Characterisation of GST-GRP1PH
GST-GRP1PH was characterized using SDS–PAGE with sensitive Coomassie stain followed by automated in-gel digestion and LC-MS/MS analysis (results not shown) . An 85% peptide coverage of the PH domain was obtained using MS/MS analysis (results not shown).
GST-GRP1PH binding specificity toward PI(3,4,5)P3 was analysed using both overlay phosphoinositide assay and Biosensor analysis (Figure 2). GST-GRP1PH was found to recognize specifically immobilized PI(3,4,5)P3 when compared to immobilized PI(4,5)P2 (Figure 2A) and 2B). A standard curve generated by plotting the average intensity of the dot blot versus the amount of immobilized PI(3,4,5)P3 showed that approximately 4ng (3.3pmoles) of PI(3,4,5)P3 could be detected in this assay.
Binding specificity was also characterized using biosensor analysis. GST-GRP1-PH was injected at various concentrations (1.5µM, 750nM, 375nM, 188nM, 94nM) over immobilized PC/PE/PI(4,5)P2 and PC/PE/PI(3,4,5)P3 liposomes (Figure 2C and 2D). GST-GRP1PH was observed to bind specifically to immobilized PC/PE/PI(3,4,5)P3 liposomes (Figure 2C) as compared to immobilized PC/PE/PI(4,5)P2 PC/PE (Figure 2D).
Kinetic constants were extracted from the biosensor curves from global analysis using 1/1 Langmuir binding with mass transfer. Analysis of the interaction between GST-GRP1PH and immobilized PI(3,4,5)P3 gave an apparent association rate (ka) of 4.5 x 104M-1s-1 and apparent dissociation rate (kd) of 1.6 x 10-3s-1 resulting in an equilibrium dissociation constant (KD) of 35.5nM.
Fluorescent imaging of GST-GRP1PH in resting and EGF stimulated HEK293T, LIM1215 and LIM2550 cells
Confocal microscopy was used to analyse PI(3,4,5)P3 localisation using GST-GRP1PH in resting and EGF stimulated HEK293T, LIM1215 and LIM2550 colonic carcinoma cells (Figure 3). EGF stimulation was performed for 1, 3, 5, 10 and 20 mins prior to cell fixation. Optimisation experiments indicated that 5 mins fixation in 4% formaldehyde was necessary and that the entire process of stimulation, staining and mounting should be completed within one day at room temperature. Unstimulated HEK293T cells showed a diffuse cytoplasmic and nuclear background staining (Figure 3). Increased fluorescence and localisation of GST-GRP1PH at the plasma membrane was observed 5 mins after EGF stimulation, with a concomitant reduction in cytoplasmic and nuclear staining (Figure 3). Membrane fluorescence and diffuse background staining returned to pre-stimulation levels 10 mins after stimulation. No background staining was observed when control experiments were performed with GST protein instead of GST-GRP1PH (results not shown).
Unstimulated LIM1215 cells also showed a diffuse cytoplasmic staining with evidence of some basal membrane staining (Figure 3). Increased fluorescence intensity and localisation of GST-GRP1PH at the plasma membrane, with concomitant decrease in background , were observed following 5 mins EGF stimulation. In these...