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. 2009 Mar 27;284(13):8855-65.
doi: 10.1074/jbc.M808463200. Epub 2009 Jan 26.

{beta}-Arrestin-2 Mediates Anti-apoptotic Signaling through Regulation of BAD Phosphorylation

Seungkirl Ahn  1 , Jihee Kim, Makoto R Hara, Xiu-Rong Ren, Robert J Lefkowitz
Affiliations

{beta}-Arrestin-2 Mediates Anti-apoptotic Signaling through Regulation of BAD Phosphorylation

Seungkirl Ahn et al. J Biol Chem. .

Abstract

beta-Arrestins, originally discovered as terminators of G protein-coupled receptor signaling, have more recently been appreciated to also function as signal transducers in their own right, although the consequences for cellular physiology have not been well understood. Here we demonstrate that beta-arrestin-2 mediates anti-apoptotic cytoprotective signaling stimulated by a typical 7-transmembrane receptor the angiotensin ATII 1A receptor, expressed endogenously in rat vascular smooth muscle cells or by transfection in HEK-293 cells. Receptor stimulation leads to concerted activation of two pathways, ERK/p90RSK and PI3K/AKT, which converge to phosphorylate and inactivate the pro-apoptotic protein BAD. Anti-apoptotic effects as well as pathway activities can be stimulated by an angiotensin analog (SII), which has been previously shown to activate beta-arrestin but not G protein-dependent signaling, and are abrogated by beta-arrestin-2 small interfering RNA. These findings establish a key role for beta-arrestin-2 in mediating cellular cytoprotective functions by a 7-transmembrane receptor and define the biochemical pathways involved.

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Figures

FIGURE 1.
FIGURE 1.
β-Arrestin-2, but not activation of G proteins, is essential for AngII-promoted cytoprotection against apoptotic challenges in rat VSMCs. A-G, primary cultured VSMCs were transfected with either control (CTL) or simultaneously two different β-arrestin-2 (βarr2) siRNAs. A and B, cells were stimulated with AngII before either H2O2 (A) or etoposide (B) treatment as described under “Experimental Procedures.” Pro- and cleaved caspase-3 were visualized by immunoblotting (IB). C and D, contents of cleaved caspase-3 in the immunoblots were quantified and expressed as percent of the level obtained in H2O2-(C) or etoposide- (D) treated cells in the absence of stimulation with AngII. Data represent the mean ± S.E. from at least five independent experiments. E, 50 μm Z-VAD-fmk was treated to block caspase-dependent cell death before stimulation with AngII. At ∼6 h after H2O2 treatment, cellular distribution of cytochrome c was visualized by immunostaining. Images shown represent similar results obtained from three independent experiments. F and G, the amounts of H2O2-(F) or etoposide- (G) induced DNA fragmentation in the absence or presence of stimulation with AngII were measured as described under “Experimental Procedures.” Values were expressed as folds over basal in non-stimulated, non-treated (NT), CTL-siRNA-transfected cells and represent the mean ± S.E. from at least six independent experiments. H-J, VSMCs were stimulated with SII prior to treatment with either H2O2 or etoposide as indicated. The amounts of cleaved caspase-3 (H and I) and fragmented DNA (J) were determined and expressed as described above. Data represent the mean ± S.E. from at least 5 (I) or 6 (J) independent experiments. All statistical analyses were performed as described under “Experimental Procedures.”
FIGURE 2.
FIGURE 2.
Effects of silencing β-arrestin-2 expression on ERK-RSK-BAD phosphorylation as well as AKT activation in response to AngII in rat VSMCs. A-G, indicated siRNA-transfected VSMCs were serum-starved for ∼24 h and then stimulated with 100 nm AngII at 37 °C for the indicated periods. After stimulation, expression of β-arrestins (A) and phosphorylation of ERK1/2 (B), p90RSK at Thr359/Ser363 (D), and BAD at Ser113 (F) were visualized by immunoblotting (IB). C, contents of ERK1/2 phosphorylation in the immunoblots (left panels) were quantified and normalized by the amount of total ERK1/2 (right panels). Values were expressed as percent of the maximal phosphorylation of ERK1/2 obtained in control (CTL) siRNA-transfected cells. Data were obtained from five independent experiments. E and G, the amounts of p90RSK (E) and BAD (G) phosphorylation shown in the immunoblots were measured and determined as described for C. Data were obtained from six independent experiments. H and I, serum-starved VSMCs were stimulated with 10 μm SII for 5 min. The level of SII-induced p90RSK and BAD phosphorylation were determined as described above and expressed folds over basal phosphorylation in non-stimulated (NS) cells. Values were obtained from seven independent experiments (I). J and K, siRNA-transfected VSMCs were serum-starved and then stimulated with 100 nm AngII for the indicated periods. AKT phosphorylation at Ser473 was visualized (J) and determined (K) as described for C from six independent experiments. In all graphs, each data point represents the mean ± S.E., and statistical analyses were carried out as described under “Experimental Procedures.”
FIGURE 3.
FIGURE 3.
Kinase activities of both RSK and AKT as well as BAD phosphorylation are required for AngII-promoted cellular protection in rat VSMCs. A-D, serum-starved VSMCs were incubated with either 100 μm SL0101 for 2 h or 1 μm LY294004 for 1 h at 37 °C before stimulation with AngII and following etoposide treatment. A and C, contents of pro- and cleaved caspase-3 were visualized (A), and the amounts of cleaved caspase-3 were determined (C) as described in the legend to Fig. 1. Values were expressed as percent of the level obtained in vehicle alone (dimethyl sulfoxide, DMSO) and then etoposide-treated cells in the absence of stimulation with AngII and represent the mean ± S.E. from seven independent experiments (C). B, after stimulation with AngII for 5 min, phosphorylation of AKT at Ser473 and BAD at Ser113 were visualized by immunoblotting (IB). D, levels of fragmented DNA were measured and expressed as described for C. Data represent the mean ± S.E. from seven independent experiments. E-G, VSMCs were transfected with each plasmid expressing the indicated BAD. Empty pcDNA3 was used as a transfection control. E, cells were treated with 50 μm Z-VAD-fmk during transfection. Within 24 h after transfection, cellular distribution of cytochrome c (green) and expression of hemagglutinin-tagged BAD (red) were visualized by immunostaining. Images shown represent similar results obtained from four independent experiments. F, approximately 6 h after transfection, cells were stimulated with 300 nm AngII and incubated overnight. Contents of pro- and cleaved caspase-3 were visualized by immunoblotting. G, the amounts of cleaved caspase-3 were measured and expressed as percent of the maximum level obtained from each 10 independent experiment. Values represent the mean ± S.E. All statistical analyses were carried out as described under “Experimental Procedures.”
FIGURE 4.
FIGURE 4.
β-Arrestin-2 also mediates the ERK-RSK-BAD phosphorylation cascade upon stimulation of the AT1A receptor stably expressed in HEK-293 cells. A-E, cells were transfected with the indicated siRNAs and serum starved for ∼6 h. During starvation, inhibitors were pre-treated before stimulation: 1 μm Ro31-8425, 2.5 μm GF109203X (GFX), and 5 μm U0126 for 30 min; 100 μm SL0101 for 2 h. After stimulation with 100 nm AngII at 37 °C for the indicated periods, whole cell extracts were prepared to visualize expression β-arrestins (A) and phosphorylation of p90RSK at Thr359/Ser363 (B) and BAD at Ser75 (D) by immunoblotting (IB). Levels of p90RSK (C) and BAD (D) phosphorylation in each sample were determined as described for Fig. 2 and expressed as percent of the maximal phosphorylation in control (CTL) siRNA-transfected cells. Data were obtained from at least three independent experiments. F and G, serum-starved AT1AR-expressing HEK-293 cells were stimulated with 10 μm SII for the indicated periods. Contents of p90RSK (left panel) and BAD (right panel) phosphorylation were visualized (F) and determined (G). Levels of phosphorylation in each time point were expressed as percent of the maximal phosphorylation obtained in three independent experiments (G). In all graphs, each data point represents the mean ± S.E. and statistical analyses were carried out as described under “Experimental Procedures.”
FIGURE 5.
FIGURE 5.
Effects of β-arrestin-2 knocking down on AngII-induced interaction of BAD with its partners in AT1AR-stably expressing HEK-293 cells. Cells were transfected with either control (CTL) or β-arrestin-2 (βarr2) siRNA. After transfection, cells were serum-starved for ∼6 h and then stimulated with 100 nm AngII for 30 min. A, cell lysates were prepared and used for immunoprecipitation (IP) with a 14-3-3 antibody. The BAD protein co-immunoprecipitated with 14-3-3 was visualized by immunoblotting (IB) (upper blot), and its amount in each sample was quantified and normalized by the amounts of BAD in input lysates (lower blot). B, values were expressed as folds over the basal level obtained in non-stimulated (NS), CTL siRNA-transfected cells. Data were obtained from four independent experiments. C-F, after immunoprecipitation with a BAD antibody, co-immunoprecipitated 14-3-3 (C) and Bcl-xL (E) proteins with BAD were visualized, and their amounts were determined as described for A. D, data were expressed as folds over the basal level and obtained from three independent experiments. F, values were expressed as percent of the basal level and obtained from four independent experiments. G and H, phosphorylation of BAD at Ser75 in each lysate used for immunoprecipitation experiments were visualized (G) and measured (H). Data were expressed as percent of the response to AngII in CTL siRNA-transfected cells and obtained from four independent experiments (H). In all graphs, values represent the mean ± S.E., and statistical analyses were carried out as described under “Experimental Procedures.”
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