![Figure 4. Cbl-R420Q inhibits ubiquitylation and endocytosis of RTKs. (A) Identification of a novel Cbl mutant from an AML patient. The cDNA from 150 AML patients was screened for Cbl mutations by direct sequencing as described in “Materials and methods.” c-Cbl of one patient showed a heterozygous mutation at position 1259 (G>A), resulting in replacement of arginine 420 by glutamine of c-Cbl. (B,C) Cbl-R420Q binds to Flt3 and inhibits ubiquitylation of Flt3. COS-7 cells were transiently transfected with the indicated plasmids together with a plasmid for HA-tagged ubiquitin. Forty-eight hours later, cell lysates were prepared and equal amounts of lysate were immunoprecipitated using anti-Flt3 or anti-HA antibodies. The immunoprecipitates were resolved on SDS-PAGE and analyzed with anti-HA or anti-Cbl (panel B) or anti-Flt3 (panel C) antibodies. (D) The R420Q mutation results in loss of Cbl E3 ubiquitin ligase activity. The 293T cells were transiently transfected with Flt3 and Flt3 was immunoprecipitated. The immunoprecipitated Flt3 was incubated with His-tagged ubiquitin and the indicated Cbl RING finger constructs and in vitro ubiquitylation was performed as described in “Materials and methods.” The reaction mixtures were separated on SDS-PAGE and ubiquitylation of Flt3 was analyzed by immunoblotting the membrane with anti-His antibodies. (E) Internalization of EGFR, PDGFR, and Flt3 is inhibited by the mutant Cbl. The surface level of receptors following time lapse (up to 60 minutes) after EGF, PDGF, or FL stimulation was analyzed by receptor down-regulation assays. The results are expressed as a percentage of the [125I]-labeled EGF or PDGF bound to cell surface receptor after stimulation by nonlabeled EGF or PDGF for the indicated times. For Flt3, the surface expression of HA-tagged Flt3 was analyzed by flow cytometry after staining with an anti-HA antibody. The total level of steady-state surface EGFR after 2.5 days of transfection was detected using [125I]-EGF without preceding incubation with nonlabeled EGF. Results are expressed as means plus or minus SD of 3 independent experiments.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/110/3/10.1182_blood-2007-01-066076/4/zh80150705380004.jpeg?Expires=1735818461&Signature=Xr9X~np-VZsJVaph401jt3xW~BrMa312IoKvbokBtUqIk9cbhPmVgbXTdaRkYNBzg2FZI4kYvRoufBJVRduud8IX0eOGQdTTqDGPfe4CKu5SHJQ4qEuKxtt5LpbJqM9DlF7LP9ewgkYzrz1mS4AsFYJRtRAkiHNMYdpDJE-uBBb5IfPSs9prxsxzKyYMINII-vWN6RzbU~QAtWTGseyIcWuTFIXMhhx-R2qa~mhiQATD0c2nQ~Opv7ltWnaN4D~Qlyu3Guz4fnJwVP81rDUfzJQjgPT2Edk1JZ2~VK4Casc4LEVXTjX4FrJCAiEViuhM2bJnEqEsD7yMrz4o81XlVA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Cbl-R420Q inhibits ubiquitylation and endocytosis of RTKs. (A) Identification of a novel Cbl mutant from an AML patient. The cDNA from 150 AML patients was screened for Cbl mutations by direct sequencing as described in “Materials and methods.” c-Cbl of one patient showed a heterozygous mutation at position 1259 (G>A), resulting in replacement of arginine 420 by glutamine of c-Cbl. (B,C) Cbl-R420Q binds to Flt3 and inhibits ubiquitylation of Flt3. COS-7 cells were transiently transfected with the indicated plasmids together with a plasmid for HA-tagged ubiquitin. Forty-eight hours later, cell lysates were prepared and equal amounts of lysate were immunoprecipitated using anti-Flt3 or anti-HA antibodies. The immunoprecipitates were resolved on SDS-PAGE and analyzed with anti-HA or anti-Cbl (panel B) or anti-Flt3 (panel C) antibodies. (D) The R420Q mutation results in loss of Cbl E3 ubiquitin ligase activity. The 293T cells were transiently transfected with Flt3 and Flt3 was immunoprecipitated. The immunoprecipitated Flt3 was incubated with His-tagged ubiquitin and the indicated Cbl RING finger constructs and in vitro ubiquitylation was performed as described in “Materials and methods.” The reaction mixtures were separated on SDS-PAGE and ubiquitylation of Flt3 was analyzed by immunoblotting the membrane with anti-His antibodies. (E) Internalization of EGFR, PDGFR, and Flt3 is inhibited by the mutant Cbl. The surface level of receptors following time lapse (up to 60 minutes) after EGF, PDGF, or FL stimulation was analyzed by receptor down-regulation assays. The results are expressed as a percentage of the [125I]-labeled EGF or PDGF bound to cell surface receptor after stimulation by nonlabeled EGF or PDGF for the indicated times. For Flt3, the surface expression of HA-tagged Flt3 was analyzed by flow cytometry after staining with an anti-HA antibody. The total level of steady-state surface EGFR after 2.5 days of transfection was detected using [125I]-EGF without preceding incubation with nonlabeled EGF. Results are expressed as means plus or minus SD of 3 independent experiments.
