Biotinylated phosphatidylinositol 3,4,5-trisphosphate as affinity ligand

Phosphatidylinositol 3,4,5-trisphosphate (PIPs), a primary output signal of phosphoinositide (PI) 3-kinase, plays a crucial role in diverse cellular processes. Evidence indicates that PIP₃ exerts downstream signaling, in part, by recruiting effector proteins to plasma membranes. Consequently, identification of signaling enzymes with PIP₃-binding motifs represents a viable approach to understand the mechanism by which specificity of the PI 3- kinase-mediated signaling network is maintained. To address this issue, we have developed biotinylated derivatives of PIP₃ as affinity probes for the purification and characterization of PIP₃-binding proteins. Considering the relaxed requirement for the acyl moiety in PIP₃ recognition, these biotinylated PIP₃ analogues display two structural features. First, they contain short acyl side chains (C₄ and C₈), allowing them to be soluble in aqueous milieu. This desirable feature avoids the formation of lipid aggregates, which minimizes nonspecific hydrophobic interactions with proteins. Second, the appended biotin is located at the terminus of the sn-1 acyl side chain, thereby maintaining the integrity of the phosphoinositol head group essential for selective recognition. The utility of these affinity ligands is validated by the purification of recombinant PIP₃-binding proteins, expressed as GST fusion proteins, to homogeneity from bacterial lysates. These include the C-terminal SH2 domain of the p85 subunit of PI 3- kinase and the N-terminal PH domain of PLCγ1. The efficiency of biotinylated PIP₃ analogues in the purification of these recombinant proteins was approximately 20% of that of glutathione beads (C) 2000 Academic Press.