Figure 1.
ALKWT and ALKF1174L expression and phosphorylation in the SH-SY5Y neuroblastoma cell line.
A. IMR-32 (WT) and SH-SY5Y (WT/F1174L) cells were untreated or stimulated with 6 nM agonist mAb 46 for 15 min. ALK immunoprecipitates were immunoblotted with polyclonal anti-ALK (REAB) and antiphosphotyrosine (4G10 platinium). Right panel: Quantification of the ratio P-ALK/ALK in IMR-32 and SH-SY5Y cells, results are expressed in mean and s.e.m. B. Total RNA of IMR-32 (WT) and SH-SY5Y (WT/F1174L) were extracted and cDNA were obtained by reverse transcription. Sequencing chromatograms of the cDNAs obtained after RT-PCR are shown. C. SH-SY5Y cells (WT/F1174L) were treated or not with the ALK specific tyrosine kinase inhibitor NVP-TAE684 at 50 nM for two days. After ALK immunoprecipitation proteomics analysis and quantifications of interested peptides (carrying or not the mutation spot) were done by ESI-MS, after normalization.
Figure 2.
Proteasome dependent degradation of receptor retained in intracellular compartment.
A. NIH3T3 cells stably expressing either the WT ALK or F1174L mutated ALK were non-treated or treated with Bafilomycin A1 (0.25 µM) or with Lactacystin (10 µM) for 16 hours. ALK immunoprecipitates from 1 mg of total cell lysate proteins were subjected to western blot analysis. ALK was immunoblotted with polyclonal REAB antibody. B. SH-SY5Y were non-treated or treated with Bafilomycin A1 (0.25 µM) or with Lactacystin (10 µM) for 16 hours. ALK immunoprecipitates from 1.5 mg of total cell lysate proteins were subjected to western blot analysis using polyclonal REAB antibody. The experiment was done in triplicates and quantified with LiCor Odyssey system. Results were expressed in percentage of control +/− s.e.m.
Figure 3.
Agonist mAb treatment induced lysosome targeting and antagonist mAb treatment induced cell surface recyling of ALK receptor.
A. CHO cells were transiently transfected with construct encoding for ALK WT. Cells were pulsed 10 minutes on ice at 4°C with 6 nM of agonist mAb 46 and transferrin coupled to AlexaFluor 546, then chased at 37°C in a time course manner. Direct immunodetection of agonist mAb 46 were done with anti-mouse secondary antibody coupled to Alexa Fluor 488. Cellular localization of ALK receptors were shown by immunofluorescence using confocal laser scanning microscopy. Merge images were mounted thanks to Photoshop software. B. CHO cells were transiently transfected with construct encoding for ALK wild type. Cells were pulsed 10 minutes on ice at 4°C with 6 nM of antagonist mAb 30 and transferring coupled to AlexaFluor 546, then chased at 37°C in a time course manner. Direct immunodetection of antagonist mAb 30 were done with anti-mouse secondary antibody coupled to Alexa Fluor 488. Cellular localization of ALK receptors were shown by immunofluorescence using confocal laser scanning microscopy. Merged images were mounted thanks to photophop software. C. Experiment identical to B but with a cell pre-treatment with monensin (50 µM) for 30 min.
Figure 4.
Agonist mAb treatment induced ALK degradation whereas antagonist mAb induced ALK internalization without down-regulation.
A. SH-SY5Y cells were untreated (-) or treated with agonist mAb 46 for 15 min, 60 min or 3 hours. At the end of the agonist treatment, cells were subjected to cell surface protein biotinylation. ALK immunoprecipates from 1.5 mg of total cells lysate proteins were analyzed by western blot. Biotinalyted cell surface ALK were detected with streptavidin coupled to IRdye 800, and total ALK with REAB antibody. Experiments were done in triplicates, total and biotinylated (cell surface) ALK were quantified with LiCor Odyssey software. Results are expressed in percentage of control +/− s.e.m. B. SH-SY5Y cells were untreated or treated with agonist mAb 30 for 15 min, 1 h or 3 h. At the end of mAb treatment, cells were subjected to cell surface protein biotinylation. ALK immunoprecipates from 1.5 mg of total cells lysate proteins were analyzed by western blot. Biotinalyted cell surface ALK were detected with streptavidin coupled to IRdye 800, and total ALK with REAB antibody. Experiments were done in triplicates, total and biotinylated (cell surface) ALK were quantified with LiCor Odyssey software. Results are expressed in percentage of control +/− s.e.m.
Figure 5.
ALK down-regulation after agonist mAb 46 treatment.
A. IMR-32 (WT) and B. SH-SY5Y (WT/F1174L) were treated with mAb 46 for 15 min to 6 h. ALK immunoprecipitates from 1.5 mg of total cell lysate proteins were submitted to western blot. Total ALK were immunoblotted with polyclonal REAB and phosphorylated ALK were detected with monoclonal phosphotyrosine antibody 4G10 platinium. Tubulin acted as a loading control. C. SH-SY5Y cells were pre-treated or not with 50 nM NVP-TAE684 one hour before cell stimulation by agonist mAb 46 at 6 nM in time course manner (0 min to 6 h). ALK immunoprecipitates from 1.5 mg of total cell lysate proteins were submitted to western-blot analysis and immunoblotted as described. Lower panel: Experiments were done in triplicates and the 220 kD forms (the doublet was impossible to separate) and the 140 kD form of ALK were quantified with LiCor Odyssey software. Results are expressed in percentage of control +/− s.e.m. D. SH-SY5Y cells were pre-treated with either bafilomycin (0.25 µM) or lactacystin (10 µM) for 15′, then non treated or treated with agonist mAb 46 at 6 nM during 6 hours. ALK immunoprecipitates from 1.5 mg of total cell lysate proteins were submitted to Western-blot analysis as described. Lower panel: Experiments were done in triplicates total ALK (220 kD forms+140 kD form) was quantified with LiCor Odyssey software. Results are expressed in percentage of control +/− s.e.m.
Figure 6.
Kinase activation dependent down-regulation was regulated by Cbl ubiquitin ligase and ALK ubiquitylation.
A. SH-SY5Y cells were treated or not with either agonist mAb 46 or antagonist mAb 30 at 6 nM during 15 or 60 min. Cbl immunoprecipitates from 2 mg total cell lysate proteins were submitted to western-blot analysis, and then immunoblotted with polyclonal anti Cbl and ALK recruitment with polyclonal REAB antibody. Phosphorylated ALK and Cbl were revealed with 4G10 antibody. B. SH-SY5Y cells were serum starved for 16 hours and non-treated or treated with either agonist mAb 46 or antagonist mAb 30 at 6 nM during 15 or 60 min. ALK immunoprecipitates from 2 mg total cell lysate proteins were submitted to western-blot analysis and then immunoblotted with polyclonal REAB. Cbl recruitment was revealed with polyclonal anti Cbl antibody. Phosphorylated ALK and Cbl were revealed with 4G10 antibody. C. SH-SY5Y were serum starved for 16 hours, and then non-treated or treated with either mAb 46 or mAb 30 at 6 nM for 15 minutes or 60 minutes. ALK immunoprecipitates were submitted to Western-blot analysis as described.