Proteomic investigation of Cbl and Cbl-b in neuroblastoma cell differentiation highlights roles for SHP-2 and CDK16
Anna-Kathrine Pedersen,
Anamarija Pfeiffer,
Gopal Karemore,
Vyacheslav Akimov,
Dorte B. Bekker-Jensen,
Blagoy Blagoev,
Chiara Francavilla,
Jesper V. Olsen
Affiliations
Anna-Kathrine Pedersen
Proteomics Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark
Anamarija Pfeiffer
Proteomics Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark
Gopal Karemore
Proteomics Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark
Vyacheslav Akimov
Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense, Denmark
Dorte B. Bekker-Jensen
Proteomics Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark
Blagoy Blagoev
Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense, Denmark
Chiara Francavilla
Division of Molecular and Cellular Functions, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester M13 9PL, UK; Corresponding author
Jesper V. Olsen
Proteomics Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark; Corresponding author
Summary: Neuroblastoma is a highly heterogeneous embryonal solid tumor of the sympathetic nervous system. As some tumors can be treated to undergo differentiation, investigating this process can guide differentiation-based therapies of neuroblastoma. Here, we studied the role of E3 ubiquitin ligases Cbl and Cbl-b in regulation of long-term signaling responses associated with extracellular signal-regulated kinase phosphorylation and neurite outgrowth, a morphological marker of neuroblastoma cell differentiation. Using quantitative mass spectrometry (MS)-based proteomics, we analyzed how the neuroblastoma cell line proteome, phosphoproteome, and ubiquitylome were affected by Cbl and Cbl-b depletion. To quantitatively assess neurite outgrowth, we developed a high-throughput microscopy assay that was applied in combination with inhibitor studies to pinpoint signaling underlying neurite outgrowth and to functionally validate proteins identified in the MS data sets. Using this combined approach, we identified a role for SHP-2 and CDK16 in Cbl/Cbl-b-dependent regulation of extracellular signal-regulated kinase phosphorylation and neurite outgrowth, highlighting their involvement in neuroblastoma cell differentiation.
