Figure 5: PtdIns5P increases Tiam1 DH-PHc intrinsic exchange activity.
From: Phosphatidylinositol 5-phosphate regulates invasion through binding and activation of Tiam1
(a) The assay was performed on 0.1 μm of Ni2+-NTA-containing liposomes (with the different PI species) bound to Rac-His loaded with Bodipy FL-GDP. Recombinant Tiam1 DH-PHc was added and the nucleotide exchange reaction was started by adding an excess of GTP. The exchange reaction was followed by fluorospectroscopy. (b) Rac1-His-binding capacity of Ni2+-NTA-containing liposomes. A total of 300 μM liposomes PC:PE:PEfluorescein:DGS-NTA(Ni) (67:28:2:3 molar ratio) were incubated with increasing concentrations of Rac1-His. Liposome-associated Rac1-His was quantified by SDS–PAGE followed by Coomassie staining and plotted as a function of Rac-His input concentration using ImageJ. Data are shown as mean±s.e.m. (n=3 experiments). (c) Recombinant Rac1-His loaded with Bodipy FL-GDP (1 μM final) was bound to liposomes via DGS-NTA(Ni). Liposomes were composed of PC:PE:DGS-NTA(Ni):PI (61:30:3:6 molar ratio). DH-PHc Tiam1 (100 nM) was then preincubated and the exchange reaction (Bodipy FL-GDP/GTP) was started by adding 200 μM final GTP. The data were fit as a single exponential decay and all experiments were performed three times with identical results. (d) Quantification of the experiment described in (c). Results are expressed as maximum Rac1 exchange at t=40 min assuming 100% exchange was obtained with EDTA. Data are shown as mean±s.e.m. (n=3; *P<0.05 using non-parametric t-test)
