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Fig. 1. The PX domain of PLD1 specifically binds to PtdIns(3,4,5)P₃. (A) The sequence alignment of regions from several PX domains. The conserved Arg residue mutated in this study is marked as ${blacktriangledown}$ . (B) Specificities of PLD1 PX, PLD1 PX(R179K), PLD1 PX(R179A) and PLD2 for a range of phosphatidylinositols were analyzed by liposome-binding assay. Binding of GST-PLD PX domains to liposomes containing PE (phosphatidylethanolamine), PC (phosphatidylcholine) and indicated PtdIns (phosphatidylinositol) were analyzed. Liposomes (see Materials and Methods) were incubated with 10 ng of purified GST-PLD PX for 15 minutes at 37°C. The reaction mixtures were then centrifuged and the resulting supernatant (S) and pellets (P) were subjected to SDS-PAGE. Western blotting was performed with anti-GST antibody. A representative result of three individual experiments is shown. (C) The selectivity of PtdIns(3,4,5)P₃ for the PLD1 PX domain was confirmed by competition analysis. Water-soluble inositolphosphates were added to the reaction mixture as described in (B). (D) The affinity of PLD1 PX for PtdIns(3,4,5)P₃ was compared with those of PH domains from Btk and PDK-1.

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