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First published online 6 February 2007
doi: 10.1242/jcs.03373

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PI-3-kinase-dependent membrane recruitment of centaurin-₂ is essential for its effect on ARF6-mediated actin cytoskeleton reorganisation

Department of Pharmacology, The University of Bristol, Bristol, BS8 1TD, UK

View larger version (21K): [in this window] [in a new window]   Fig. 1. centaurin-2 interacts with PI 3,4-P2 and PIP3 via the C-terminal PH domain in vitro. COS7 cells were transiently transfected with GFP, GFP-centaurin-2 or its mutants [the N-PH mutant (R152C), the C-PH mutant (R276C) and the double mutant (R152C/R276C)]. After 2 days, the cells were lysed and the lysates were incubated with the indicated biotinylated PI lipids coupled to avidin beads either in the presence (B) or absence (A,C) of water soluble non-biotinylated PI lipids for 2 hours at 4°C. Protein that remained bound to the PI beads after washing with 0.5% NP-40 was analysed by immunoblotting with an anti-GFP antibody.

View larger version (72K): [in this window] [in a new window]   Fig. 2. centaurin-2 is recruited to the plasma membrane in EGF-stimulated PC12 cells. PC12 cells were transiently transfected with the indicated expression vector for 2 days. (A) The cells expressing either GFP or GFP-centaurin-2 were serum starved and live cells were imaged by confocal microscopy in the absence or presence of 0.1 µg/ml EGF (images were captured 2 minutes after EGF addition). Similar data were obtained from five other imaged cells. (B) The cells expressing FLAG-centaurin-2 were serum starved and stimulated with or without EGF (0.1 µg/ml) for 5 minutes. The cells were then fixed, immunostained with an anti-FLAG antibody and imaged using a confocal microscope. Images are representative of 100-150 transfected cells from three experiments.

View larger version (35K): [in this window] [in a new window]   Fig. 3. The membrane recruitment of centaurin-2 is mediated through PI 3-kinase activation. (A) PC12 cells transiently transfected with GFP-centaurin-2 alone or with the PI 3-kinase regulatory subunit (p85). After 2 days, the cells were serum starved and stimulated with 0.1 µg/ml EGF for 5 minutes. The cells expressing GFP-centaurin-2 alone were preincubated with PI 3-kinase inhibitors (0.1 µM wortmannin or 50 µM LY294002) prior to EGF-stimulation. The cells were fixed and imaged by confocal microscopy. (B) PC12 cells were co-transfected with GFP-centaurin-2 and Myc-tagged membrane-targeted PI 3-kinase (membPI3K) or its inactive form (kinase dead; membPI3K-KD). After 2 days, cells were serum starved, fixed, immunostained with an anti-Myc antibody (not shown) and imaged using a confocal microscope. (C) The EGF-stimulated cells expressing GFP-centaurin-2 were scored for the plasma membrane localisation of centaurin-2. The number of cells (n) counted for each condition are shown in brackets in the graph. Data are from three independent experiments.

View larger version (27K): [in this window] [in a new window]   Fig. 4. centaurin-2 interacts with the PI 3-kinase lipids products via the C-terminal PH domain in vivo. (A) Schematic of the mutant domains. (B) PC12 cells were transiently transfected with GFP, GFP-tagged centaurin-2 (wild type; WT) or its mutants {R53C, the GAP mutant; R152C, the N-PH mutant; R276C, the C-PH mutant; R152C/R276C, the double PH domains mutant (DM)]. After 2 days, the cells were serum starved and live cells were imaged by confocal microscopy in the absence or presence of 0.1 µg/ml EGF. Images were captured 2 minutes after EGF addition. (C) The cells expressing GFP-tagged proteins were scored for the plasma membrane localisation. The number of cells (n) counted for each condition are shown in brackets in the graph. Data are from three experiments.

View larger version (69K): [in this window] [in a new window]   Fig. 5. centaurin-2 shows sustained kinetics of membrane recruitment after stimulation with EGF. (A) PC12 cells transiently transfected with either GFP-centaurin-2, GFP-cytohesin 3, GFP-TAPP1 or GFP-PLC1 PH expression plasmid for 2 days were serum starved and live cells were imaged by confocal microscopy in the presence of 0.1 µg/ml EGF. Time points (top) indicate minutes of EGF stimulation. (B) PC12 cells were transiently transfected with expression plasmids encoding GFP-cytohesin 3, GFP-TAPP1 or GFP-centaurin-2 with HA alone (vector), HA-PIPP or FLAG-4-phosphatase. After 2 days, cells were serum starved and stimulated with 0.1 µg/ml EGF for 2 minutes, fixed, immunostained with an anti-HA or an anti-FLAG antibody (not shown) and imaged using confocal microscopy. (C) The transfected cells, as shown in B, were scored for GFP-tagged protein as an indicator of plasma membrane localisation. A minimum of 200 transfected cells from three independent experiments were scored for each condition.

View larger version (71K): [in this window] [in a new window]   Fig. 6. Effect of centaurin-2 and its mutants on ARF6 localisation and cortical actin formation in EGF-stimulated cells. HeLa cells were transiently transfected with GFP, GFP-centaurin-2 or its mutants (R53C; DM), with or without ARF6-HA. After 2 days, the cells were serum starved and stimulated with EGF (0.1 µg/ml). The cells were then fixed, immunostained with an anti-HA antibody or Rhodamine-conjugated phalloidin (for actin) and imaged using a confocal microscope. The images are representative of 175-180 transfected cells from three experiments.

View larger version (41K): [in this window] [in a new window]   Fig. 7. The constitutively active mutant of ARF6, ARF6Q67L, prevents inhibition of cortical actin formation by centaurin-2 in EGF-stimulated cells. HeLa cells were transiently transfected with either GFP or GFP-centaurin-2 with ARF6Q67L-HA or ARF1Q71L-HA. After 2 days, the cells were serum starved before stimulation with EGF. The cells were then fixed, immunostained with an anti-HA antibody and Rhodamine-conjugated phalloidin (for actin), and imaged using a confocal microscope. The images are representative of 90-95 transfected cells from three experiments.

View larger version (52K): [in this window] [in a new window]   Fig. 8. Effect of membrane targeted centaurin-2 and its mutants on ARF6 activation by cytohesin 2CAAX. HeLa cells were transiently transfected with the indicated expression vectors. After 2 days, the cells were serum starved, fixed, immunostained with an anti-HA antibody and imaged using a confocal microscope. Images are representative of 90-95 transfected cells from three experiments.