Fig. 5
FAT phosphorylation affects LD2 binding to the H14 site in FAT. (A) Model of FAT phosphorylated on Y925 (pY925) with LD2 bound to the H14 site. pY925 is shown in space fill with carbons in yellow. The modelling suggests that Y925 phosphorylation impedes LD2 binding. (B) Time course of FAT phosphorylation on Y925 by Src, monitored by ELISA using a phosphospecific anti-FAK pY925 antibody. Assays were performed with the Src kinase domain (Src-Kin; residues 254–536) or Src SH3-SH2-kinase (Src-SH3SH2Kin; residues 84–536); without or with preheating the FAT domain to 95 °C prior to addition of Src. Error bars represent SD from 4 measurements (2 independent reactions). (C-F) Binding curves for peptides LD1 (C), LD2 (D), LD4 (E) and LD2/4 (F) to FAT-WT, phospho-deficient FAT-Y925F and phosphomimetic FAT-Y925E. Apparent KD values are obtained from fitting a one-site binding model. Both Y925 mutations affect binding of LD2 and LD2/4, but not LD1 and LD4. Error bars represent SD from 4 measurements. (G) ECM mediated FAK activation in cells requires Y925 phosphorylation. FAK-WT or the phosphodeficient FAK-Y925F mutant were stably expressed into FAK -/- MEFs. ECM mediated FAK activation was induced by keeping cells in suspension (Susp) prior to plating them on fibronectin (FN). FAK activation was monitored by western blot as in Fig. 4. A representative experiment is shown in the left panel and the quantification is plotted in the right panel. For quantifications signals were normalized to FAK-WT signals observed in MEFs on FN. Error bars represent SD from three independent experiments. *p = 0.02; **p < 0.006 (unpaired Student t test)