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

Abstract

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 Bruton's 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.

FIGURE 1. Specific staining of EGF-stimulated PI[3,4,5]P3 at the leading edge of lamellipod

Quiescent MTLn3 cells were stimulated with 5nM EGF for 1min prior to fixation. A. Cells were stained with anti-PI[3,4,5]P3 antibody pre-absorbed with either PI, PI[4,5]P2, or PI[3,4,5]P3 lipid vesicles as indicated. Alternatively, cells were treated with 100 nM wortmannin prior to EGF stimulation, fixation and anti-PI[3,4,5]P3 staining (lower right). B. Quantified edge fluorescence intensity of unstimulated or EGF-stimulated cells stained with anti-PI[3,4,5]P3 antibody that had been preabsorbed with the indicated lipid vesicles. Data are the mean ± SEM from three separate experiments. C. MTLn3 cells were transfected the eGFP-PTEN. Quiescent cells were stimulated as above, fixed and stained with anti-PI[3,4,5]P3 antibodies. Left panels show GFP, right panels show anti-PI[3,4,5]P3. [17]

FIGURE 2. Kinetics of PI[3,4,5]P3 production in EGF-stimulated cells using both PH-domain-dependent and -independent assays

A. Unstimulated or EGF-stimulated (3 min) MTLn3 cells were stained with anti- PI[3,4,5]P3 antibodies. B. Cells were stimulated with EGF for various times, fixed and stained with anti- PI[3,4,5]P3 antibodies. Fluorescence intensity of anti- PI[3,4,5]P3 staining at the cells edge (0.66 μm thickness) was quantified as described in Experimental Procedures. The data show the mean ± SD from two experiments. C. MTLn3 cells were labeled with [3H]-myo-inositol for 2 days, then stimulated with EGF as indicated. Lipids were extracted and deacylated, and glycerophosphoinositides were analyzed by anion-exchange HPLC. Left-hand panel: typical profile showing the PIP2/PIP3 region of the chromatograms from cells stimulated with EGFG for 0, 1,3 or 5 min. Right-hand panel: PIP3 levels in EGF-stimulated cells. The data are the mean ± SEM from three experiments. D. Unstimulated or EGF-stimulated (3 min) ^BTKPH-transfected MTLn3 cells were stained with anti-myc antibodies. E. ^BTKPH-transfected MTLn3 cells were stimulated with EGF for various times, fixed and stained with anti-myc antibodies. Staining was quantified as in (B). Data show mean values ± SEM from four independent experiments.

Figure 3. Comparison of membrane PI[3,4,5]P3 hydrolysis as measured by anti- PI[3,4,5]P3 staining versus ^BTKPH translocation

PI[3,4,5]P3 hydrolysis was measured in control MTLn3 cells (AD) or cells transfected with ^BTKPH (E–H). A, E. Representative images of unstimulated cells (right panel) and cell stimulated with EGF for 1 min (left panel), stained with either anti-PI[3,4,5]P3 antibody (A) or Anti-Myc antibody (E). B, F. Cells were stimulated with EGF for 1 min and then treated with DMSO or LY294002 for various times. The cells were fixed and stained with either anti-PI[3,4,5]P3 antibody (B) or Anti-Myc antibody (F). C, G. Magnification of the leading edge PI[3,4,5]P3 in DMSO or LY294002-treated cells as measured by anti- PI[3,4,5]P3 staining (C) or anti-Myc staining (E). D, H. Quantification of leading edge PI[3,4,5]P3 levels as measured by anti-PI[3,4,5]P3 staining (D) versus anti-Myc staining (H). The data are mean ± SEM from three independent experiments, and are presented as % of maximal edge intensity, which is observed at 1 min of EGF stimulation. The image scale bars represent 10 μm in all panels.

Figure 4. Overexpression of ^BTKPH decreases the apparent rate of PI[3,4,5]P3 hydrolysis

Control or Myc-^BTKPH-transfected cells were stimulated with EGF for 1 min, treated with DMSO or LY294002 for various times, then fixed and stained with anti- PI[3,4,5]P3 antibodies. A. Representative images of ^BTKPH-transfected cells stimulated with EGF for 1 min. Left panel: Anti-myc staining identify transfected cells. Right panel: anti PI[3,4,5]P3 staining. B. Quantification of leading edge PI[3,4,5]P3 in ^BTKPH-transfected cells (BTK-PH) versus non-transfected cells (Ctl), treated with DMSO carrier or LY294002. The data are mean + SEM from three independent experiments, and are presented as % of maximal edge intensity, which is observed at 1 min of EGF stimulation under each condition.

Figure 5. PTEN is inhibited by PH domains from GRP-1

PTEN 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).