Fig. 4.

High Ca²⁺ concentrations abolishes the electrostatic interactions between PH domains and PIPs through the formation of Ca²⁺-PIPs. (A) Schematic representation of the various biological phospholipids (PIP strips, Echelon Biosciences), including LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; and S1P, sphingosine 1-phosphate. (B–D) Binding of the PH domains of Akt (B), PLCδ (C), and IRS1 (D) to immobilized phospholipids under the indicated Ca²⁺ concentrations. (E) Schematic representation of electrostatic interactions between the PH domain of Akt (Protein Data Bank accession code:1H10) and Ins(1,3,4,5)P₄. (F–I) ITC results for Ca²⁺ binding to the PH domain of Akt (F), PI(3,4)P₂ (G), PI(4,5)P₂ (H), or PI(3,4,5)P₃ (I) liposomes. K_d values were determined by curve fitting. (J) Schematic representation of electrostatic repulsion between basic residues in the Akt PH domain and Ca²⁺-Ins(1,3,4,5)P₄. Positive charges of basic residues in the PH domain will repel the positively charged Ca²⁺-Ins(1,3,4,5)P₄ and thus inhibit the electrostatic interactions in E.