PMC

Overexpression of APS blocks insulin-stimulated tyrosine phosphorylation of Cbl and Crk-Cbl interaction. 3T3-L1 adipocytes were transfected with vector alone, Myc-tagged wild-type APS (Myc-APS/WT), or Myc-APS/Y⁶¹⁸F. (A) Following treatment with or without 100 nM insulin for 2 min, cells were lysed and immunoprecipitation (i.p.) was performed with an anti-Cbl antibody. Immunoprecipitates were analyzed by antiphosphotyrosine and anti-Cbl immunoblotting (upper panels). Cell lysates were immunoblotted with anti-Myc and anti-phospho-ERK antibodies (lower panels). (B) Following treatment with or without 100 nM insulin for 2 min, cells were lysed and immunoprecipitation was performed with an anti-Crk antibody. Immunoprecipitates were analyzed by anti-Cbl and anti-Crk immunoblotting (upper panels). Cell lysates were immunoblotted with anti-Myc, anti-Cbl, and anti-phospho-ERK antibodies (lower panels).

APS facilitates the phosphorylation of tyrosines 371, 700, and 774 in Cbl by the insulin receptor. CHO-IR cells were cotransfected with Myc-tagged wild-type APS (Myc-APS/WT) plus HA-tagged Cbl/WT, Cbl/G³⁰⁶E, or Cbl/1-700 (A) or Cbl/WT, Cbl/Y⁷⁰⁰F/Y⁷⁷⁴F, Cbl/Y³⁷¹F/Y⁷⁰⁰F/Y⁷⁷⁴F, Cbl/Y⁷⁰⁰F/Y⁷³¹F/Y⁷⁷⁴F, or Cbl/Y³⁷¹F/Y⁷⁰⁰F/Y⁷³¹F/Y⁷⁷⁴F (B). After treatment with or without 100 nM insulin for 2 min, cells were lysed and immunoprecipitation (i.p.) was performed using an anti-HA antibody. Immunoprecipitates were analyzed by antiphosphotyrosine and anti-HA immunoblotting (upper panels). Cell lysates were immunoblotted with anti-Myc and anti-phospho-ERK antibodies (lower panels).

Association with APS is necessary for the tyrosine phosphorylation of Cbl by the insulin receptor. CHO-IR cells (A) or 3T3-L1 adipocytes (B) were cotransfected with HA-Cbl and vector DNA, Myc-tagged wild-type APS (Myc-APS/WT) or APS/Y⁶¹⁸F. Separately, CHO-IR cells were also cotransfected with HA-Cbl and empty vector or FLAG-CAP (C). After treatment with or without 100 nM insulin for 2 min, cells were lysed and immunoprecipitation (i.p.) was performed using an anti-HA antibody. Immunoprecipitates were analyzed by antiphosphotyrosine, anti-HA, anti-Myc, and anti-Crk immunoblotting (upper panels). Cell lysates were immunoblotted with anti-Myc, anti-FLAG, anti-HA, and anti-phospho-ERK antibodies (lower panels).

Phosphorylation of tyrosine 618 in APS is required for its association with Cbl in response to insulin. 3T3-L1 adipocytes were cotransfected with HA-Cbl and a Myc-tagged wild-type APS (APS/WT) or Y⁶¹⁸F mutant APS (APS/YF). After treatment with or without 100 nM insulin for 2 min, cells were lysed and immunoprecipitation (i.p.) was performed using an anti-Myc antibody. Myc-APS and insulin receptor (IR) were detected in the immunoprecipitates by immunoblotting with antiphosphotyrosine, anti-Myc, and anti-insulin receptor β chain antibodies (upper panels). HA-Cbl was detected by anti-HA immunoblotting in both immunoprecipitates and cell lysates. Phosphorylated ERK was detected in cell lysates by immunoblotting with an anti-phospho-ERK antibody (lower panels).

Dose-dependent effects of ectopic APS expression on insulin-stimulated Cbl phosphorylation. 3T3-L1 adipocytes were cotransfected with various amount of vector DNA and Myc-tagged wild-type APS (Myc-APS/WT) or Myc-APS/Y⁶¹⁸F. Lanes 1 and 6, 300 μg of vector plus 0 μg of APS; lanes 2 and 7, 285 μg of vector plus 15 μg of APS; lanes 3 and 8, 270 μg of vector plus 30 μg of APS; lanes 4 and 9, 200 μg of vector plus 100 μg of APS; lanes 5 and 10, 0 μg of vector and 300 μg of APS. Following treatment with or without 100 nM insulin for 2 min, cells were lysed and immunoprecipitation (i.p.) was performed with an anti-Cbl antibody. Immunoprecipitates were analyzed by antiphosphotyrosine and anti-Cbl immunoblotting (upper panels). Cell lysates were immunoblotted with anti-Myc antibody (lower panels).

In vivo and in vitro interactions between APS and CAP. (A) COS-1 cells were cotransfected with Myc-APS and FLAG-tagged wild-type CAP (WT), CAPΔSH3, or CAPΔSoHo. Cells were lysed 20 h after transfection, and immunoprecipitation (i.p.) was performed using an anti-FLAG antibody. Immunoprecipitates were analyzed by anti-Myc, anti-Cbl, and anti-FLAG immunoblotting (upper panels). Myc-APS was detected in cell lysates by immunoblotting with an anti-Myc antibody (lower panels). (B) 3T3-L1 cells were used for the same experiment as described for panel A except that cells were treated with or without 100 nM insulin for 2 min prior to the preparation of lysates. (C) Myc-APS was overexpressed in COS-1 cells. Lysates were incubated with glutathione-Sepharose-bound GST or GST-CAPSH3 fusion proteins. Precipitates were subjected to immunoblotting with anti-Myc or anti-Cbl antibodies. The GST fusion proteins used were stained with Coomassie blue. (D) 3T3-L1 adipocytes were electroporated with Myc-tagged wild-type APS or FLAG-CAP and allowed to recover for 30 h. The cells were treated with or without 100 nM insulin for 2 min. FLAG-CAP and Myc-APS were visualized by indirect immunofluorescent staining.

Overexpression of APS blocks insulin-stimulated GLUT4 translocation to the plasma membrane. (A) 3T3-L1 adipocytes were electroporated with 100 μg of GLUT4-EGFP plus 300 μg of vector, Myc-tagged wild-type APS (Myc-APS/WT), or Myc-APS/Y⁶¹⁸F and allowed to recover for 30 h. The cells were treated with or without 100 nM insulin for 30 min. Cells were fixed, and fluorescence was visualized by confocal microscopy. GLUT4-EGFP was visualized by direct fluorescence, and Myc-APS was visualized by indirect immunofluorescence. These are representative images of middle sections of cells obtained from three independent experiments. (B) Numbers of GLUT4-EGFP-transfected cells displaying visually detectable plasma membrane (PM) rim fluorescence. These data were obtained by blind counting of more than 80 cells from three independent experiments. Error bars indicate standard deviations. (C) 3T3-L1 adipocytes were electroporated with 100 μg of GLUT4-EGFP plus 100 μg of Myc-APS/Y⁶¹⁸F. After treatment with 100 nM insulin for 30 min, cells were fixed and fluorescence was visualized by confocal microscopy as described for panel A. This is a representative field of a middle section of cells obtained from three independent experiments.