Model for EGFR-mediated regulation of β2-chimaerin.

Enhanced Rac association of the Rac-GAP-deficient mutant ΔEIE-β2-chimaerin. (A) FRET in cells coexpressing YFP-ΔEIE-β2-chimaerin and CFP-Rac1 (wild type) after 30 ng/ml EGF treatment, measured every 3 s. A representative experiment comparing wild type and ΔEIE-β2-chimaerin is shown. (B) Quantitative analysis of FRET from (A) at 5 min. Data are expressed as mean±s.e. of six cells. (C) FRET in cells coexpressing YFP-ΔEIE-β2-chimaerin and CFP-Rac1 (wild type) after 10 nM PMA treatment, measured every 3 s. A representative experiment comparing wild type and ΔEIE-β2-chimaerin is shown. (D) Quantitative analysis of FRET from (C) at 5 min. Data are expressed as mean±s.e. of six cells.

Redistribution of β2-chimaerin by activated Rac1 in PLC/DAG-dependent manner. HeLa cells were cotransfected with plasmids encoding for various forms of HA-tagged Rac1 and GFP-fusion proteins. After 48 h, cells were serum starved for 20 h and fixed. Cells were stained with a mouse anti-HA antibody followed by donkey anti-mouse-IgG conjugated to Cy3. (A) Localization of GFP-β2-chimaerin (wild type or mutants). (B) Localization of GFP-PKCα and GFP-PKCα C1 domain. U73122 (10 μM) was added for 60 min.

Analysis of β2-chimaerin–Rac1 interaction by FRET. (A) Schematic representation of the FRET assay to assess Rac1-β2-chimaerin association. A typical cell section selected for analysis and the peripheral translocation of YFP-β2-chimaerin in serum-starved (20 h) COS-7 cells in response to EGF (100 ng/ml) is shown. (B) FRET was measured every 6 s after EGF treatment (100 ng/ml). Representative experiments in cells expressing various CFP and YFP constructs are shown. Each experiment was repeated at least three times. (C) Effect of different concentrations of EGF in cells expressing CFP-Rac1 (wild type) and YFP-β2-chimaerin (wild type). Data are expressed as mean±s.e. of nine cells.

EGF induces translocation of β2-chimaerin in a PLCγ/DAG-dependent manner. (A) Schematic representation of the GFP-fused constructs used in these experiments. (B, C, E, G) Localization of the various GFP-fused proteins in HeLa cells was assessed 48 h after transfection. Cells were treated with either PMA (1 μM) or EGF (100 ng/ml) for the times indicated in the figure. U73122 or U73433 (10 μM) were added 30 min before and during the incubation with EGF or PMA. In the case of EGF treatment, cells were previously serum-starved for 20 h. Cells were monitored by time-lapse confocal microscopy for the times indicated in the figure. Similar results were observed in at least three different experiments. Similar results have been observed in at least more than 10 individual cells. Colocalization of GFP-β2-chimaerin (green) and the membrane marker RFP-CAAX (red) in response to EGF (100 ng/ml) is also shown in (E). (D) Colocalization of GFP-β2-chimaerin (green) and the membrane marker RFP-CAAX (red) in response to EGF (100 ng/ml). (F) Inhibition of PLCγ1 expression in HeLa cells 48 h after transfecting the dsRNA.

The β2-chimaerin C1 domain and DAG generation are required for the β2-chimaerin-Rac1 association in response to EGF. (A) Representative experiment showing the absence of FRET in response to EGF (100 ng/ml) in COS-7 cells coexpressing CFP-Rac1 (wild type) and YFP-β-GAP or YFP-C246A-β2-chimaerin. Each experiment was carried out at least three times. (B) FRET in cells coexpressing CFP-Rac1 (wild type) and YFP-β2-chimaerin (wild type) was determined in response to EGF (300 ng/ml), either in the presence of the PLC inhibitor U73122 or its inactive analog U73433 (10 μM, added 30 min before and during EGF treatment). Left panel, representative experiment. FRET was measured every 3 s. Central panel, quantitative analysis of maximum FRET. Right panel, quantitative analysis of FRET 5 min after EGF treatment, expressed as the percentage of maximum obtained in each case. Data are expressed as mean±s.e. of six cells. (C) FRET in PLCγ1 knocked-down COS-7 cells that coexpress CFP-Rac1 (wild type) and YFP-β2-chimaerin (wild type) was determined in response to EGF (300 ng/ml). Left panel, knock down of PLCγ1 in COS-7 cells. Expression was assessed by Western blot 48 h after dsRNA transfection. Central panel, a representative experiment using 300 ng/ml EGF is shown. Right panel, quantitative analysis of maximum FRET. Data are expressed as mean±s.e. of 10 cells. (D) Enhanced FRET in cells coexpressing CFP-Rac1 (wild type) and C1 domain exposed β2-chimaerin mutants fused with YFP in response to 10 ng/ml EGF. Data are expressed as mean±s.e. of 10 cells.

β2-Chimaerin inhibits EGF-induced activation of Rac1 and Rac-mediated responses. (A) HeLa cells were infected with a recombinant HA-β2-chim-AdV or a LacZ-AdV (MOI 10 PFU/cell, 16 h). Cells were serum-starved for 20 h and then treated with EGF (100 ng/ml) for the times indicated in the figure. Active Rac1, Cdc42, and RhoA were measured using pull-down assays. Lower panels, representative experiments. Expression of HA-tagged β2-chimaerin was assessed using an anti-HA antibody. Upper panels, densitometric analysis of active GTPase levels, normalized in each case to the total immunoreactivity in cell lysates. Results are expressed as % of control (LacZ-AdV, without EGF), and represent the mean±s.e. (n=3). (B) Analysis of EGFR phosphorylation by Western blot using antibodies that recognized various phosphotyrosine sites. (C) Inhibition of actin reorganization by β2-chimaerin. HeLa cells transfected with vectors encoding for various GFP-β2-chimaerin forms (green cells) were serum-starved for 20 h and then treated with EGF (100 ng/ml, 10 min). Cells were fixed, stained with rhodamine–phaloidin for actin staining (red), and DAPI (blue), and analyzed by microscopy. Similar results have been observed in at least 10 cells. (D) Rac1-GTP levels in MCF-7 cells stably expressing G12V-Rac1. (E) Migration assay. Control (vector-transfected) MCF-7 cells and G12V-Rac1-MCF-7 cells were infected overnight in serum-free medium with either β2-chim-AdV or LacZ-AdV at different MOIs. AdVs were removed 20 h later by extensive washing in serum-free medium. Migration was assessed in response to EGF (50 ng/ml) using a Boyden chamber for a period of 5 h. Values corresponding to cell number per HMF are expressed as the mean±s.e. of four independent experiments. (F) At 48 h after transfection with dsRNA for β2-chimaerin or control duplex (CTL), HEK 293 cells were stimulated with EGF (100 ng/ml) for different times, and Rac-GTP levels were determined using the PBD pull-down assay. Left panel, representative experiment. Middle panel, densitometric analysis of Rac-GTP levels normalized to total Rac in each case (n=3). Results were expressed as fold-increase relative to control in the absence of EGF. Right panel, RT–PCR for β2-chimaerin in HEK 293 cells.