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.
Guillou
Hervé
H
The Babraham Institute, Cambridge, UK.
Lécureuil
Charlotte
C
Anderson
Karen E
KE
Suire
Sabine
S
Ferguson
G John
GJ
Ellson
Chris D
CD
Gray
Alexander
A
Divecha
Nullin
N
Hawkins
Phillip T
PT
Stephens
Len R
LR
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002478:Cells, Cultured; D058544:Class Ib Phosphatidylinositol 3-Kinase; D006801:Humans; D015151:Immunoblotting; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera
D007527:Isoenzymes; D018129:Phosphatidylinositol Phosphates; D018160:Receptors, Cytoplasmic and Nuclear; C060974:phosphatidylinositol 3,4,5-triphosphate; C040985:phosphatidylinositol receptors; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019869:Phosphatidylinositol 3-Kinases; D058544:Class Ib Phosphatidylinositol 3-Kinase; C546760:PIK3CG protein, human; C546764:Pik3cg protein, mouse