JCS035691 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. STI571 inhibits basal SHP-2 phosphorylation in 10T1/2-EGFR fibroblasts. Serum-starved 10T-1/2-EGFR cells were pretreated with STI571 (10 µM) or vehicle (water) for 4 hours, and either left unstimulated, or stimulated with EGF (100 ng/ml) for 15 seconds. SHP-2 was immunoprecipitated, and probed with anti-phosphotyrosine antibody (top). The blot was stripped and reprobed with antibody to SHP-2. Lysates were blotted with a phospho-specific SHP-2 antibody to Y580, and the blot was stripped and reprobed with SHP-2 antibody.

• Supplemental Figure S2 -

  Fig. S2. Abl kinases bind SHP-2. (A,B) SHP-2 (A-top, B) or c-Abl (A-bottom) was immunoprecipitated from 293T cells transfected with wild-type SHP-2 (+) and either wild-type c-Abl or Arg (WT) or constitutively active forms (PP). SHP-2 immunoprecipitates were probed with antibody that recognizes both c-Abl and Arg (A-top, B), and c-Abl immunoprecipitates were probed with SHP-2 antibody (A-bottom). Results are representative of three independent experiments. (C) SHP-2 was immunoprecipitated from lysates obtained from PDGF-BB (12.5 ng/ml)-stimulated fibroblasts (c-Abl/Arg double null fibroblasts reconstituted with c-Abl and Arg) and blotted with pan Abl antibody. Rabbit IgG was used as a control. SHP-2 is highly phosphorylated following PDGF stimulation, as evidenced by a shift in the SHP-2 reactive bands. (D) Lysates from PDGF-BB-stimulated (12.5 ng/ml) NIH3T3 cells were incubated with GST-fusion proteins and glutathione sepharose, and precipitates were probed with SHP-2 antibody (top). The blot was Ponceau-stained to visualize fusion proteins (bottom). Results are representative of three independent experiments. (E) NIH3T3 lysates were prepared as in D, SHP-2 was immunoprecipitated, and the blot was incubated with GST fusion proteins (2 µg/ml), followed by anti-GST primary antibody, and HRP-conjugated secondary antibody. Results are representative of three independent experiments.

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• Supplemental Figure S6 -

  Figs S3-S6. Mass spectra. Representative MS-MS fragmentation spectra for the four identified phosphopeptides. b- and y- ions are marked, and the phosphotyrosine immonium ion at m/z 216 is denoted by an asterisk.

• Supplemental Figure S7 -

  Fig. S7. SHP-2 phosphorylation mutants have altered activities towards phosphotyrosine-containing substrates. Wild-type or mutant forms of SHP-2 were immunoprecipitated from serum-starved NIH3T3 cells expressing wild-type or mutant SHP-2 proteins, incubated with 250 µM substrate (R-R-L-I-E-D-A-E-pY-A-A-R-G) for 30 minutes at 37°C, and the amount of phosphate released into the supernatant was quantified by Malachite Green detection. A₆₂₀ was 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 as mean ± s.e.m. from three independent experiments. Some error bars are too small to be visible. ***P≤0.001 using one-way ANOVAs followed by Bonferroni post-hoc tests. Immunoprecipitates were blotted with SHP-2 antibody (representative experiments are shown).