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First published online 30 September 2008
doi: 10.1242/jcs.035691

Journal of Cell Science 121, 3335-3346 (2008)
Published by The Company of Biologists 2008
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SHP-2 is a novel target of Abl kinases during cell proliferation

Sayan Mitra1, Carol Beach2, Gen-Sheng Feng3 and Rina Plattner1,*

1 Department of Molecular and Biomedical Pharmacology, University of Kentucky, Lexington, KY 40536, USA
2 Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY 40536, USA
3 Burnham Institute for Medical Research, La Jolla, CA 92037, USA


Figure 1
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  		Fig. 1. Abl kinases phosphorylate SHP-2. (A) SHP-2 was immunoprecipitated from 293T cells transfected with SHP-2 and kinase-inactive (KR), wild-type (WT), or constitutively active forms (PP) of Abl or Arg, and blotted with phosphotyrosine antibody (4G10/PY99; top). The blot was stripped and reprobed with SHP-2 antibody (middle). Results are representative of three independent experiments. (B) Immunoprecipitated Abl kinases were incubated in a `cold' kinase assay with GST, GST-SHP-2, or GST-Crk, and probed with anti-phosphotyrosine antibody. Arg-WT was expressed to a greater extent than Arg-PP (data not shown). Data are representative of three independent experiments. (C) Serum-starved 10T1/2-EGFR cells were pretreated with STI571 (10 µM) or vehicle (water) for 4 hours, stimulated with EGF (100 ng/ml), SHP-2 was immunoprecipitated from the lysates, and probed with anti-phosphotyrosine antibody (top). Percentage phosphorylation is relative to total immunoprecipitated SHP-2 protein, and is expressed as a percentage of phosphorylation observed in untreated cells. (D) SHP-2 phosphorylation (relative to total protein levels) from STI571-treated cells (as in C) was compared to untreated cells and expressed as a percentage of untreated. Results from three independent experiments are shown (mean ± s.e.m.). *P≤0.05 using a ratio paired t-test. (E) 10T1/2-EGFR cells, transfected with Abl or Arg siRNAs (40 nM), were starved and stimulated with EGF for 30 minutes, SHP-2 was immunoprecipitated and blotted with phosphotyrosine antibody. The blot was stripped and reprobed with SHP-2 antibody. SHP-2 phosphorylation (relative to total protein levels) from Abl or Arg siRNA-transfected cells was compared with scrambled control-transfected cells and expressed as a percentage of scrambled control. Since Arg antibodies do not work well for western blots, an in vitro kinase assay was utilized to determine knockdown efficiency (Srinivasan and Plattner, 2006Go). `Percentage knockdown' is the decrease in protein expression or activity relative to the scrambled control. (F) SHP-2 phosphorylation (relative to total protein levels) from Abl or Arg siRNA-transfected cells (E) was compared with scrambled control-transfected cells and expressed as a percentage of scrambled. Results from three independent experiments are shown; values are mean ± s.e.m. *P≤0.05, **P<0.005 using a one-way ANOVA followed by a Bonferroni post-hoc test.

 

Figure 2
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  		Fig. 2. Abl kinases phosphorylate SHP-2 on Y580 and induce phosphorylation of SHP-2 on Y63 and Y279. (A) GST-SHP-2 was phosphorylated by Abl kinases in vitro, and analyzed by mass spectrometry. For identification of SHP-2 phosphorylation sites in cells, Abl and Arg were co-expressed with SHP-2 in 293T cells, and immunoprecipitated SHP-2 was analyzed by mass spectrometry. Coverage is the percentage of protein recovered as peptides. ND, not done. (B) Wild-type (WT) or mutant forms of SHP-2 and Abl or Arg were co-expressed in 293T cells, SHP-2 was immunoprecipitated and probed with phosphotyrosine antibody (top). Percentage phosphorylation is relative to total immunoprecipitated SHP-2 protein obtained from reprobed blots. Results shown are representative of three independent experiments. (C) Structure of SHP-2 and putative Abl kinase phosphorylation sites. (D) Immunoprecipitated Abl kinases were incubated in an in vitro kinase assay with wild-type or mutant forms of GST-SHP-2. Coomassie Blue staining showed that SHP-2 proteins were equivalent (data not shown). Results shown are representative of three independent experiments.

 

Figure 3
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  		Fig. 3. Endogenous Abl kinases phosphorylate SHP-2 on Y580, and are required for sustained ERK activation in response to PDGF. (A) Serum-starved 10T1/2-EGFR cells were pretreated with STI571 (10 µM), stimulated with EGF, and lysates were probed with phospho-specific SHP-2 antibodies (Y580, Y542). Percentage phosphorylation is relative to total SHP-2 protein levels obtained from reprobed blots. (B) Percentage phosphorylation in lysates probed with phospho-SHP-2-Y580 antibody (as in A). Values are mean ± s.e.m. of three independent experiments. *P≤0.05 using a ratio paired t-test. (C) 10T1/2-EGFR or NIH3T3 cells, transfected with Abl or Arg siRNAs (40 nM), were serum-starved, stimulated with EGF (100 ng/ml; 10T1/2-EGFR) or PDGF-BB (12.5 ng/ml; NIH3T3) for 30 or 10 minutes, respectively, and lysates were probed with the indicated antibodies. SHP-2-Y580 phosphorylation in Abl or Arg siRNA-transfected cells was expressed as a percentage of SHP-2-Y580 phosphorylation observed in scrambled control-transfected cells and is relative to total SHP-2 protein levels obtained from reprobed blots. (D) Results from three independent experiments as in C. Values are mean ± s.e.m.*P≤0.05, ***P≤0.005 using a one-way ANOVA followed by a Bonferroni post-hoc test. (E) NIH3T3 cells, transfected with siRNAs, were serum-starved, stimulated with PDGF-BB (12.5 ng/ml) for 30 minutes, and phosphorylation of ERK1/2 was assessed by western blotting with a phospho-specific antibody [T(P)EY(P)]. ERK phosphorylation shown is relative to total ERK protein levels obtained from reprobed blots, and is expressed as a percentage of phosphorylation observed in scrambled control-transfected cells. (F) Results from three independent experiments as in E. Values are mean ± s.e.m.*P≤0.05, **P≤0.005, ***P≤0.001 using a one-way ANOVA followed by a Bonferroni post-hoc test.

 

Figure 4
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  		Fig. 4. SHP-2 phosphorylation mutants have altered activities towards phosphotyrosine-containing substrates. (A) Wild-type or mutant forms of SHP-2 were immunoprecipitated from serum-starved, overexpressing 293T cells, incubated with pNPP substrate and supernatant absorbance at 405 nm (A405) was measured. Some error bars are too small to be visible. (B) Wild-type or mutant forms of SHP-2 were immunoprecipitated from serum-starved overexpressing 293T cells, incubated with 250 µM substrate [R-R-L-I-E-D-A-E-Y(P)-A-A-R-G] for 30 minutes at 37°C, and phosphate release into the supernatant was quantified by incubation with Malachite Green, and measurement of A620. Absorbance values were compared to a standard phosphate curve to determine pmoles of phosphate released. Values obtained for mutant forms of SHP-2 were expressed as a percentage of wild-type; values are mean ± s.e.m. from three independent experiments. ***P≤0.001 using one-way ANOVAs followed by Bonferroni post-hoc tests. Immunoprecipitates were blotted with SHP-2 antibody (representative experiments are shown). (C) SHP-2 immunoprecipitates from expressing 293T cells (as in B) were incubated in a phosphatase assay with increasing concentrations of peptide substrate. Velocity values were converted to pmol/minute/pmol of enzyme utilized. A representative experiment is shown. (D) Velocity values from two independent experiments (as in C) were standardized by converting the values to percentage of the maximum obtained in each experiment; values are mean ± s.e.m. (some error bars are too small to be visible).

 

Figure 5
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  		Fig. 5. Expression of SHP-2 Y63F and Y279F mutants increases EGF-induced sustained ERK phosphorylation in 293T cells. (A,C) 293T cells were transfected with plasmids encoding wild-type or mutant forms of SHP-2 (10 µg), Gab1 (4 µg) and HA-tagged ERK2 (1 µg), cells were serum-starved, stimulated with EGF for the indicated times, and lysates were probed with phospho-ERK1/2 antibody. Only HA-tagged ERK2, which migrates slower than endogenous ERK1/2, is shown. Blots were stripped and reprobed with HA antibody. (B,D) Values for three unstimulated (as in A) or four EGF-stimulation experiments (as in C). Levels of phosphorylated HA-ERK2 relative to total HA-ERK2 for cells expressing SHP-2 mutants were expressed as a percentage of HA-ERK2 phosphorylation observed in cells expressing wild-type SHP-2. Values are mean ± s.e.m. *P≤0.05, **P≤0.03, ***P≤0.001, t-tests.

 

Figure 6
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  		Fig. 6. Expression of SHP-2-Y279F increases PDGF-induced sustained ERK phosphorylation and proliferation in fibroblasts. (A,B) Lysates from serum-starved, PDGF-stimulated NIH3T3 cells, infected with wild-type or mutant SHP-2 retroviruses, were blotted with phospho-ERK1/2 antibody. Changes in ERK1/2 phosphorylation shown are relative to total ERK1/2 levels obtained from reprobed blots. Different exposures are shown for each time point, so that differences between mutants can easily be seen. Results are expressed as a percentage of wild type. (B) Results from three independent experiments (as in A); values are mean ± s.e.m. *P≤0.05, **P≤0.02, ***P≤0.005, using t-tests. (C,D) Tritiated thymidine incorporation was assessed in infected NIH3T3 cells labeled with tritiated thymidine for 24 hours. (C,D) One representative experiment (C; mean ± s.d.), and (D) a composite of three independent experiments in which tritiated thymidine incorporation was compared in cells expressing SHP-2 mutants and in cells expressing wild-type SHP-2. Values are mean ± s.e.m. (some error bars are too small to be visible). Aliquots of infected cells were lysed and probed with SHP-2 antibody. *P≤0.05, ***P≤0.001 using a one-way ANOVA followed by a Bonferroni post-hoc test.

 

Figure 7
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  		Fig. 7. SHP-2 lies downstream of Abl kinases during PDGF-mediated mitogenesis. Abl/Arg double null fibroblasts, infected with vectors (Migr1, MigCD4, PK1), Abl and Arg (MigCD4-Abl, PK1-Arg), and/or wild-type or mutant forms of SHP-2 (Migr1-SHP2-C459S, Migr1-SHP-2-E76K), were serum-starved, stimulated with PDGF-BB (12.5 ng/ml) for 16-20 hours, pulsed with tritiated thymidine, and harvested. (A) Results from one representative experiment; values are mean ± s.d. (some error bars are too small to be visible). Lysates from infected cells were probed with the indicated antibodies. (B) Composite of three independent experiments; values are mean ± s.e.m. Tritiated thymidine incorporation is expressed as a percentage of vector-transfected cells. *P≤0.05, **P≤0.01, ***P≤0.001, using a one-way ANOVA followed by a Bonferroni post-hoc test.

 

Figure 8
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  		Fig. 8. Model of Abl-dependent proliferation in fibroblasts. Endogenous Abl kinases directly phosphorylate SHP-2 on Y580, which increases sustained ERK activation in response to PDGF (Figs 3 and 6) (Araki et al., 2003Go). Abl kinases also induce phosphorylation of Y63 and Y279 (Fig. 2B) presumably by activating unknown tyrosine kinases (X and Y). Phosphorylation of Y63 potentiates mitogenic signaling in fibroblasts (Fig. 6C,D), and phosphorylation of Y279 negatively regulates ERK activation (Fig. 6A,B) and proliferation (Fig. 6C,D). Abl kinases also activate a Rac-JNK pathway (Boureux et al., 2005Go), which may be dependent or independent of SHP-2 (Yu et al., 2006Go) (dotted lines).

 

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