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Originally published In Press as doi:10.1194/jlr.D600038-JLR200 on November 27, 2006

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Journal of Lipid Research, Vol. 48, 726-732, March 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

Methods

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{dagger}, Alexander Gray§, Nullin Divecha**, Phillip T. Hawkins* and Len R. Stephens1,*

* The Babraham Institute, Cambridge, UK
{dagger} MIT, Boston, MA
§ University of Dundee, UK
** The Inositide Laboratory, Department of Cellular Biochemistry, Netherlands Cancer Institute, Amsterdam, Netherlands

Published, JLR Papers in Press, November 27, 2006.

1 To whom correspondence should be addressed. e-mail: len.stephens{at}bbsrc.ac.uk

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-methionyl-leucyl-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 PI3K{gamma} (p110{gamma}DASAA/DASAA) is substantially dependent on the Ras binding domain of PI3K{gamma}.

Supplementary key words phosphatidylinositol (3,4,5)-trisphosphate • neomycin • phosphoinositide 3-kinase • general receptor for phosphoinositides-1 • protein-lipid overlay • neutrophil • p110{gamma} • Ras

Abbreviations: DAPP1, dual adaptor for phosphotyrosine and 3-phosphoinositide 1; fMLP, N-formyl-methionyl-leucyl-phenylalanine; GRP1, general receptor for phosphoinositides-1; PH, pleckstrin homology; PI, phosphoinositide; PI3K, phosphoinositide 3-kinase; PtdIns(3,4,5)P3, phosphatidylinositol (3,4,5)-trisphosphate; PPP, platelet-poor plasma; PtdS, phosphatidylserine; RBD, Ras binding domain; TNF{alpha}, tumor necrosis factor {alpha}


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Copyright © 2007 by the American Society for Biochemistry and Molecular Biology.