Fig. 6: MAP4K4 and STRN3 mediate VASPS157 phosphorylation.
From: Cooperation of Striatin 3 and MAP4K4 promotes growth and tissue invasion
a Heat map of increased (red) and decreased (blue) phosphorylation of Ser/Thr (STK, left) or Tyr (PTK, right) consensus peptides associated with lysates from siRNA transfected DAOY cells treated ± 100 ng/ml bFGF for 15 min. Log2 of the fold change between siTarget and siCTRL (n = 3). b Venn diagram displaying the number of Ser/Thr and Tyr peptides with significantly changed phosphorylation (p < 0.1) in each siTarget versus siCTRL with bFGF stimulation (n = 3). c Volcano plots representing the changes in phosphorylation of STK (circles) and PTK (diamonds) peptides in siMAP4K4 (upper) or siSTRN3 (lower) versus siCTRL with bFGF stimulation. Blue: peptides with significantly decreased (fold change < 0.6), red: peptides with significantly increased phosphorylation (fold change > 1.67). The p-values were calculated versus siCTRL by ANOVA and post hoc Dunnett’s test in the BioNavigator software. n = 3. d Single confocal section of VASP localization in DAOY cells seeded on collagen-coated plates ± 100 ng/ml bFGF for 15 min. 3× magnifications of boxed areas are shown. Red: VASP, Green: Lifeact-EGFP. Blue: DNA Hoechst. Scale bar: 20 μm. e IB and quantification of VASPS157 phosphorylation in DAOY cells 72 h after siRNA transfection ± 100 ng/ml bFGF for 15 min (n = 4, means ± SEM). f IB and quantification of VASPS157 phosphorylation in DAOY cells expressing full-length or truncated (KD-ID1-ID2) MAP4K4-FLAG ± 100 ng/ml bFGF for 15 min (n = 3, means ± SEM). g In vitro VASP S157 phosphorylation of FLAG-immunoprecipitated DAOY cells expressing FLAG-tagged MAP4K4 and transfected for 48 h with the indicated siRNA ± 100 ng/ml bFGF for 15 min. The in vitro kinase reaction was conducted incubating the IP fraction with a recombinant VASP protein. Parental WT DAOY cells were used as a negative control for the IP. h Quantification and representative images of SIA of siCTRL or siVASP transfected DAOY cells ± 100 ng/ml bFGF (n = 4, means ± SEM). Scale bar: 300 µm. Statistical analysis in e, f was performed by unpaired Student’s t-test, in h by one-way ANOVA. *p < 0.05, **p < 0.01, ****p < 0.0001. See also Fig. S9. Source data for quantifications shown in (e), (f), and (h) are available in Supplementary Data 10.
