AIP2 interacts with EGR2. (A and B) Domain structures of AIP2 and EGR2. (C) An EGR2 expression plasmid was transfected with or without the AIP2 plasmid into HEK293 cells. AIP2 in the lysates of transfected cells was immunoprecipitated with anti-AIP2 antibody. Binding EGR2 to AIP2 was determined with anti-EGR2 antibody (top). The same membrane was reprobed with anti-AIP2 antibody (middle). Protein levels of EGR2 in whole-cell lysates were analyzed as a control (bottom). (D) Purified primary T cells were stimulated with anti-CD3 or anti-CD3 plus anti-CD28 for 2 h. Interactions occurring in those cells were analyzed, as shown in panel A. (E) Myc-tagged AIP2 expression plasmids were cotransfected with Flag-EGR2 or each of its YF mutants into HEK293 cells. The interaction of AIP2 with EGR2 or its YF mutants was determined by coimmunoprecipitation of EGR2 with anti-Flag antibody and Western blotting with anti-Myc antibody (top). Protein expression of EGR2 (middle) and AIP2 (bottom) was analyzed by Western blotting using anti-Myc and anti-Flag, respectively. (F) GST or GST-WW proteins (5 μg) were incubated with His-EGR2 or His-EGR2/YF proteins. GST or GST-WW proteins were pulled down by adding GST-Sepharose beads. Binding of EGR2 was detected with anti-EGR2 antibody (top). The same membrane was reprobed with anti-GST antibodies (middle). The levels of EGR2 in the whole reaction mixtures were determined by Western blotting using anti-EGR2 antibody (bottom).

AIP2 expression promotes EGR2 degradation. (A) Flag-tagged EGR2 or its YF mutant expression plasmids were cotransfected with or without Myc-AIP2 expression plasmids into HEK293 cells. Transfected cells were treated with cycloheximide (CHX; 50 μg/ml) for different amounts of time, as indicated. The protein stability of EGR2 was examined by Western blotting using anti-Flag antibody (top panels). Protein levels of actin were determined as loading controls (bottom panels). (B) The densities of each EGR2 band were quantified using NIH Image 1.63 software. The half-lives of EGR2 and its YF mutant were calculated as shown in panel A. (C) AIP2 and EGR2 expression plasmids were cotransfected into HEK293T cells. Cells were treated with or without cycloheximide in the absence or presence of MG132 or with bafilomycin A (Baf) for 4 h. EGR2 protein levels were analyzed by Western blotting (top). The same membrane was reprobed with anti-actin antibody as a control (bottom). (D) Mouse primary T cells were infected with retrovirus carrying control vector (MIG) or with MIG-AIP2. Protein levels of EGR2 in infected T cells were analyzed by Western blotting. (E) Total RNA in these infected T cells shown in panel C was isolated, and the levels of EGR2 mRNA were determined by reverse transcriptase PCR. (F) T cells infected with virus carrying the MIG empty vector or MIG-AIP2 were cultured with anti-CD3 plus anti-CD28 for 2 days. EGR2 ubiquitination was detected by immunoprecipitation with anti-EGR2 antibody and Western blotting with anti-Ub antibody (top). Protein expression of EGR2 was analyzed with anti-EGR2 antibody (bottom).

AIP2 knockdown upregulates FasL expression in T cells. (A) The selected sequences for AIP2 siRNA. (B) Mouse CD4⁺ T cells were infected with lentiviruses that carry empty control vector or each of these three API2 siRNAs. The expression of AIP2 (top) and EGR2 (middle) were analyzed by Western blotting. Actin expression was analyzed as a control (bottom). (C) EGR2 ubiquitination in AIP2 knockdown or control T cells was determined by immunoprecipitation of EGR2 and Western blotting with anti-ubiquitin (Ub) antibody. (D) Mouse CD4⁺ T cells were infected with control or AIP2 siRNA viruses. Infected cells were restimulated with anti-CD3 plus anti-CD28, and the percentages of apoptotic cells (left panels) and FasL expression cells (right panels) were analyzed by flow cytometry. (E) Whole-cell lysates of AIP2 knockdown and control cells were subjected to Western blot analysis of Cbl-b (top) and actin (middle) protein expression. The mRNA levels of Cbl-b in these cells were analyzed by real-time reverse transcriptase PCR in parallel (bottom). Error bars indicate data from three independent experiments (means ± standard deviations).

AIP2 expression enhances T-cell activation. (A) Mouse primary T cells were infected with retrovirus carrying AIP2, Itch, or GFP only as a control. Infected cells were cultivated with anti-CD3 or anti-CD3 plus anti-CD28 for 2 days. Partial supernatants were collected from each well for the analysis of IL-2 production by enzyme-linked immunosorbent assay. (B) Cells were then chased with 0.5 μCi [³H]thymidine (³H-TdR) for an additional 12 h, and the [³H]thymidine incorporation was analyzed. Error bars represent data from three independent experiments (means ± standard deviations). (C) Infected cells were stained with phycoerythrin-conjugated annexin V. GFP-positive cells were gated for the analysis of annexin V, and representative data are shown. (D) The average percentages of annexin V-positive cells from three independent experiments are shown. The Student t test was used for the statistic analysis.

AIP2 is an E3 ubiquitin ligase of EGR2. (A) Myc-AIP2, Flag-EGR2, and HA-ubiquitin (Ub) expression plasmids were transfected into HEK293 cells. The EGR2 protein was immunoprecipitated with anti-Flag antibody. Ubiquitin conjugation onto EGR2 was detected by Western blotting using anti-HA antibody (top). The same membrane was reprobed with anti-EGR2 antibody (middle). Protein expression of AIP2 in whole-cell lysates was detected with anti-Myc antibody (bottom). (B) Flag-tagged EGR2 or its YF mutant was cotransfected with HA-ubiquitin and AIP2. Ubiquitination of EGR2 or its mutant was determined as described in the legend to panel A. (C) Flag-EGR2 and HA-ubiquitin expression plasmids were cotransfected into HEK293 cells with Myc-tagged AIP2 or with the AIP2/ΔHECT mutant. Ubiquitination of EGR2 was determined as described in the legend to panel A. (D) The interaction of Myc-AIP2 or Myc-AIP2/ΔHECT with Flag-EGR2 was analyzed by immunoprecipitation of Myc-tagged AIP2 or AIP2/ΔHECT with anti-Myc antibody and Western blotting with anti-EGR2 antibody (top). The expression levels of AIP2, AIP2/ΔHECT (middle), and EGR2 (bottom) were determined as controls. (E) Mouse primary T cells were stimulated with anti-CD3 or anti-CD3 plus anti-CD28 for 24 h. EGR2 ubiquitination was detected by immunoprecipitation of EGR2 from the lysates of stimulated T cells and Western blotting with anti-ubiquitin antibody (top). The expression of EGR2 was analyzed as a control (bottom).

AIP2 inhibits FasL expression in T cells. (A) FasL luciferase, AIP2, and EGR2 expression plasmids were cotransfected into HEK293 cells, with combinations as indicated. Luciferase activities were determined. Error bars represent data from three independent experiments. (B) Mouse primary T cells were infected with retroviruses that carry the pMIG control plasmid, pMIG-AIP2, or pMIG-Itch. Infected cells were restimulated with anti-CD3 plus antiCD28 for 24 h. FasL expression in the gated GFP⁺ cells was analyzed by flow cytometry. (C) The percentage of FasL⁺ cells, as analyzed in panel B, is shown. Error bars indicate data from four independent experiments (means ± standard deviations).