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First published online 24 April 2007
doi: 10.1242/jcs.03444

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Src-dependent phosphorylation of 2-adaptin dissociates the -arrestin–AP-2 complex

Delphine Fessart^1,*, May Simaan^1,*, Brandon Zimmerman^1,2, Jonathan Comeau³, Fadi F. Hamdan⁴, Paul W. Wiseman^3,5, Michel Bouvier⁴ and Stéphane A. Laporte^1,2,

¹ Hormones and Cancer Research Unit, Department of Medicine, Royal Victoria Hospital, 687, Pine Avenue West, Montréal, QC, H3A 1A1, Canada
² Department of Pharmacology and Therapeutics, Royal Victoria Hospital, 687, Pine Avenue West, Montréal, QC, H3A 1A1, Canada
³ Department of Chemistry, McGill University, 801 Sherbrooke West, Montréal, QC, H3A 2K6, Canada
⁴ Department of Biochemistry and Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, QC, H3C 3J7, Canada
⁵ Department of Physics, McGill University, 3600 University Street, Montréal, QC, H3A 2T8, Canada

View larger version (37K): [in this window] [in a new window]   Fig. 1. Ang II induces the tyrosine phosphorylation of 2-adaptin. (A) VSMCs were stimulated with Ang II (1 µM) for the indicated time. Endogenous -adaptins were immunoprecipitated using the anti--adaptin antibody (AP-1/2), and the immunoprecipitates were analyzed by western blot as described in Materials and Methods. (B,C) COS-7 (B) or HEK293 cells (C) transfected with HA-AT1R and Flag-2-adaptin were stimulated with Ang II (1 µM) for the indicated time and immunoprecipitated Flag-2-adaptin was analyzed as described above. Densitometry analyses are presented as the mean ± s.e.m. of at least three independent experiments. They represent the relative amount of 2-adaptin phosphorylated as compared with non-stimulated cells after normalizing for equal amounts of 2-adaptin.

View larger version (25K): [in this window] [in a new window]   Fig. 2. Ang II-mediated phosphorylation of 2-adaptin involves Src. (A) COS-7 cells transfected with HA-AT1R and Flag-2-adaptin were incubated for 30 minutes with either the vehicle (DMSO), the Src inhibitor (PP2, 4 µM), the EGFR inhibitor (PD158780, 50 nM) or the less active form of PP2 (PP3, 4 µM), before adding Ang II (1 µM). Flag-2-adaptin was immunoprecipitated using an anti-Flag antibody, and the immunoprecipitates were analyzed by western blot as described in Materials and Methods. Data are presented as the mean ± s.e.m. of three independent experiments. They represent the percent of 2-adaptin phosphorylation as compared to vehicle, after normalizing for equal amounts of -adaptin. ** indicates P<0.01 vs DMSO determined by one-way ANOVA, and was considered significant. (B) COS-7 cells transfected with AT1R, Flag-2-adaptin and either Src wild type or Src K298R (HA-Src and HA-Src K298R) were left untreated (–) or stimulated (+) with Ang II (1 µM). Detection of the phosphorylated 2-adaptin in the immunoprecipitates was performed as described in (A).

View larger version (37K): [in this window] [in a new window]   Fig. 3. -arrestin–AP-2–Src complex is involved in 2-adaptin phosphorylation. (A,B) HEK293 cells were transfected with AT1R, Flag-2-adaptin and either pcDNA3.1, HA-SH1 or HA-SH1-KD (A) or with AT1R and either Flag-2-adaptin wild type or Flag-2-adaptin E902A (B), before either being left untreated (–) or stimulated (+) with Ang II (1 µM). Flag-2-adaptin were immunoprecipitated from cell lysates using an anti-Flag antibody, and the immunoprecipitates were analyzed by western blot as previously described. Whole cell extracts (Total: HA) were probed for the detection of HA-SH1 and HA-SH1-KD using an anti-HA antibody (A). Data are presented as the mean ± s.e.m. for three independent experiments.

View larger version (42K): [in this window] [in a new window]   Fig. 4. Phosphorylation of 2-adaptin by Src reduces its ability to bind -arrestins. (A) GST-fusion proteins of the ear domain of 2-adaptin were left unphosphorylated (–) or phosphorylated (+) in vitro with purified Src. The amounts of GST-2-adaptin were assessed by Ponceau Red, and Src-phosphorylated proteins were detected by western blot using anti-phosphotyrosine antibody 4G10 (P-Tyr). (B) After removing Src from the reaction, the unphosphorylated or phosphorylated GST-proteins were incubated with increasing amounts of Flag--arrestin1 or 2 from cell lysates. The amounts of -arrestin associated with the GST-proteins were determined by western blot using an anti-Flag antibody. Whole cell extracts (Total, right panels) were also blotted for detecting the level of Flag--arrestin1 or 2 expression using the anti-Flag antibody. Data are representative of three to five independent experiments.

View larger version (27K): [in this window] [in a new window]   Fig. 5. Y737 in 2-adaptin is a regulatory site for Src-mediated binding of -arrestin in vitro. (A) GST-fusion proteins of 2-adaptin with single substitutions (Y737F, Y874F and Y926F) were either left unphosphorylated (–) or phosphorylated (+) with purified Src. The amounts of GST-2-adaptin were assessed by Ponceau Red and Src-phosphorylated proteins were detected by western blot. (B) After removing Src from the reaction, the unphosphorylated or phosphorylated GST-proteins were incubated with increasing amounts of Flag--arrestin1. The amounts of GST-proteins were detected using an anti-GST antibody and -arrestin associated with the GST proteins was determined by western blot using anti-Flag antibody. Data are representative of three to five independent experiments.

View larger version (51K): [in this window] [in a new window]   Fig. 6. Y737 in 2-adaptin is a Src regulatory site for -arrestin–AP-2 dissociation in cells. (A) Expression of 2-adaptin-YFP in HEK293 cells. Cells were transfected with pcDNA3 (Mock), non-silencing siRNA (siRNA-NS) or siRNA targeting 2-adaptin (siRNA--ad) and either 2-adaptin-YFP (WT) or 2-adaptin-Y737F-YFP (Y737F) as described in Materials and Methods. Shown is the expression of endogenous and transfected 2-adaptin, and ERK1/2. (B) Colocalization of -arrestin2 with wild type and mutant 2-adaptin in HEK293 cells. Cells expressing HA-AT1R, -arrestin2-CFP, and either 2-adaptin-YFP wild type or Y737F mutant were treated with Ang II (1 µM) for 2 minutes and imaged live by confocal microscopy as described in Materials and Methods. Shown are representative images of -arrestin2-CFP (cyan) and 2-adaptin-YFP proteins (yellow). (C) Ang II-promoted BRET between -arrestin2 and 2-adaptin. HEK293 cells expressing Flag-AT1R, -arrestin2-Rluc and either 2-adaptin-YFP wild-type or Y737F mutant were stimulated with increasing concentrations of Ang II before BRET measurements. (D) Effects of Src on agonist-promoted maximal values of BRET (BRETmax). HEK293 cells expressing Flag-AT1R, -arrestin2-Rluc and either 2-adaptin-YFP wild type or mutant, in absence (black bars) or presence (white bars) of Src were stimulated with Ang II (1 µM). BRET measurements were performed as described in Materials and Methods. Data represent the average of three to five independent experiments.

View larger version (43K): [in this window] [in a new window]   Fig. 7. Y737 in 2-adaptin regulates the dissociation of -arrestin–AP-2 complexes in clathrin-coated pits and the internalization of AT1R. (A) Representative image of a cell expressing -arrestin2 and 2-adaptin that was analyzed by ICCS. HEK293 cells transfected with HA-AT1R, -arrestin2-CFP, and 2-adaptin-YFP wild-type were treated with Ang II (1 µM) and imaged live by confocal microscopy as described in Materials and Methods. Shown is a representative overlay confocal image taken after 1 minute of Ang II treatment and illustrated as pseudo color of -arrestin2-CFP (green), 2-adaptin-YFP (red), and the colocalization of both proteins (yellow) (left panel). The region that was analyzed by ICCS is outlined in the white rectangle. The right panel represents the spatial autocorrelation (solid) and best-fit function (mesh) calculated for the image region outlined in the left panel. (B) Fraction of colocalization between -arrestin2 and 2-adaptin wild type or its mutant was calculated from several image regions for different time points after Ang II treatment (1 µM) using ICCS as described in Materials and Methods. The dissociation rates were calculated from linear fits to the decay of the interaction fraction after reaching its maximum for five different cells from at least three different sets of experiments. (C) AT1R internalization was performed in HEK293 cells transfected with HA-AT1R and either 2-adaptin wild-type or Y737F mutant using [125I]-Ang II. Percent of receptor internalization was calculated as described in Materials and Methods. Data represent the mean ± s.e.m. of at least eight independent experiments.