FIG. 3.

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).