M. of PI[3,4,5]P3 hydrolysis by measuring the decay of the PI[3,4,5]P3 signal after LY294002 treatment of EGF-stimulated cells. SJA6017 LY294002 abolished anti-PI[3,4,5]P3 membrane staining within 10 sec of treatment, suggesting that PI[3,4,5]P3 turnover occurs within seconds of synthesis. In contrast, BTKPH membrane recruitment, once initiated SJA6017 by EGF, was relatively insensitive to LY294002. These data suggest that sequestration of PI[3,4,5]P3 by PH domains may affect the apparent kinetics of PI[3,4,5]P3 accumulation and turnover; consistent with this hypothesis, we found that GRP1 PH domains (which like BTK are specific for PI[3,4,5]P3) inhibit PTEN dephosphorylation of PI[3,4,5]P3 in vitro. These data suggest that anti-PI[3,4,5]P3 antibodies are a useful tool to detect localized PI[3,4,5]P3, and illustrate the importance of using multiple approaches for the estimation of membrane phosphoinositides. and in cells. Open in a separate window Figure 5 PTEN is inhibited by PH domains from GRP-1PTEN activity was measured in the presence of recombinant PH domains from GRP-1 or dynamin, using the Malachite Green assay kit from Echelon Biosciences (Salt Lake City, UT). DISCUSSION We have examined the kinetics of PI[3,4,5]P3 synthesis and turnover in EGF-stimulated MTLn3 cells, where Class IA PI3-kinases are required for barbed end formation and protrusion . Membrane PI[3,4,5]P3 levels increase rapidly after EGF stimulation and remain elevated for up to 3 min in EGF-stimulated cells. Furthermore, we display that membrane PI[3,4,5]P3 turnover is extremely quick, having a half-life of less than 10 sec. Given the rate of PI[3,4,5]P3 turnover, the kinetics of PI[3,4,5]P3 levels in the plasma membrane directly reflect the SJA6017 kinetics of PI 3-kinase activity. Thus, the sustained elevation of leading edge PI[3,4,5]P3 production in EGF-stimulated cells requires prolonged PI3-kinase activation in the leading edge of protruding cells. Recent advances in defining the mechanisms that travel chemotaxis have focused on the asymmetric production of PI[3,4,5]P3 in the leading edge. These studies possess widely used the PH-domain-containing proteins to detect membrane localization of phosphoinositide lipids [25, 26]. While the use of GFP-linked PH-domain probes is clearly advantageous for live-image microscopy, our data suggest that the kinetics of PI[3,4,5]P3 build up in live cell PH-domain assays may be inaccurate. When we directly compared measurements of PI[3,4,5]P3 turnover using different methods, we found that SJA6017 PH-domain-dependent assays showed a slow rate of PI[3,4,5]P3 turnover, having a half-life of approximately 60 sec. This is consistent with a earlier study in which the half-life of PI[3,4,5]P3 turnover in insulin-stimulated 3T3-L1 cells was estimated to be 1 min, based on the membrane translocation of an AKTPH-CFP construct . Our data suggest that PH-domain-dependent assays overestimate the lifetime of PI[3,4,5]P3 relative to anti- PI[3,4,5]P3 immunostaining assays. This is likely to be the reason for the fact that both pAkt and PH-domain-based assays display that PI[3,4,5]P3 levels remain elevated for over 5 min in EGF-stimulated cells, whereas anti- PI[3,4,5]P3 staining demonstrates PI[3,4,5]P3 results to basal levels by 5 min. Interestingly, the BTKPH probe responded to EGF-stimulation with related initial kinetics to that seen in anti- PI[3,4,5]P3 immunostained cells (Number 2). These results suggest that even though the protective effects of the PH-domain probes are likely to distort the decay rates of PI[3,4,5]P3 signaling, these probes may accurately measure initial rates of PI[3,4,5]P3 build up. It should be noted that these problems do not apply to the recently explained use of PH domains for electron microscopic analysis of phosphoinositide localization and large quantity [27C29]. While manifestation of PH domains may alter the apparent kinetics of lipid build up/degradation, they may be a less perturbing and more Rabbit polyclonal to DCP2 accurate method of analyzing the spatial distribution of membrane phosphoinositides. As mentioned by Downes, Lucocq and coworkers, membrane lipids are less likely to become immobilized that membrane proteins by aldehyde-based fixation methods . This increases the query of potential lateral diffusion of membrane-bound PI[3,4,5]P3 using the antibody-based method. Furthermore, the fixation method used here requires the presence of saponin (0.15 mg/ml). Triton concentrations as low as 0.0025% induce.