Differential regulation of tyrosine phosphorylation on Eps8.

<p>Heavy, medium and light SILAC labelled HEK 293T cells were treated with either 25 nM dasatinib, 20 µM SU5402, or no inhibitor, prior to FGF2 stimulation (20 ng/ml; 15 min). Myc-Eps8 was immunoprecipitated and the resulting sample was run on an SDS-PAGE gel and, following in-gel trypsin digestion and phophopeptide enrichment, analysed by mass spectrometry. Each graph represents specific residues on Eps8 as indicated. Each data point represents a single peptide identification. <i>P</i> values were calculated by an unpaired t-test (0.01–0.05 =  *; 0.001–0.01 =  **; <0.001 =  ***). +, the median of the ratios isMethod S1). A) Experiment was carried out in the absence of sodium pervanadate. B) Experiment was carried out in the presence of 2 mM sodium pervanvadate.</p

Chemical inhibition of Src kinase and FGFR kinase activity.

<p>A) HEK 293T cells were treated with SU5402, SU6656, or dasatinib 30 min prior to addition of FGF2 for 15 min. Cells were lysed and analysed by western blotting. B) HEK 293T cells were treated with increasing concentrations of dasatinib for 30 min prior to addition of FGF2 for 15 min. Cells were lysed and analysed by western blotting.</p

Eps8 and IRS4 interact in an FGF2 dependent manner that correlates with an increase in their tyrosine phosphorylation.

<p>A) HEK293T cells transfected with Myc-Eps8 were stimulated with 20 ng/ml FGF2 for 15 min either following 30 min pretreatment with SU5402 or dasatinib or in the absence of inhibitors. Anti-Myc immunoprecipitation and whole cell lysate samples were analysed by western blotting. B) HEK293T cells were stimulated with 20 ng/ml FGF2 for different lengths of time and tyrosine phosphorylated proteins were immunoprecipitated. Anti-pY immunoprecipitation and whole cell lysate samples were analysed by western blotting. C) HEK 293T cells were stimulated with 20 ng/ml FGF2 for 15 min and immnoprecipitations carried out using antibodies to either Eps8 or rabbit IgG. Resulting IP samples were analysed by western blotting. d) Following 30 min treatment with SU5402 or dasatinib and stimulation with 20 ng/ml FGF2 for a further 15 min, endogenous IRS4 was immunoprecipitated from HEK293T cells. Resulting IP samples were analysed by western blotting.</p

Full-length Eps8 interacts with a range of proteins identified in the peptide pull down assays.

<p>HEK 293T cells were either transfected with Myc-Eps8 or left untransfected. Cells were stimulated with 20 ng/ml FGF2 for 15 min and immunoprecipitated using an anti-Myc antibody. Western blot analysis was carried out on whole cell lysate and immunoprecipitation samples using antibodies against the indicated proteins.</p

Eps8 and IRS4 colocalise within cells in an FGF2 dependent manner.

<p>NIH 3T3 cells were co-transfected with IRS4-GFP and Eps8-mCherry. Cells stimulated with FGF2 (20 ng/ml) in the presence and absence of SU5402 (25 µM) were compared to unstimulated cells (control). A) Confocal microscopy was used to visualise the localisation of IRS4 and Eps8. B) The colocalisation (Pearson’s coefficient) between IRS4 and Eps8 is significantly increased in the presence of FGF2 and absence of SU5402 (Pearson’s coefficient, mean±SEM, n = 42 cells. Scale bars = 5 µm. ***, P<0.001).</p

Protein-peptide interaction network for proteins binding specifically to phosphotyrosine-containing Eps8 peptides.

<p>A) Schematic diagram showing locations of the pY residues within the domain structure of Eps8. B) Using SILAC we carried out quantitative peptide pull-down assays from FGF2 stimulated (20 ng/ml; 15 min) HEK 293T cells to compare protein-peptide interactions for phosphotyrosine versus non-phosphotyrosine containing Eps8 peptides. Proteins interacting preferentially to phosphotyrosine peptides have been plotted in an interaction network.</p

Representative mass spectra for identification and site localisation of tyrosine phosphorylation on Eps8.

<p>A) Eps8 is phosphorylated on residue 525. B) Eps8 is phosphorylated on residue 540. pY indicates phosphotyrosine.</p