Use of the GRP1 PH domain as a tool to measure the relative levels of PtdIns(3,4,5)P3 through a protein-lipid overlay approach
Use of the GRP1 PH domain as a tool to measure the relative levels of PtdIns(3,4,5)P3 through a protein-lipid overlay approach
We describe a novel approach to the relative quantification of phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)] and its application to measure, in neutrophils, the activation of phosphoinositide 3-kinase (PI3K). This protein-lipid overlay-based assay allowed us to confirm and extend the observations, first, that N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation of primed human neutrophils leads to a transient and biphasic increase in PtdIns(3,4,5)P(3) levels and, second, that the ability of fMLP to stimulate PtdIns(3,4,5)P(3) accumulation in neutrophils isolated from mice carrying a Ras-insensitive ('DASAA') knock-in of PI3Kgamma (p110gamma(DASAA/DASAA)) is substantially dependent on the Ras binding domain of PI3Kgamma.
Animals Binding Sites Cell Line Cells, Cultured Class Ib Phosphatidylinositol 3-Kinase Humans Immunoblotting Isoenzymes/genetics/metabolism Mice Mice, Knockout N-Formylmethionine Leucyl-Phenylalanine/pharmacology Neutrophils/cytology/drug effects/metabolism Phosphatidylinositol 3-Kinases/genetics/*metabolism Phosphatidylinositol Phosphates/*metabolism Receptors, Cytoplasmic and Nuclear/genetics/metabolism Spodoptera
726-732
Guillou, H.
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Lecureuil, C.
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Anderson, K. E.
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Suire, S.
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Ferguson, G. J.
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Ellson, C. D.
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Gray, A.
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Divecha, N.
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Hawkins, P. T.
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Stephens, L. R.
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31 March 2007
Guillou, H.
8e88bbf0-d311-43e1-b19d-374d54f98769
Lecureuil, C.
72540bed-b23d-490f-892d-93faa9ba1e7e
Anderson, K. E.
313f3b96-9a0a-4ca4-ae36-01415e0ac6f8
Suire, S.
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Ferguson, G. J.
0a7ab90d-84c6-4d10-8f74-ee469223eb12
Ellson, C. D.
df08d701-80f4-472e-90f3-51471fdae282
Gray, A.
ddee2f17-676c-447e-af7b-83354f779c17
Divecha, N.
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Hawkins, P. T.
b5d0d96f-d2a0-49af-be31-2661dd29f734
Stephens, L. R.
ebe5482a-9375-44a1-b7e1-e7a82c21a753
Guillou, H., Lecureuil, C., Anderson, K. E., Suire, S., Ferguson, G. J., Ellson, C. D., Gray, A., Divecha, N., Hawkins, P. T. and Stephens, L. R.
(2007)
Use of the GRP1 PH domain as a tool to measure the relative levels of PtdIns(3,4,5)P3 through a protein-lipid overlay approach.
Journal of Lipid Research, 48 (3), .
(doi:10.1194/jlr.D600038-JLR200).
Abstract
We describe a novel approach to the relative quantification of phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)] and its application to measure, in neutrophils, the activation of phosphoinositide 3-kinase (PI3K). This protein-lipid overlay-based assay allowed us to confirm and extend the observations, first, that N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation of primed human neutrophils leads to a transient and biphasic increase in PtdIns(3,4,5)P(3) levels and, second, that the ability of fMLP to stimulate PtdIns(3,4,5)P(3) accumulation in neutrophils isolated from mice carrying a Ras-insensitive ('DASAA') knock-in of PI3Kgamma (p110gamma(DASAA/DASAA)) is substantially dependent on the Ras binding domain of PI3Kgamma.
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e-pub ahead of print date: 26 November 2006
Published date: 31 March 2007
Additional Information:
Guillou, Herve Lecureuil, Charlotte Anderson, Karen E Suire, Sabine Ferguson, G John Ellson, Chris D Gray, Alexander Divecha, Nullin Hawkins, Phillip T Stephens, Len R eng BB/D013593/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom G9403619/MRC_/Medical Research Council/United Kingdom Research Support, Non-U.S. Gov't 2006/11/30 J Lipid Res. 2007 Mar;48(3):726-32. doi: 10.1194/jlr.D600038-JLR200. Epub 2006 Nov 27.
Keywords:
Animals Binding Sites Cell Line Cells, Cultured Class Ib Phosphatidylinositol 3-Kinase Humans Immunoblotting Isoenzymes/genetics/metabolism Mice Mice, Knockout N-Formylmethionine Leucyl-Phenylalanine/pharmacology Neutrophils/cytology/drug effects/metabolism Phosphatidylinositol 3-Kinases/genetics/*metabolism Phosphatidylinositol Phosphates/*metabolism Receptors, Cytoplasmic and Nuclear/genetics/metabolism Spodoptera
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Local EPrints ID: 480126
URI: http://eprints.soton.ac.uk/id/eprint/480126
ISSN: 0022-2275
PURE UUID: ee510445-d6ba-4658-9062-a209633f5076
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Date deposited: 01 Aug 2023 16:51
Last modified: 17 Mar 2024 02:59
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Contributors
Author:
H. Guillou
Author:
C. Lecureuil
Author:
K. E. Anderson
Author:
S. Suire
Author:
G. J. Ferguson
Author:
C. D. Ellson
Author:
A. Gray
Author:
P. T. Hawkins
Author:
L. R. Stephens
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