Figure 5. PtdIns(3,4,5)P₃-dependent Tiam1 stimulation in vitro is inhibited by inclusion of PtdIns(4,5)P₂.

Nucleotide-exchange reactions were carried out as described in the Experimental section, using [³H]GDP-loaded GST–Rac1 as a substrate, for 25 min. Tiam1 GDP/GTP-exchange assays were carried out in the presence of PC/PS vesicles (final concentrations of 233 μM and 90 μM respectively), containing various concentrations of phosphoinositide(s). (A) Assays were carried out with no phosphoinositide (treatment 1), 10 μM PtdIns(3,4,5)P₃ (treatment 2), 10 μM PtdIns(4,5)P₂ (treatment 3), 100 μM PtdIns(4,5)P₂ (treatment 4), 1000 μM PtdIns(4,5)P₂ (treatment 5), 10 μM PtdIns(3,4,5)P₃+10 μM PtdIns(4,5)P₂ (treatment 6), 10 μM PtdIns(3,4,5)P₃+100 μM PtdIns(4,5)P₂ (treatment 7), or 10 μM PtdIns(3,4,5)P₃+1000 μM PtdIns(4,5)P₂ (treatment 8). Experiments were also performed using vesicles containing 10 μM PtdIns(4,5)P₂ and PtdIns(3,4,5)P₃. (B) The GDP/GTP-exchange activity of CamKII-phosphorylated, and non-phosphorylated Tiam1, was determined in the absence (treatment 1), or presence (treatment 2), of 1000 μM PtdIns(4,5)P₂. GDP released (%) is the percentage of [³H]GDP that is released from [³H]GDP-Rac1 during the assay. Results are means±S.E.M. for four independent experiments.