(A) COS-1 cells were transiently transfected with wild-type c-Kit expression plasmids and stimulated with SCF as indicated. After lysis, cell lysates were subjected to either an immunoprecipitation (IP) with c-Kit antibody or a pulldown (Pd) with GST fusion proteins of either the N-terminal or C-terminal half of Cbl. Immunocomplexes were separated on an SDS/8% polyacrylamide gel, blotted and sequentially probed for c-Kit (upper panel) or anti-phosphotyrosine (lower panel). (B) COS-1 cells were co-transfected with expression plasmid for c-Kit and Myc-tagged APS and stimulated with SCF as indicated. After lysis, cell lysates were subjected to immunoprecipitation with anti-c-Kit as well to a pulldown with GST–Cbl (N-term) fusion protein. Complexes were analysed by Western blotting and the membranes were sequentially probed for c-Kit, phosphorylated c-Kit and APS. (C) COS-1 cells were transfected with the c-Kit mutant I571A/L939A and samples were treated as described above. (D) Cbl was immunoprecipitated from COS-1 cells transfected with either wild-type c-Kit or the I571A/L939A mutant and samples were analysed by Western blotting.

(A) COS-1 cells were transfected with wild-type c-Kit (lanes 1 and 2) as well as the I571A/L939A (lanes 3 and 4) double mutant and stimulated with SCF for the indicated periods of time (min; values above lanes) and concentrations. Lysates were immunoprecipitated with a c-Kit antibody and analysed by Western blotting. The membrane was probed for tyrosine-phosphorylated residues (4G10) and reprobed for c-Kit as loading control. (B) Densitometric analysis of an Src in vitro kinase assay. Src was immunoprecipitated from an aliquot of cell lysate prepared as described above, followed by incubation (3 h at 4 °C) with 50 μM [γ-³²P]ATP in kinase buffer (described in detail in the subsection ‘In vitro kinase assay’ in the Experimental section). Samples were separated on an SDS/8% polyacrylamide gel and blotted on to Immobilon. The filter was incubated with 1 M KOH for 1 h at 55 °C to hydrolyse non-specific phosphoserine present in the proteins, exposed to the PhosphoImager and densitometrically analysed. Src activity upon stimulation in the respective mutant (grey bars) is expressed as fold activity in unstimulated cells (white bars). Mean values and standard deviations for three independent experiments are shown.

(A) PAE cells expressing wild-type, Y568F/Y570F or I571A/L939A c-Kit were stimulated with SCF for the indicated time points, followed by lysis and immunoprecipitation with c-Kit antibody. Samples were subjected to a Western-blot analysis for ubiquitin (P4D1) and c-Kit. (B) An identical experiment was performed, with the exception that the membrane was probed with a selective polyubiquitin antibody (FK1) as well as the P4D1 ubiquitin antibody recognizing both poly- and mono-ubiquitin. Immunoprecipitated Src was included as a positive control for the polyubiquitin antibody. IB, immunoblot.

Activation of SFKs, initiated by Src binding to pY568 of ligand-stimulated c-Kit, leads to phosphorylation of Cbl. Phosphorylated Cbl is then recruited to pY568 and pY936 of c-Kit, which in turn results in monoubiquitination of the receptor and its subsequent internalization and degradation in the lysosomes.

(A) Phosphopeptides pY568 and pY936 (sequences in the Experimental section) immobilized to Affigel 10 beads were incubated (3 h at 4 °C) with the indicated purified GST fusion proteins in the presence of either unphosphorylated or phosphorylated peptides in solution as indicated. Samples were analysed by SDS/PAGE and Western blotting, and membranes were probed with an anti-GST antibody. (B) Pulldown experiment performed as described above with competition by soluble peptides (100 μM) as indicated.

Transiently transfected COS-1 cells expressing wild-type c-Kit or one of the two double mutants Y568F/Y570F or I571A/L939A respectively were stimulated with SCF for the indicated periods of time, immunoprecipitated with a Cbl antibody and subjected to Western blotting. The membrane was probed for tyrosine phosphorylation and Cbl respectively.

(A) PAE cells expressing wild-type (◆), Y568F/Y570F (▲) or I571A/L939A (●) c-Kit were incubated with ¹²⁵I-SCF for 60 min on ice, followed by incubation at 37 °C for the indicated periods of time. Cells were washed with an acidic buffer (see the subsection ‘Internalization and degradation experiments’ in the Experimental section) and surface-released radioactivity was determined. Loss of surface-bound radioactivity was taken as a measure of internalization. The medium was collected and subjected to precipitation with 10% (w/v) trichloroacetic acid. Trichloroacetic acidsoluble radioactivity was taken as a measure of degradation of ¹²⁵I-SCF. As a negative control, wild-type c-Kit-expressing PAE cells were treated with 2 μM SU6656 (■) to inhibit Src activity before examining internalization and SCF degradation. (B) Flow cytometry analysis of internalization. Ba/F3 cells transfected with either wild-type (◆), Y568F/Y570F (▲) or I571A/L939A (●) mutant c-Kit were incubated with 100 ng/ml SCF for the indicated periods of time, followed by flow cytometry analysis using an antibody against the extracellular part of c-Kit. The following colour coding of the histograms was used: grey: negative control; black; 0 min; blue: 2 min; red: 5 min; green: 15 min; purple: 30 min. (C) PAE cells expressing wild-type c-Kit were pre-incubated in the absence of inhibitor (◆) or in the presence of either 100 μM lactacystin (■) or 20 μM chloroquine (▲) for 4 h, followed by the assessment of binding, internalization and degradation of ¹²⁵I-SCF as described above. Mean values and standard deviations for three independent experiments are shown.