1. 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 (November 2001)
DOI: /S (01)

2. 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, DOI: ( /S (01) )

3. 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, DOI: ( /S (01) )

4. 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, DOI: ( /S (01) )

5. 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, DOI: ( /S (01) )

6. 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, DOI: ( /S (01) )

7. Figure 6 A Model for the Recruitment of c-Cbl to the Met Receptor
Molecular Cell 2001 8, DOI: ( /S (01) )