![Figure 2. c-Cbl induces ubiquitin-dependent degradation of mature and phosphorylated BCR-ABL proteins, while CHIP degrades immature BCR-ABL proteins. (A) Structure of BCR-ABL, BCR, ABL, and BCR-ABL mutants used in this study. Deletions of functional domains of p185 BCR-ABL are shown, including the coiled-coil oligomerization domain (Δ40), SH2-binding domain (ΔSH2 bind), SH3 domain (ΔSH3), ΔSH2 domain (ΔSH2), kinase domain (Δkina), SH2-binding domain and SH3 domain (ΔSH2biΔSH3), kinase-negative mutant (ABL K290R, KD), imatinib-resistant mutants (ABL T315I, E255K), ABL, BCR, and BCR 1-413. (B) Effects of CHIP and c-Cbl on the stabilities of BCR-ABL, BCR, ABL, and BCR-ABL mutants. COS7 cells were transiently transfected with the wt p185 BCR-ABL or its mutants [KD, T315I, E255K, Δ40, Δkina, ΔSH2, ΔSH2 bind, ΔSH3, ΔSH2biΔSH3, BCR 1-413], BCR, and ABL (type 1b) together with CHIP, c-Cbl, or control vector (−) under the tet regulatory expression system. The cells were incubated with or without tet for 24 hours and analyzed by immunoblotting with antibodies against ABL, BCR, CHIP, c-Cbl, and actin. The top graphs indicate the intensities of the signals of tet (−) cells relative to those of tet (+) cells that were normalized against those of the control vector. The data were expressed as percentage of the signals obtained with each control. The red lines, at 53.2% and 73.3% in the CHIP and c-Cbl panels, respectively, are drawn to make a statistically significant discrimination into 2 groups (Akaike Information Criterion and t test, P < .001). (C) Immunoprecipitated Flag-tagged BCR-ABL proteins that were in vitro–transcribed/translated with (+) or without (−) GA (10μM) were incubated with biotin-labeled ubiquitin, GST-CHIP, GST-c-Cbl, E1, mixtures of E2s as indicated. The numbers indicate the signals of ubiquitinated BCR-ABL. (D) Immunoprecipitated BCR-ABL proteins from p185 wt- and Δkina-expressing Ba/F3 cell lysates were incubated with GST-CHIP, GST-c-Cbl, E1, E2s (Ubc4 and UbcH5c), and biotin-labeled ubiquitin. Ubiquitinated proteins were detected by streptavidin-HRP (C-D).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/116/18/10.1182_blood-2009-10-249623/4/zh89991059850002.jpeg?Expires=1725055066&Signature=oFC6emLFkO-2i28LhDODE7URgNWVqS7DsjVQddymtX3vyKqaP3jzEVXWYcmI1yMgLuqTCZL3GZMoMHEQTX5Fs~9jbMWwF8hMR4nx1BVXzuA2bhPtDpx0cALGpeOmD93QtXVljSWkirCQnBxX4eGhlqWy~Spi-2E72uE1bKjyXwUU7rJxeBreK7vQfwS8keueyDXmW-wN9UBgTbQ99Jbd8~JHGrRTxAs-ZVAI4OmzGAZNKVMIRyxQzbMBUziH-OMmLnGney2NjCHyHPtKUOFx5xCAjfP6ZcKmMWYOigHC0SQBrTgx767IN7AoHpvuKJsTbsTu88PE6avZhaRpIj1Z9w__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
c-Cbl induces ubiquitin-dependent degradation of mature and phosphorylated BCR-ABL proteins, while CHIP degrades immature BCR-ABL proteins. (A) Structure of BCR-ABL, BCR, ABL, and BCR-ABL mutants used in this study. Deletions of functional domains of p185 BCR-ABL are shown, including the coiled-coil oligomerization domain (Δ40), SH2-binding domain (ΔSH2 bind), SH3 domain (ΔSH3), ΔSH2 domain (ΔSH2), kinase domain (Δkina), SH2-binding domain and SH3 domain (ΔSH2biΔSH3), kinase-negative mutant (ABL K290R, KD), imatinib-resistant mutants (ABL T315I, E255K), ABL, BCR, and BCR 1-413. (B) Effects of CHIP and c-Cbl on the stabilities of BCR-ABL, BCR, ABL, and BCR-ABL mutants. COS7 cells were transiently transfected with the wt p185 BCR-ABL or its mutants [KD, T315I, E255K, Δ40, Δkina, ΔSH2, ΔSH2 bind, ΔSH3, ΔSH2biΔSH3, BCR 1-413], BCR, and ABL (type 1b) together with CHIP, c-Cbl, or control vector (−) under the tet regulatory expression system. The cells were incubated with or without tet for 24 hours and analyzed by immunoblotting with antibodies against ABL, BCR, CHIP, c-Cbl, and actin. The top graphs indicate the intensities of the signals of tet (−) cells relative to those of tet (+) cells that were normalized against those of the control vector. The data were expressed as percentage of the signals obtained with each control. The red lines, at 53.2% and 73.3% in the CHIP and c-Cbl panels, respectively, are drawn to make a statistically significant discrimination into 2 groups (Akaike Information Criterion and t test, P < .001). (C) Immunoprecipitated Flag-tagged BCR-ABL proteins that were in vitro–transcribed/translated with (+) or without (−) GA (10μM) were incubated with biotin-labeled ubiquitin, GST-CHIP, GST-c-Cbl, E1, mixtures of E2s as indicated. The numbers indicate the signals of ubiquitinated BCR-ABL. (D) Immunoprecipitated BCR-ABL proteins from p185 wt- and Δkina-expressing Ba/F3 cell lysates were incubated with GST-CHIP, GST-c-Cbl, E1, E2s (Ubc4 and UbcH5c), and biotin-labeled ubiquitin. Ubiquitinated proteins were detected by streptavidin-HRP (C-D).
