Figure 4: The C-terminal PH domain of Tiam1 binds to PtdIns5P.

(a) Structure of the Tiam1 protein. PHn, N-terminal pleckstrin homology domain; CC, coiled-coil region; RBD, Ras-binding domain; PDZ, postsynaptic density-96/Discs large/Zonaoccludens-1; DH, Dbl homology domain (the catalytic domain); PHc, C-terminal pleckstrin homology domain. Amino-acid sequence of Tiam1 PHc-WT and mutants are presented below. Lysine to glutamine mutations in PH*L2 and PH*L1/L2 are in red. (b) GST or GST-Tiam1-DH-PHc (0.5 μg ml⁻¹) was used to probe PIP array. Weak binding to phosphatidylserine was sometimes observed but was not reproduced in all experiments. Data are representative of more than five experiments done with commercial or homemade membranes. (c) A total of 0.5 μg ml⁻¹ of each GST-tagged protein was used to probe nitrocellulose spotted with serial dilutions of PtdIns3P, PtdIns4P and PtdIns5P, as described under ‘Methods’. Lipid–protein overlays are representative of five independent experiments. (d) A total of 0.5 μg ml⁻¹ of GST-tagged PHn of Tiam1 was used to probe nitrocellulose spotted with 100 pmol of each PI as described under ‘Methods’. The result is representative of three independent experiments. (e) GST or Tiam1 GST-DH-PHc was incubated with liposomes containing the indicated PIs. Liposomes were recovered by centrifugation and protein content was analysed by SDS-PAGE and silver staining. Bottom panel: quantitative analysis of liposome binding experiments. Results are presented as mean±s.e.m. (n=3; *P<0.05 using one sample t-test for the deviation from mean value of 100 in the PI5P group and using non-parametric t-test).