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

Hervé Guillou; Charlotte Lécureuil; Karen E. Anderson; Sabine Suire; G. John Ferguson; Chris D. Ellson; Alexander Gray; Nullin Divecha; Phillip T. Hawkins; Len R. Stephens

doi:10.1194/jlr.D600038-JLR200

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

Hervé Guillou, Charlotte Lécureuil, Karen E. Anderson, Sabine Suire, G. John Ferguson, Chris D. Ellson, Alexander Gray, Nullin Divecha, Phillip T. Hawkins, Len R. Stephens

Research output: Contribution to journal › Article › peer-review

24 Citations (Scopus)

Abstract

We describe a novel approach to the relative quantification of phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P₃] 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-methionylleucyl-phenylalanine (fMLP) stimulation of primed human neutrophils leads to a transient and biphasic increase in PtdIns(3,4,5)P₃ levels and, second, that the ability of fMLP to stimulate PtdIns(3,4,5)P₃ accumulation in neutrophils isolated from mice carrying a Ras-insensitive ('DASAA') knock-in of PI3Kγ (p110γ^DASAA/DASAA) is substantially dependent on the Ras binding domain of PI3Kγ.

Original language	English
Pages (from-to)	726-732
Number of pages	7
Journal	Journal of Lipid Research
Volume	48
Issue number	3
Early online date	27 Nov 2006
DOIs	https://doi.org/10.1194/jlr.D600038-JLR200
Publication status	Published - Mar 2007

Keywords

General receptor for phosphoinositides-1
Neomycin
Neutrophil
p110γ
Phosphatidylinositol (3,4,5)-trisphosphate
Phosphoinositide 3-kinase
Protein-lipid overlay
Ras

ASJC Scopus subject areas

Endocrinology

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10.1194/jlr.D600038-JLR200

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@article{8548f2d43ece414cb6e0023ace60a6a8,

title = "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",

abstract = "We describe a novel approach to the relative quantification of phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] 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-methionylleucyl-phenylalanine (fMLP) stimulation of primed human neutrophils leads to a transient and biphasic increase in PtdIns(3,4,5)P3 levels and, second, that the ability of fMLP to stimulate PtdIns(3,4,5)P3 accumulation in neutrophils isolated from mice carrying a Ras-insensitive ('DASAA') knock-in of PI3Kγ (p110γDASAA/DASAA) is substantially dependent on the Ras binding domain of PI3Kγ.",

keywords = "General receptor for phosphoinositides-1, Neomycin, Neutrophil, p110γ, Phosphatidylinositol (3,4,5)-trisphosphate, Phosphoinositide 3-kinase, Protein-lipid overlay, Ras",

author = "Herv{\'e} Guillou and Charlotte L{\'e}cureuil and Anderson, {Karen E.} and Sabine Suire and Ferguson, {G. John} and Ellson, {Chris D.} and Alexander Gray and Nullin Divecha and Hawkins, {Phillip T.} and Stephens, {Len R.}",

year = "2007",

month = mar,

doi = "10.1194/jlr.D600038-JLR200",

language = "English",

volume = "48",

pages = "726--732",

journal = "Journal of Lipid Research",

issn = "0022-2275",

publisher = "American Society for Biochemistry and Molecular Biology",

number = "3",

}

TY - JOUR

T1 - 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

AU - Guillou, Hervé

AU - Lécureuil, Charlotte

AU - Anderson, Karen E.

AU - Suire, Sabine

AU - Ferguson, G. John

AU - Ellson, Chris D.

AU - Gray, Alexander

AU - Divecha, Nullin

AU - Hawkins, Phillip T.

AU - Stephens, Len R.

PY - 2007/3

Y1 - 2007/3

N2 - We describe a novel approach to the relative quantification of phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] 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-methionylleucyl-phenylalanine (fMLP) stimulation of primed human neutrophils leads to a transient and biphasic increase in PtdIns(3,4,5)P3 levels and, second, that the ability of fMLP to stimulate PtdIns(3,4,5)P3 accumulation in neutrophils isolated from mice carrying a Ras-insensitive ('DASAA') knock-in of PI3Kγ (p110γDASAA/DASAA) is substantially dependent on the Ras binding domain of PI3Kγ.

AB - We describe a novel approach to the relative quantification of phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] 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-methionylleucyl-phenylalanine (fMLP) stimulation of primed human neutrophils leads to a transient and biphasic increase in PtdIns(3,4,5)P3 levels and, second, that the ability of fMLP to stimulate PtdIns(3,4,5)P3 accumulation in neutrophils isolated from mice carrying a Ras-insensitive ('DASAA') knock-in of PI3Kγ (p110γDASAA/DASAA) is substantially dependent on the Ras binding domain of PI3Kγ.

KW - General receptor for phosphoinositides-1

KW - Neomycin

KW - Neutrophil

KW - p110γ

KW - Phosphatidylinositol (3,4,5)-trisphosphate

KW - Phosphoinositide 3-kinase

KW - Protein-lipid overlay

KW - Ras

U2 - 10.1194/jlr.D600038-JLR200

DO - 10.1194/jlr.D600038-JLR200

M3 - Article

C2 - 17130283

AN - SCOPUS:33947174209

SN - 0022-2275

VL - 48

SP - 726

EP - 732

JO - Journal of Lipid Research

JF - Journal of Lipid Research

IS - 3

ER -

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

Abstract

Keywords

ASJC Scopus subject areas

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