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Identification of ARAP3, a novel PI3K effector regulating both Arf and Rho GTPases, by selective capture on
S Krugmann1, K E Anderson, S H Ridley
1Inositide Laboratory, The Babraham Institute, Cambridge, CB2 4AT, United Kingdom.
Molecular Cell
|January 24, 2002
Summary
Researchers developed a method using phosphoinositide matrices to capture and identify phosphoinositide-binding proteins from cell extracts. This approach identified over 20 proteins, including novel ones like ARAP3, involved in cellular processes.
Area of Science:
- Biochemistry
- Cell Biology
- Proteomics
Background:
- Phosphoinositides are key signaling lipids regulating diverse cellular processes.
- Identifying proteins that bind specific phosphoinositides is crucial for understanding cellular signaling.
- Existing methods for phosphoinositide-binding protein identification have limitations.
Purpose of the Study:
- To develop and validate a novel method for capturing and identifying phosphoinositide-binding proteins.
- To discover novel proteins with phosphoinositide-binding capabilities.
- To characterize the function of a newly identified phosphoinositide-binding protein, ARAP3.
Main Methods:
- Utilizing matrices with tethered phosphoinositide analogs to capture binding proteins from cell and tissue extracts.
- Employing mass spectrometry for the identification of captured proteins.
- Biochemical and cellular assays to characterize the function of identified proteins, specifically ARAP3.
Main Results:
- Successfully captured and identified over 20 phosphoinositide-binding proteins, primarily from leukocyte extracts.
- Identified known proteins with established phosphoinositide-binding domains, as well as novel proteins with unexpected binding properties.
- Characterized ARAP3, a novel PtdIns(3,4,5)P3/PtdIns(3,4)P2-stimulated Arf6 GTPase-activating protein (GAP) with five PH domains, demonstrating its role in PI3K-dependent cytoskeletal rearrangements.
Conclusions:
- Tethered phosphoinositide matrices provide an effective platform for capturing and identifying phosphoinositide-binding proteins.
- The method has revealed novel proteins and unexpected binding properties, expanding the known interactome of phosphoinositides.
- ARAP3 is a novel, multifunctional GAP involved in regulating cytoskeleton dynamics and cell shape through phosphoinositide-mediated signaling.
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