Src family tyrosine kinases play an important role in Cbl-70Z–mediated transformation. (A) Phosphorylation of SFKs is enhanced by Cbl mutants. The 32D-Kit-WT (top 2 blots) or 32D-Kit-KD (bottom 2 blots) cells stably overexpressing the indicated Cbl proteins were starved from IL-3 overnight and subsequently exposed to the indicated cytokines for 10 minutes. Western blot analyses using the phosphospecific antibody recognizing the activated form of SFK members (anti-pSrc Y₄₁₆) and anti–β-actin antibody were performed as described. (B) Cbl-70Z binds stronger to p-SFKs and Cbl mutants physically interact with the KD Kit receptor. The 32D cells with or without Kit-KD stably transfected with HA-tagged Cbl proteins (Cbl-R420Q and Cbl-70Z) were treated as in panel A. Cbl proteins were immunoprecipitated by anti-HA antibodies, and immunoprecipitates were resolved on SDS-PAGE. Coimmunoprecipitation of Kit and p-SFKs was analyzed using anti-Kit or anti–phospho-SFK antibodies. (C) Interaction of SFKs and Cbl proteins is completely inhibited by the Src inhibitor PP-2. The 32D-Kit-KD cells stably overexpressing HA-tagged Cbl-70Z were starved from IL-3 in the presence of DMSO or PP-2 (15 μM) for 12 hours. Immunoprecipitation and Western blot analyses were performed as in panel B with anti–phospho-SFK and anti-HA antibodies. (D) Fyn physically binds to Cbl and its binding is phosphorylation dependent. The 32D-Kit-KD cells stably overexpressing HA-tagged Cbl-70Z were starved from IL-3 in the presence of DMSO or PP-2 (15 μM) for 12 hours. Cbl proteins were immunoprecipitated by anti-HA antibodies, and immunoprecipitates were resolved on SDS-PAGE. Coimmunoprecipitation of Fyn was analyzed using anti-Fyn antibody. (E) Src inhibitors also inhibit phosphorylation of Akt. Kit-KD cells overexpressing Cbl-70Z were treated as in panel C. In addition, cells were also starved in the presence of dasatinib (150 nM). Western blot analyses were performed with anti–phospho-Akt and anti-Akt antibodies. (F) Cbl-70Z–mediated ligand-independent DNA synthesis is blocked by Src inhibitors. The 32D cells stably expressing Kit or Flt3 and/or Cbl-70Z were starved from IL-3 for 12 hours, treated with the indicated cytokines and PP-1/PP-2 (15 μM) or DMSO for 6 hours, and incubated with ³[H]-thymidine. After 15 hours of incubation, proliferation was measured in ³[H]-thymidine incorporation assays. Data are shown as percentage of thymidine incorporation relative to the thymidine incorporation of the respective cell line with IL-3 supplementation. The data represent the average and SD of 3 independent experiments. (G) Colony growth is blocked by the Src inhibitor PP-2. The 32D cells stably overexpressing Kit or Flt3 constructs and/or Cbl-70Z were serum starved for 12 hours and then plated at a concentration of 1000 cells per dish in the presence or absence of PP-2 (15 μM) and in the absence of any growth factors. Colonies were counted on day 6. The assays were plated as triplicates. The numbers given show the results of 1 of at least 3 independent experiments per construct, which all gave similar results. (H) Imatinib and dasatinib inhibit cell proliferation substantially differently. Indicated cell lines were treated as in Figure 1A. In addition, either Kit inhibitor imatinib (2.5 μM) or dual kinase inhibitor (Kit and SFKs) dasatinib (150 nM) was added, and cells were counted at the indicated time points by the trypan blue exclusion method. The data represent the average and SD of 3 independent experiments.