Temporal proteomics of NGF-TrkA signaling identifies an inhibitory role for the E3 ligase Cbl-b in neuroblastoma cell differentiation.
SH-SY5Y neuroblastoma cells respond to nerve growth factor (NGF)-mediated activation of the tropomyosin-related kinase A (TrkA) with neurite outgrowth, thereby providing a model to study neuronal differentiation. We performed a time-resolved analysis of NGF-TrkA signaling in neuroblastoma cells using mass spectrometry-based quantitative proteomics. The combination of interactome, phosphoproteome, and proteome data provided temporal insights into the molecular events downstream of NGF binding to TrkA. We showed that upon NGF stimulation, TrkA recruits the E3 ubiquitin ligase Cbl-b, which then becomes phosphorylated and ubiquitylated and decreases in abundance. We also found that recruitment of Cbl-b promotes TrkA ubiquitylation and degradation. Furthermore, the amount of phosphorylation of the kinase ERK and neurite outgrowth increased upon Cbl-b depletion in several neuroblastoma cell lines. Our findings suggest that Cbl-b limits NGF-TrkA signaling to control the length of neurites.
Proteomics Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark.
Kristina B
KB
Emdal
Proteomics Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark.
Anna-Kathrine
AK
Pedersen
Proteomics Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark.
Dorte B
DB
Bekker-Jensen
Disease Systems Biology Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark.
Kalliopi P
KP
Tsafou
Disease Systems Biology Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark.
Heiko
H
Horn
Department of Pediatric Oncology and Hematology, University Children's Hospital Essen, Hufelandstrasse 55, 45122 Essen, Germany.
Sven
S
Lindner
Department of Pediatric Oncology and Hematology, University Children's Hospital Essen, Hufelandstrasse 55, 45122 Essen, Germany. Department of Pediatric Oncology and Hematology, Charité Berlin, Charitéplatz 1, 10117 Berlin, Germany. German Cancer Consortium (DKTK), 13353 Berlin, Germany.
Johannes H
JH
Schulte
Department of Pediatric Oncology and Hematology, University Children's Hospital Essen, Hufelandstrasse 55, 45122 Essen, Germany. Department of Pediatric Oncology and Hematology, Charité Berlin, Charitéplatz 1, 10117 Berlin, Germany. German Cancer Consortium (DKTK), 13353 Berlin, Germany.
Angelika
A
Eggert
Disease Systems Biology Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark.
Lars J
LJ
Jensen
Proteomics Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark. chiara.francavilla@cpr.ku.dk jesper.olsen@cpr.ku.dk.
Chiara
C
Francavilla
Proteomics Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark. chiara.francavilla@cpr.ku.dk jesper.olsen@cpr.ku.dk.
Jesper V
JV
Olsen
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D006801:Humans; D020935:MAP Kinase Signaling System; D020932:Nerve Growth Factor / Q000235:genetics / Q000378:metabolism*; D016501:Neurites / Q000378:metabolism / Q000473:pathology; D009447:Neuroblastoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D050721:Proto-Oncogene Proteins c-cbl / Q000235:genetics / Q000378:metabolism*; D020917:Receptor, trkA / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; C502240:NGF protein, human; D020932:Nerve Growth Factor; C492211:CBLB protein, human; D050721:Proto-Oncogene Proteins c-cbl; D020917:Receptor, trkA