Quantification of PtdIns(3,4,5)P(3) dynamics in EGF-stimulated carcinoma cells: a comparison of PH-domain-mediated methods with immunological methods.

Class IA PI3Ks (phosphoinositide 3-kinases) generate the secondary messenger PtdIns(3,4,5) P 3 , which plays an important role in many cellular responses. The accumulation of PtdIns(3,4,5) P 3 in cell membranes is routinely measured using GFP (green fluorescent protein)-labelled PH (pleckstrin homology) domains. However, the kinetics of membrane PtdIns(3,4,5) P 3 synthesis and turnover as detected by PH domains have not been validated using an independent method. In the present study, we measured EGF (epidermal growth factor)-stimulated membrane PtdIns(3,4,5) P 3 production using a specific monoclonal anti-PtdIns(3,4,5) P 3 antibody, and compared the results with those obtained using PH-domain-dependent methods. Anti-PtdIns(3,4,5) P 3 staining rapidly accumulated at the leading edge of EGF-stimulated carcinoma cells. PtdIns(3,4,5) P 3 levels were maximal at 1 min, and returned to basal levels by 5 min. In contrast, membrane PtdIns(3,4,5) P 3 production, measured by the membrane translocation of an epitope-tagged BTK PH (PH domain of Bruton9s tyrosine kinase), remained approx. 2-fold above basal level throughout 4–5 min of EGF stimulation. To determine the reason for this disparity, we measured the rate of PtdIns(3,4,5) P 3 hydrolysis by measuring the decay of the PtdIns(3,4,5) P 3 signal after LY294002 treatment of EGF-stimulated cells. LY294002 abolished anti-PtdIns(3,4,5) P 3 membrane staining within 10 s of treatment, suggesting that PtdIns(3,4,5) P 3 turnover occurs within seconds of synthesis. In contrast, BTK PH membrane recruitment, once initiated by EGF, was relatively insensitive to LY294002. These data suggest that sequestration of PtdIns(3,4,5) P 3 by PH domains may affect the apparent kinetics of PtdIns(3,4,5) P 3 accumulation and turnover; consistent with this hypothesis, we found that GRP-1 (general receptor for phosphoinositides 1) PH domains [which, like BTK, are specific for PtdIns(3,4,5) P 3 ] inhibit PTEN (phosphatase and tensin homologue deleted on chromosome 10) dephosphorylation of PtdIns(3,4,5) P 3 in vitro . These data suggest that anti-PtdIns(3,4,5) P 3 antibodies are a useful tool to detect localized PtdIns(3,4,5) P 3 , and illustrate the importance of using multiple approaches for the estimation of membrane phosphoinositides.