Journal
CHEMISTRY AND PHYSICS OF LIPIDS
Volume 165, Issue 2, Pages 207-215Publisher
ELSEVIER IRELAND LTD
DOI: 10.1016/j.chemphyslip.2011.12.001
Keywords
Phospholipids; Membranes; Phosphoinositides; Cell surface; Microarray
Categories
Funding
- National Science Foundation [CHE-0954297]
- National Institutes of Health [GM68849]
- Division Of Chemistry
- Direct For Mathematical & Physical Scien [0954297] Funding Source: National Science Foundation
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Signaling lipids control many of the most important biological pathways, typically by recruiting cognate protein binding targets to cell surfaces, thereby regulating both their function and subcellular localization. A critical family of signaling lipids is that of the phosphatidylinositol polyphosphates (PIP(n)s), which is composed of seven isomers that vary based on phosphorylation pattern. A key protein that is activated upon PIPn binding is Akt, which then plays important roles in regulating the cell cycle, and is thus aberrant in disease. Characterization of protein-PIPn binding interactions is hindered by the complexity of the membrane environment and of the PIPn structures. Herein, we describe two rapid assays of use for characterizing protein-PIPn binding interactions. First, a microplate-based binding assay was devised to characterize the binding of effectors to immobilized synthetic PIPn headgroup-biotin conjugates corresponding to all seven isomers. The assay was implemented for simultaneous analysis of Akt-PH domain, indicating PI(3,4,5)P-3 and PI(3,4)P-2 as the primary ligands. In addition, density-dependant studies indicated that the amount of ligand immobilized on the surface affected the amplitude of protein binding, but not the affinity, for Akt-PH. Since the PIPn, ligand motifs used in this analysis lack the membrane environment and glycerolipid backbone, yet still exhibit high-affinity protein binding, these results narrow down the structural requirements for Akt recognition. Additionally, binding detection was also achieved through microarray analysis via the robotic pin printing of ligands onto glass slides in a miniaturized format. Here, fluorescence-based detection provided sensitive detection of binding using minimal amounts of materials. Due to their high-throughput and versatile attributes, these assays provide invaluable tools for probing and perturbing protein-membrane binding interactions. (C) 2011 Elsevier Ireland Ltd. All rights reserved.
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