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First published online October 11, 2005
doi: 10.1242/10.1242/jcs.02606

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Selective cellular effects of overexpressed pleckstrin-homology domains that recognize PtdIns(3,4,5)P₃ suggest their interaction with protein binding partners

¹ Endocrinology and Reproduction Research Branch, National Institutes of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA
² Department of Physiology, Faculty of Medicine, Semmelweis University, 1085 Budapest, Hungary
³ Department of Medical Chemistry, Faculty of Medicine, Semmelweis University, 1085 Budapest, Hungary

View larger version (36K): [in a new window]   Fig. 1. Localization and wortmannin-sensitive translocation of expressed PH-GFP chimeras that recognize PIP3 in NIH 3T3 cells. NIH 3T3 cells were transfected with the indicated PH-GFP constructs and live cells were examined by confocal microscopy after 1 day of transfection. Note the recruitment of the PH domains (except that of the 3G variant ARNO) to the plasma membrane after PDGF stimulation, and the reversal of this by the PI 3-kinase inhibitor wortmannin.

View larger version (39K): [in a new window]   Fig. 2. Inhibition of PIP3-dependent Akt activation by the various PH domains. COS-7 cells were transfected with a HA-tagged full-length Akt construct with or without a construct encoding a plasma-membrane-targeted PI 3-kinase (PI3K-CAAX) and the indicated PH-GFP constructs. One day after transfection and after 6-8 hours of serum deprivation, cells were lysed and the activity of HA-Akt was measured after immunoprecipitation as detailed under Materials and Methods. Equal expression of HA-Akt, as well as the other expressed proteins, was ensured by keeping the total transfected DNA equal by complementing with the respective empty plasmid DNAs during transfection and were determined from the total cell lysates. In each experiment, the Akt activities were normalized to the level observed with PI3K-CAAX in the presence of GFP alone, which gave an average of tenfold increase over the basal. Mean±s.e.m. of three similar observations are shown.

View larger version (31K): [in a new window]   Fig. 3. Inhibition of cell attachment by overexpressed PH-GFP chimeras in COS-7 cells. COS-7 cells were transfected with the indicated PH-GFP constructs for 1 day. Cells were removed by mild trypsinization and divided into three aliquots. One aliquot was centrifuged and the cells were immediately lysed in Laemmli buffer (labeled c). The other two aliquots were plated into 35mm culture dishes and the cells were allowed to attach for 30 minutes (labeled a). After this, the medium was removed and the cells were washed twice with 2 ml of ice-cold Dulbecco's PBS before lysis in the same volume of Laemmli buffer that was added to the first aliquot of cells. Samples were then sonicated (but not boiled) before separation by SDS-PAGE. Electrophoresis gels were analyzed in a Storm 860 Phosphorimager (Molecular Dynamics) using the blue fluorescent laser for quantitation of the GFP fusion protein band in the gel. The fraction of GFP signal found in the attached cells relative to the total amount of cells seeded was calculated for each construct including GFP alone, which served as a control. Representative gel samples are shown in (A), and the mean±s.e.m. from five experiments performed in duplicates are shown in (B).

View larger version (39K): [in a new window]   Fig. 4. Inhibition of cell spreading by overexpressed PH domain chimeras in COS-7 cells. COS-7 cells transfected with the indicated PH-GFP constructs for 24 hours were removed from the culture dishes with gentle trypsinization and re-plated onto fibronectin-coated glass cover slips as detailed under Materials and Methods. After 10 minutes, cells were fixed and stained with TRITC-phalloidin. Cells overexpressing the GFP fusion proteins were identified by fluorescent microscopy and were classified into three groups: unspread (adherent with no projections), partially spread (adherent with limited lamellipodia) and fully spread. The percentage of fully spread cells was then calculated for each group of cells expressing the GFP fusion proteins and related to the untransfected controls. When added, the PI 3-kinase inhibitor, LY 424002 (Ly) was added 10 minutes before plating on fibronectin. Mean±s.e.m. of 3-6 experiments are shown.

View larger version (25K): [in a new window]   Fig. 5. Inhibition of EGF-stimulated InsP formation by overexpressed PH domain chimeras in COS-7 cells. COS-7 cells were transfected with cDNA encoding the human EGF receptor, together with selected GFP-PH domain fusion constructs as described under Materials and Methods. One day after transfection, cells were labeled with myo-[3H]inositol for 24 hours. Cells were stimulated by EGF for 30 minutes in the presence of 10 mM LiCl, and [3H]-labeled inositol phosphates were separated by Dowex minicolumns and measured by liquid scintillation counting. The relatively small activation of PLC by EGF in these cells did not permit direct analysis of the 1,4,5-isomer of InsP3 and the pooled InsP3 and InsP2 samples were used as an indicator of overall PLC activity. The InsP response of the cells was normalized to the value observed after EGF stimulation of cells expressing only GFP, which was about a twofold increase in average. The PI 3-kinase inhibitor wortmannin (wm) was added 10 minutes before EGF treatment. Mean±s.e.m. of 3-5 experiments are shown performed in duplicate (except for Akt-PH, where n=2).

View larger version (18K): [in a new window]   Fig. 6. The dymanics of expression of various PH-GFP chimeras in COS-7 cells. COS-7 cells were transfected with the indicated PH-GFP constructs and the GFP fluorescence was monitored as a function of time using a fluorescence plate reader. Fluorescence values were normalized to the first reading in each group (20 hours after transfection) as shown for a representative experiment in (A). To determine the PIP3-dependent component of the effects of the expressed PH domains on the balance of proliferation/apoptosis, the ratios of the fluorescence of cells expressing the wild-type PH domains were calculated using the mutant forms of the same domain, which are incapable of lipid binding, as a control at each time point for each PH domain (B). Mean±s.e.m., n=3.

View larger version (51K): [in a new window]   Fig. 7. The effects of Akt-PH–GFP mutations on PIP3 binding, cellular localization and inhibition of cellular responses. (A) COS-7 (upper) and HEK 293 cells (lower) were transfected with the indicated mutant Akt-PH–GFP construct and examined by confocal microscopy without stimulation (HEK 293 cells) or after stimulation with peroxyvanadate (30 µM peroxide, 100 µM ortho-vanadate) for 5-10 minutes (COS-7 cells). (B) Binding of recombinant Akt-PH–GFP mutants to PIP3 conjugated to agarose beads (sn, unbound fraction in the supernatant; pellet, bound fraction associated with the beads); bars represent the % bound fraction determined by Phosphorimager analysis from three separate experiments (±s.e.m.), one of which is shown as a representative. (C) Inhibition of endogenous Akt activation by Akt-PH–GFP mutants expressed in COS-7 cells. After 24 hours of transfection, cells were stimulated by EGF (100 ng/ml) for 5 minutes. Total cell lysates were analysed by SDS PAGE followed by western blotting using an anti-phospho-Akt antibody and densitometric analysis. In (B) and (C), the R25C mutant, unable to bind the lipid, is indicated by darker columns. (D) Expression kinetics of the various Akt-PH–GFP mutants in COS-7 cells as described in Fig. 6. Fluorescence values were related to those of the non-binding R25C mutant at each time points (mean±s.e.m., n=3). (E) The position of the mutated residue (Thr34) within the Akt-PH domain relative to the PIP3 binding site in the crystal structure of Akt-PH (1H10).

View larger version (43K): [in a new window]   Fig. 8. The effects of GRP1-PH–GFP mutations on PIP3 or InsP4 binding, cellular localization and inhibition of cellular spreading in COS-7 cells. (A) COS-7 (upper) and HEK 293 cells (lower) were transfected with the indicated mutant GRP1-PH–GFP construct and examined by confocal microscopy without stimulation (HEK 293 cells) or after stimulation with peroxyvanadate (30 µM peroxide, 100 µM ortho-vanadate) for 5-10 minutes (COS-7 cells). (B) Binding of recombinant GRP1-PH–GFP mutants to PIP3 conjugated to agarose beads (sn, unbound fraction in the supernatant; pellet, bound fraction associated with the beads); bars represent the % bound fraction determined by Phosphorimager analysis from two separate experiments (mean±range), one of which is shown as a representative. (C) Binding of InsP4 to GRP1-PH–GFP mutants. Recombinant proteins were incubated with [3H]InsP4 in the presence of increasing concentration of unlabeled InsP4 for 10 minutes. Protein-bound radioactivity was determined as described under Materials and Methods and expressed as % B0 (means±s.e.m., n=3). (D) Inhibition of cell spreading by the various GRP1-PH–GFP constructs as described in Fig. 4. (mean±s.e.m., n=3). In (B) and (D), the R284C mutant, unable to bind the lipid, is indicated by darker columns. (E) The position of the mutated residues (I307 and K340) within the GRP1-PH domain relative to the PIP3 binding site in the crystal structure of GRP1-PH (1FHX).

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