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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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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.
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pone-0033581-g001: 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.

Mentions: We first investigated the impact of ALK mutation on receptor phosphorylation by comparing two neuroblastoma cell lines, IMR-32 cells expressing only wild-type receptor and SH-SY5Y cells exhibiting a heterozygous ALK F1174L activating mutation [11], [12]. As expected, in both cell lines, a doublet at 220 kD corresponding to the full length receptor, together with a band representing the cleaved form at 140 kD [17] were detected. The level of ALK expression in the two cell lines was roughly similar and ALK phosphorylation was barely discernible in either cell lines under basal conditions, even given, the presence of an activated mutation in the SH-SY5Y cell line (Fig. 1A). Agonist mAb stimulation performed with the mAb 46 induced an increase of ALK phosphorylation in both IMR-32 and SH-SY5Y cells (Fig. 1A, right panel: representative Western Blot, left panel: quantification). In agreement with our previous data [18] we observed an induction of ALK phosphorylation for the upper band of the 220 kD doublet and the 140 kD form.


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)

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.
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Related In: Results  -  Collection

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pone-0033581-g001: 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.
Mentions: We first investigated the impact of ALK mutation on receptor phosphorylation by comparing two neuroblastoma cell lines, IMR-32 cells expressing only wild-type receptor and SH-SY5Y cells exhibiting a heterozygous ALK F1174L activating mutation [11], [12]. As expected, in both cell lines, a doublet at 220 kD corresponding to the full length receptor, together with a band representing the cleaved form at 140 kD [17] were detected. The level of ALK expression in the two cell lines was roughly similar and ALK phosphorylation was barely discernible in either cell lines under basal conditions, even given, the presence of an activated mutation in the SH-SY5Y cell line (Fig. 1A). Agonist mAb stimulation performed with the mAb 46 induced an increase of ALK phosphorylation in both IMR-32 and SH-SY5Y cells (Fig. 1A, right panel: representative Western Blot, left panel: quantification). In agreement with our previous data [18] we observed an induction of ALK phosphorylation for the upper band of the 220 kD doublet and the 140 kD form.

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