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.
Highlights
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)
We tested the ability of neomycin beads to reproducibly purify PtdIns(3,4,5)P3 from a complex lipid extract prepared from human neutrophils
Using 32P-PI radiolabeling of neutrophils, we had shown previously that human neutrophils transiently synthesize PtdIns(3,4,5)P3 when challenged with agonists such as bacterially derived fMLP (17). Consistent with this previous report, we showed that the general receptor for phosphoinositides-1 (GRP1) probe allowed detection of PtdIns(3,4,5)P3 accumulation in human neutrophils stimulated with fMLP (Fig. 4)
Summary
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 PI3Kg (p110gDASAA/DASAA) is substantially dependent on the Ras binding domain of PI3Kg.—Guillou, H., C. PI3K signaling pathways have been extensively studied in cultured cells, the number of assays avail-
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