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

JCS03444 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Tyrosine phosphorylation of the Ear domain of β2-adaptin. (A) Representation of the different domains of β2-adaptin with the nine putative tyrosine phosphorylation sites in the aa sequence starting at residue 664-937. (B) HEK 293 cells were transfected with the empty vector pcDNA3.1 (Mock) or HA-β2-adaptin constructs of the Ear (β2-ad (E)) or the Hinge+Ear domains of β2-adaptin (β2-ad (H+E)), with or without Src. HA-tagged β2-adaptin constructs were immunoprecipitated (IP) using antibody against HA-conjugated beads (Roche); their tyrosine phosphorylation status was detected by western blotting (WB) using an antibody against phosphotyrosine residues (P-Tyr). Data are representative of at least three independent experiments.

• Supplemental Figure S2 -

  Fig. S2. Tyrosine residues 737, 874 and 926 in β2-adaptin are phosphorylated by Src. (A) Ribbon diagram of the β2-appendage showing different tyrosine residues and those involved in binding β-arrestin (Y888, E902 and E849). (B) Recombinant fusion proteins of ten amino acids encoding various portions of the Ear domain of β2-adaptin (residues 704-712, 733-741, 811-819, 869-877, 884-892, 907-915, 921-929 and 927-933) or GST alone were subjected to in vitro phosphorylation by purified Src kinase. Each reaction contained equivalent amounts of GST and GST-β2-adaptin fusion proteins as assessed by Ponceau Red. Src-phosphorylated proteins were detected by western blotting (WB) using an antibody against phosphotyrosine residues (P-Tyr). (C) In vitro Src phosphorylation of GST-fusion proteins containing residues 733-741, 869-877 or 921-929 and mutants where the tyrosine (Y) was replaced by phenylalanine (F) to obtain Y737F, Y874F or Y926F mutants, respectively. Each reaction contained equivalent amounts of GST- β2-adaptin fusion proteins as assessed by Ponceau Red. Src-phosphorylated proteins were detected by western blotting (WB) using an antibody against phosphotyrosine residues (P-Tyr). Note that the Y737F, Y874F and Y926F mutants are not phosphorylated. Data are representative of at least three independent experiments.

• Supplemental Figure S3 -

  Fig. S3. Distribution of transferrin in cells expressing β2-adaptin wild type or the Y737F mutant. HEK 293 cells, seeded on coverslips, were transfected with siRNA β2-adaptin and either wild type siRNA resistant β2-adaptin-YFP or the Y737F mutant. On the day of the experiment, cells were incubated with transferrin conjugated to Alexa Fluor 568 (Molecular Probes) for 5 minutes at 37°C. Cells were then washed with cold PBS and fixed for 15 minutes with paraformaldehyde (4% w/v in PBS). Distribution of transferrin and β2-adaptin YFP was visualized on a Zeiss LSM-510 META laser scanning microscope with a 40× oil immersion lens in a multitrack mode using a dual excitation (543 nm for Alexa Fluor 568 and 514 nm for YFP) and emission (LP 560 nm for Alexa Fluor 568 and BP 530-600 nm for YFP) filter sets. Shown are representative confocal images of transferrin (red, upper and lower left panels) and either β2-adaptin-YFP wild type (yellow, upper right panel) or the Y737F mutant (yellow, lower right panel).