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.