Volume 8, Issue 5, Pages 995-1004 (November 2001) Mutation of the c-Cbl TKB Domain Binding Site on the Met Receptor Tyrosine Kinase Converts It into a Transforming Protein  Pascal Peschard, Tanya M Fournier, Louie Lamorte, Monica A Naujokas, Hamid Band, Wallace Y Langdon, Morag Park  Molecular Cell  Volume 8, Issue 5, Pages 995-1004 (November 2001) DOI: 10.1016/S1097-2765(01)00378-1

Figure 1 c-Cbl Promotes the Ubiquitination of the Met Receptor 293T cells were transiently transfected with expression plasmids encoding CSF-Met, alone or with the indicated Cbl construct. Cell lysates (500 μg) were subjected to immunoprecipitation (IP) with Met antibodies followed by immunoblotting with anti-ubiquitin antibodies. The nitrocellulose membrane was stripped and reprobed with anti-Met. Whole-cell lysates (25 μg) were subjected to immunoblotting with HA antibodies. Molecular Cell 2001 8, 995-1004DOI: (10.1016/S1097-2765(01)00378-1)

Figure 2 Identification of the Cbl TKB Domain Binding Site in Met (A) Structure of c-Cbl, Cbl-N, and Cbl-C proteins. (B) 293T cells were transfected with expression plasmids encoding CSF-Met wild-type, mutants, or Tpr-Met. Proteins from serum-starved cells were immunoprecipitated with anti-c-Cbl and immunoblotted with anti-pTyr. In parallel, cell lysates were subjected to in vitro binding assays with GST-Cbl-N and GST-Cbl-C fusion proteins followed by immunoblotting with anti-Met. Whole-cell lysates were subjected to immunoblotting with anti-Met. (C) Lysates (500 μg) of 293T cells transfected with CSF-Met wild-type or Y1003F were subjected to in vitro binding assays as described in (B) and immunoblotted with anti-Met. (D) Proteins from 293T cells transfected with the indicated plasmids were immunoprecipitated with anti-HA and immunoblotted with anti-Met. The same lysates were immunoprecipitated with anti-Met and immunoblotted with anti-pTyr. Whole-cell lysates were immunoblotted with Met and HA antibodies. (E) Competition of HA-Cbl-N from coimmunoprecipitation with wild-type CSF-Met protein using peptides corresponding to the sequence surrounding Y1003 with either a phosphotyrosine residue (pY1003) or a phenylalanine residue (F1003) at position 1003 or using a phosphopeptide surrounding the tyrosine 1356 (pY1356). The immunoprecipitations were performed as described in (D). (F) CSF-Met was immunoprecipitated from transfected 293T cells transfected with either vector, CSF-Met wild-type, or CSF-Met Y1003F. The immunoprecipitated proteins were resolved by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was immunoblotted with purified GST-Cbl-N proteins, and then stripped and probed with Met antibody. Molecular Cell 2001 8, 995-1004DOI: (10.1016/S1097-2765(01)00378-1)

Figure 3 The Cbl TKB Domain Binding Site in Met Is Required for c-Cbl-Induced Ubiquitination of Met Lysates from 293T cells cotransfected with expression plasmids encoding c-Cbl, CSF-Met, or Tpr-Met were subjected to immunoprecipitation with either anti-Met antibodies and immunoblotted with anti-ubiquitin or anti-HA-Cbl and immunoblotted with anti-pTyr. Whole-cell lysates (25 μg) were immunoblotted with anti-Met and anti-HA. Molecular Cell 2001 8, 995-1004DOI: (10.1016/S1097-2765(01)00378-1)

Figure 4 Tyrosine 1003 to Phenylalanine Substitution in Met Promotes Its Oncogenic Activation (A) Rat1 fibroblasts were transfected with either vector alone, CSF-Met wild-type, or Y1003F (2 μg DNA/60 mm petri dish) or Tpr-Met (500 ng DNA/60 mm petri dish) and grown in DMEM containing 10% FBS for 2 weeks in the presence or absence of ligand (10 ng/ml CSF-1) as indicated. The numbers indicating the transforming frequency are indicative of four different experiments in which five different clones of CSF-Met Y1003F were tested. (B) NIH 3T3 mouse fibroblasts were infected with retroviruses encoding either Met wild-type or Met Y1003F. The cells were maintained for 2 weeks in 5% FCS under G418 selection. For each infection, ten clones were picked, grown, and pooled. Phase contrast images were taken 3 days after cells had been seeded. (C) Phase contrast images of stable lines of MDCK cells expressing either CSF-Met wild-type or Y1003F mutant. Molecular Cell 2001 8, 995-1004DOI: (10.1016/S1097-2765(01)00378-1)

Figure 5 A Met Receptor Mutated at Tyrosine 1003 Is Impaired in Receptor Downregulation (A) Metabolic labeling of stable fibroblast cell lines expressing either wild-type Met or the Y1003F mutant Met. Stable cell lines were pulse-labeled with 35S-methionine for 1 hr. At the indicated times, cell lysates were immunoprecipitated with anti-Met antibodies, resolved by 8% SDS-PAGE, and subjected to autoradiography. (B) Graphic representation of duplicate experiments. (C) Stable cell lines as in (A) were stimulated with ligand (500 ng/ml CSF-1) for the times indicated. Proteins were immunoprecipitated with anti-Met sera, separated by 8% SDS-PAGE, and transferred to a membrane. Membranes were immunoblotted with phosphotyrosine antibody, stripped, and reprobed with Met antibody. Molecular Cell 2001 8, 995-1004DOI: (10.1016/S1097-2765(01)00378-1)

Figure 6 A Model for the Recruitment of c-Cbl to the Met Receptor Molecular Cell 2001 8, 995-1004DOI: (10.1016/S1097-2765(01)00378-1)