig. 1A. As a control, data obtained with normally grown cells (10% FCS, 48 hours) and serum-starved cells (– FCS, additional 48 hours) are shown (). (C) Carcinoma 5637 cells were treated as in panel B. After treatment (96 hours post treatment), cell death (black bars) and caspase 3/7 protease activity (grey bars) of the whole cell extracts (20 µg/assay) were determined as in Fig. 1D. Data shown are representative of three independent experiments. Results are the mean ± s.d. of three independent experiments. *P<0.01 compared with levels in the control.

View larger version (5K): [in a new window]   Fig. 7. Schematic model for signal transduction pathways in serum-starved 5637 cells. In carcinoma 5637 cells, serum starvation promotes signaling events involving EGFR ligands, EGFR, SFKs (e.g. Src) and p145met to suppress activation of caspase-like proteases. Serum-starved cells can therefore survive and proliferate. In this study, several materials are used to validate this scheme: positive regulators such as conditioned medium and purified EGF, but not HGF; negative regulators such as a neutralizing anti-EGFR antibody (mAb528), PP2, SU6656, AG99 and downregulation of p145met, but not K252a. See text for details.

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