Figure 4
Figure 4. Cbl mutants bind to Kit, inhibit ubiquitination and endocytosis, and potentiate Kit-induced signaling. (A) Kit physically interacts with c-Cbl. 32D-Kit-WT cells stably transfected with HA-tagged Cbl proteins (Cbl-WT, Cbl-R420Q, and Cbl-70Z) were deprived from cytokines overnight and subsequently exposed to the indicated cytokines for 10 minutes. Cbl proteins were immunoprecipitated by anti-HA antibodies, and immunoprecipitates were resolved on SDS-PAGE. Coimmunoprecipitation of Kit was analyzed using anti-Kit antibodies. (B) Cbl mutants inhibit ubiquitination of Kit. COS-7 cells were transiently transfected with the indicated constructs together with a plasmid for HA-tagged Ubq. After 48 hours of transfection, cells were serum-starved for 12 hours and stimulated with 50 ng/mL SCF for 10 minutes or left unstimulated. Cell lysates were prepared and equal amounts of lysates were immunoprecipitated using anti-Kit antibody. The immunoprecipitates were resolved on SDS-PAGE and analyzed with anti-HA or anti-Kit antibodies. Expression of overexpressed Cbl mutants is shown in total cell lysates (TCLs) using anti-Cbl antibody. (C) Internalization of Kit is inhibited by Cbl mutants. The 32D cells stably expressing Kit in the presence or absence of different Cbl mutants were washed with PBS and then stimulated with SCF for the indicated time points. Subsequently, the amount of Kit remained on the cell surface was measured by flow cytometry after staining with a PE-labeled anti-Kit antibody. Sodium azide was used to stop the internalization. Results are expressed as mean ± SD of 3 independent experiments. (D-E) Cbl influences Kit-mediated signaling. The 32D cells with and without Kit-WT were engineered to express the indicated Cbl proteins, deprived from cytokines overnight, and subsequently exposed to the indicated cytokines for 10 minutes. Western blot analyses with the indicated antibodies were performed. (F-H) Cbl proteins change the kinetics of Kit-phosphorylation and Kit-induced Akt and Erk activity. The 32D-Kit-WT cells stably transfected with the indicated Cbl proteins were treated as in panels D and E with the exception that they were exposed to SCF for the indicated time periods. Western blot analyses using phospho-specific antibodies for Kit (F), Akt (G), and Erk1/2 (H) were performed.

Cbl mutants bind to Kit, inhibit ubiquitination and endocytosis, and potentiate Kit-induced signaling. (A) Kit physically interacts with c-Cbl. 32D-Kit-WT cells stably transfected with HA-tagged Cbl proteins (Cbl-WT, Cbl-R420Q, and Cbl-70Z) were deprived from cytokines overnight and subsequently exposed to the indicated cytokines for 10 minutes. Cbl proteins were immunoprecipitated by anti-HA antibodies, and immunoprecipitates were resolved on SDS-PAGE. Coimmunoprecipitation of Kit was analyzed using anti-Kit antibodies. (B) Cbl mutants inhibit ubiquitination of Kit. COS-7 cells were transiently transfected with the indicated constructs together with a plasmid for HA-tagged Ubq. After 48 hours of transfection, cells were serum-starved for 12 hours and stimulated with 50 ng/mL SCF for 10 minutes or left unstimulated. Cell lysates were prepared and equal amounts of lysates were immunoprecipitated using anti-Kit antibody. The immunoprecipitates were resolved on SDS-PAGE and analyzed with anti-HA or anti-Kit antibodies. Expression of overexpressed Cbl mutants is shown in total cell lysates (TCLs) using anti-Cbl antibody. (C) Internalization of Kit is inhibited by Cbl mutants. The 32D cells stably expressing Kit in the presence or absence of different Cbl mutants were washed with PBS and then stimulated with SCF for the indicated time points. Subsequently, the amount of Kit remained on the cell surface was measured by flow cytometry after staining with a PE-labeled anti-Kit antibody. Sodium azide was used to stop the internalization. Results are expressed as mean ± SD of 3 independent experiments. (D-E) Cbl influences Kit-mediated signaling. The 32D cells with and without Kit-WT were engineered to express the indicated Cbl proteins, deprived from cytokines overnight, and subsequently exposed to the indicated cytokines for 10 minutes. Western blot analyses with the indicated antibodies were performed. (F-H) Cbl proteins change the kinetics of Kit-phosphorylation and Kit-induced Akt and Erk activity. The 32D-Kit-WT cells stably transfected with the indicated Cbl proteins were treated as in panels D and E with the exception that they were exposed to SCF for the indicated time periods. Western blot analyses using phospho-specific antibodies for Kit (F), Akt (G), and Erk1/2 (H) were performed.

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