Proteomic investigation of Cbl and Cbl-b in neuroblastoma cell differentiation highlights roles for SHP-2 and CDK16

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 ERK 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 datasets. Using this combined approach, we identified a role for SHP-2 and CDK16 in Cbl/Cbl-b-dependent regulation of ERK phosphorylation and neurite outgrowth, highlighting their involvement in neuroblastoma cell differentiation.