Temporal proteomics of NGF-TrkA signaling identifies an inhibitory role for the E3 ligase Cbl-b in neuroblastoma cell differentiation

Proteomic analysis of neurotrophin signaling identifies an inhibitory E3 ubiquitin ligase. Proteomic analysis reveals an inhibitor Neurotrophins, such as nerve growth factor (NGF), control the differentiation and proliferation of neuronal precursors, and the outcome depends on the duration of the signal and neurotrophin-receptor pair. NGF binding to the receptor tyrosine kinase TrkA induces neuronal differentiation and neurite outgrowth. By mediating the attachment of ubiquitin chains, E3 ubiquitin ligases stimulate the internalization and degradation, and hence reduce the activity, of various receptors. Emdal et al. performed a temporal analysis by mass spectrometry of changes in the proteome in response to NGF in neuroblastoma cells and found that NGF not only promoted the activation of TrkA but also signaled its degradation by promoting the interaction of TrkA with the E3 ubiquitin ligase Cbl-b, which resulted in the ubiquitylation and degradation of both proteins. Neuroblastoma cells with reduced Cbl-b had increased TrkA signaling and produced longer neurites. In addition to identifying this inhibitory role for Cbl-b, the proteomic data are a resource for further investigation of TrkA signaling dynamics. 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.

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