TY - CHAP
T1 - PLD Protein–Protein Interactions With Signaling Molecules and Modulation by PA
AU - Gomez-Cambronero, J.
AU - Morris, A. J.
AU - Henkels, K. M.
N1 - Publisher Copyright:
© 2017 Elsevier Inc.
Copyright:
Copyright 2017 Elsevier B.V., All rights reserved.
PY - 2017
Y1 - 2017
N2 - We describe methods for studying phospholipase D (PLD) interactions with signaling proteins and modulation of these interactions by the PLD reaction product, phosphatidic acid (PA). PLD is fundamental to the physiological maintenance of cellular/intracellular membranes, protein trafficking, cytoskeletal dynamics, membrane remodeling, cell proliferation, meiotic division and sporulation. PA is an acidic phospholipid involved in the biosynthesis of many other lipids that affects the enzymatic activities of many different signaling proteins via protein–lipid interactions or as a substrate. The involvement of PLD as an effector of protein–protein interactions and downstream signaling via PA-mediated processes has led to the investigation of PA-binding domains in target protein partners. We present here data and protocols detailing the interaction between PLD2–Rac2 interaction and modulation of this interaction by PA. We describe biochemical techniques to measure interactions between PLD, PA, and the small GTPase Rac2, which are associated in the cell. We found two maxima concentrations of PA that contributed to association or dissociation of Rac2 with PLD2, as well as the PLD2 lipase and guanine nucleotide exchange factor (GEF) activities. Fluctuations in the Rac2–PLD2 protein–protein binding interaction facilitate shuttling of Rac2 and/or PLD2 within the cell dependent on local cellular PA concentration. Fluorescence resonance emission transfer stoichiometry for PLD2 and Rac2 binding yielded a 3:1 ratio of Rac2:PLD2. Detection of PA in mammalian cells with a new biosensor showed colocalization in and around the nucleus. We also described methods for quantitation of PA in biological materials by HPLC electrospray ionization tandem mass spectrometry.
AB - We describe methods for studying phospholipase D (PLD) interactions with signaling proteins and modulation of these interactions by the PLD reaction product, phosphatidic acid (PA). PLD is fundamental to the physiological maintenance of cellular/intracellular membranes, protein trafficking, cytoskeletal dynamics, membrane remodeling, cell proliferation, meiotic division and sporulation. PA is an acidic phospholipid involved in the biosynthesis of many other lipids that affects the enzymatic activities of many different signaling proteins via protein–lipid interactions or as a substrate. The involvement of PLD as an effector of protein–protein interactions and downstream signaling via PA-mediated processes has led to the investigation of PA-binding domains in target protein partners. We present here data and protocols detailing the interaction between PLD2–Rac2 interaction and modulation of this interaction by PA. We describe biochemical techniques to measure interactions between PLD, PA, and the small GTPase Rac2, which are associated in the cell. We found two maxima concentrations of PA that contributed to association or dissociation of Rac2 with PLD2, as well as the PLD2 lipase and guanine nucleotide exchange factor (GEF) activities. Fluctuations in the Rac2–PLD2 protein–protein binding interaction facilitate shuttling of Rac2 and/or PLD2 within the cell dependent on local cellular PA concentration. Fluorescence resonance emission transfer stoichiometry for PLD2 and Rac2 binding yielded a 3:1 ratio of Rac2:PLD2. Detection of PA in mammalian cells with a new biosensor showed colocalization in and around the nucleus. We also described methods for quantitation of PA in biological materials by HPLC electrospray ionization tandem mass spectrometry.
KW - GEF
KW - PABD
KW - Phosphatidic acid
KW - Phospholipase D
KW - Rac2
UR - http://www.scopus.com/inward/record.url?scp=85006265634&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85006265634&partnerID=8YFLogxK
U2 - 10.1016/bs.mie.2016.09.042
DO - 10.1016/bs.mie.2016.09.042
M3 - Chapter
C2 - 28063497
AN - SCOPUS:85006265634
T3 - Methods in Enzymology
SP - 327
EP - 357
BT - Methods in Enzymology
ER -