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Internalization and down-regulation of the ALK receptor in neuroblastoma cell lines upon monoclonal antibodies treatment.

Mazot P, Cazes A, Dingli F, Degoutin J, Irinopoulou T, Boutterin MC, Lombard B, Loew D, Hallberg B, Palmer RH, Delattre O, Janoueix-Lerosey I, Vigny M - PLoS ONE (2012)

Bottom Line: We further explored ALK receptor trafficking by investigating the effect of agonist and antagonist mAb (monoclonal antibodies) on ALK internalization and down-regulation, either in SH-SY5Y cells or in cells expressing only ALK(WT).We observe that treatment with agonist mAbs resulted in ALK internalization and lysosomal targeting for receptor degradation.This study provides novel insights into the mechanisms regulating ALK trafficking and degradation, showing that various ALK receptor pools are regulated by proteasome or lysosome pathways according to their intracellular localization.

View Article: PubMed Central - PubMed

Affiliation: Université Pierre et Marie Curie, Paris, France.

ABSTRACT
Recently, activating mutations of the full length ALK receptor, with two hot spots at positions F1174 and R1275, have been characterized in sporadic cases of neuroblastoma. Here, we report similar basal patterns of ALK phosphorylation between the neuroblastoma IMR-32 cell line, which expresses only the wild-type receptor (ALK(WT)), and the SH-SY5Y cell line, which exhibits a heterozygous ALK F1174L mutation and expresses both ALK(WT) and ALK(F1174L) receptors. We demonstrate that this lack of detectable increased phosphorylation in SH-SY5Y cells is a result of intracellular retention and proteasomal degradation of the mutated receptor. As a consequence, in SH-SY5Y cells, plasma membrane appears strongly enriched for ALK(WT) whereas both ALK(WT) and ALK(F1174L) were present in intracellular compartments. We further explored ALK receptor trafficking by investigating the effect of agonist and antagonist mAb (monoclonal antibodies) on ALK internalization and down-regulation, either in SH-SY5Y cells or in cells expressing only ALK(WT). We observe that treatment with agonist mAbs resulted in ALK internalization and lysosomal targeting for receptor degradation. In contrast, antagonist mAb induced ALK internalization and recycling to the plasma membrane. Importantly, we correlate this differential trafficking of ALK in response to mAb with the recruitment of the ubiquitin ligase Cbl and ALK ubiquitylation only after agonist stimulation. This study provides novel insights into the mechanisms regulating ALK trafficking and degradation, showing that various ALK receptor pools are regulated by proteasome or lysosome pathways according to their intracellular localization.

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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.
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pone-0033581-g006: 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.

Mentions: We first performed Cbl immunoprecipitation, examining co-immunoprecipitation of ALK by immunoblotting in SH-SY5Y cells. In control conditions we could not detect ALK co-immunoprecipitation with Cbl. Agonist mAb treatment led to co-immunoprecipitation of Cbl and ALK (Fig. 6A). Furthermore, we also detected the phosphorylation of both proteins strongly suggesting that the phosphorylation of Cbl resulted from ALK activation. We then tested Cbl recruitment after ALK immunoprecipitation. After ALK immunoprecipitation we detected a weak recruitment of Cbl in control conditions; however agonist mAb treatment strongly increased this interaction (Fig. 6B). Once again, agonist mAb activation triggered ALK and Cbl phosphorylation. Finally we tested ALK ubiquitylation in response to mAb treatment in SH-SY5Y cells. For this purpose we performed ALK immunoprecipitation after mAb treatment and investigated ALK ubiquitylation using anti ubiquitin antibody P4D1. We detected a basal ALK ubiquitylation, which was moderately but significantly increased by agonist mAb 46 stimulation. Altogether these results demonstrated that ALK activation by agonist mAb stimulation induced receptor phosphorylation thus allowing Cbl recruitment and receptor ubiquitylation (Fig. 6C), which is required for lysosome targeting.


Internalization and down-regulation of the ALK receptor in neuroblastoma cell lines upon monoclonal antibodies treatment.

Mazot P, Cazes A, Dingli F, Degoutin J, Irinopoulou T, Boutterin MC, Lombard B, Loew D, Hallberg B, Palmer RH, Delattre O, Janoueix-Lerosey I, Vigny M - PLoS ONE (2012)

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.
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Related In: Results  -  Collection

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pone-0033581-g006: 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.
Mentions: We first performed Cbl immunoprecipitation, examining co-immunoprecipitation of ALK by immunoblotting in SH-SY5Y cells. In control conditions we could not detect ALK co-immunoprecipitation with Cbl. Agonist mAb treatment led to co-immunoprecipitation of Cbl and ALK (Fig. 6A). Furthermore, we also detected the phosphorylation of both proteins strongly suggesting that the phosphorylation of Cbl resulted from ALK activation. We then tested Cbl recruitment after ALK immunoprecipitation. After ALK immunoprecipitation we detected a weak recruitment of Cbl in control conditions; however agonist mAb treatment strongly increased this interaction (Fig. 6B). Once again, agonist mAb activation triggered ALK and Cbl phosphorylation. Finally we tested ALK ubiquitylation in response to mAb treatment in SH-SY5Y cells. For this purpose we performed ALK immunoprecipitation after mAb treatment and investigated ALK ubiquitylation using anti ubiquitin antibody P4D1. We detected a basal ALK ubiquitylation, which was moderately but significantly increased by agonist mAb 46 stimulation. Altogether these results demonstrated that ALK activation by agonist mAb stimulation induced receptor phosphorylation thus allowing Cbl recruitment and receptor ubiquitylation (Fig. 6C), which is required for lysosome targeting.

Bottom Line: We further explored ALK receptor trafficking by investigating the effect of agonist and antagonist mAb (monoclonal antibodies) on ALK internalization and down-regulation, either in SH-SY5Y cells or in cells expressing only ALK(WT).We observe that treatment with agonist mAbs resulted in ALK internalization and lysosomal targeting for receptor degradation.This study provides novel insights into the mechanisms regulating ALK trafficking and degradation, showing that various ALK receptor pools are regulated by proteasome or lysosome pathways according to their intracellular localization.

View Article: PubMed Central - PubMed

Affiliation: Université Pierre et Marie Curie, Paris, France.

ABSTRACT
Recently, activating mutations of the full length ALK receptor, with two hot spots at positions F1174 and R1275, have been characterized in sporadic cases of neuroblastoma. Here, we report similar basal patterns of ALK phosphorylation between the neuroblastoma IMR-32 cell line, which expresses only the wild-type receptor (ALK(WT)), and the SH-SY5Y cell line, which exhibits a heterozygous ALK F1174L mutation and expresses both ALK(WT) and ALK(F1174L) receptors. We demonstrate that this lack of detectable increased phosphorylation in SH-SY5Y cells is a result of intracellular retention and proteasomal degradation of the mutated receptor. As a consequence, in SH-SY5Y cells, plasma membrane appears strongly enriched for ALK(WT) whereas both ALK(WT) and ALK(F1174L) were present in intracellular compartments. We further explored ALK receptor trafficking by investigating the effect of agonist and antagonist mAb (monoclonal antibodies) on ALK internalization and down-regulation, either in SH-SY5Y cells or in cells expressing only ALK(WT). We observe that treatment with agonist mAbs resulted in ALK internalization and lysosomal targeting for receptor degradation. In contrast, antagonist mAb induced ALK internalization and recycling to the plasma membrane. Importantly, we correlate this differential trafficking of ALK in response to mAb with the recruitment of the ubiquitin ligase Cbl and ALK ubiquitylation only after agonist stimulation. This study provides novel insights into the mechanisms regulating ALK trafficking and degradation, showing that various ALK receptor pools are regulated by proteasome or lysosome pathways according to their intracellular localization.

Show MeSH
Related in: MedlinePlus