Abstract
Protein phosphorylation plays a central role in creating a highly dynamic network of interacting proteins that reads and responds to signals from growth factors in the cellular microenvironment. Cells of the neural crest employ multiple signaling mechanisms to control migration and differentiation during development. It is known that defects in these mechanisms cause neuroblastoma, but how multiple signaling pathways interact to govern cell behavior is unknown. In a phosphoproteomic study of neuroblastoma cell lines and cell fractions, including endosomes and detergent-resistant membranes, 1622 phosphorylated proteins were detected, including more than half of the receptor tyrosine kinases in the human genome. Data were analyzed using a combination of graph theory and pattern recognition techniques that resolve data structure into networks that incorporate statistical relationships and protein-protein interaction data. Clusters of proteins in these networks are indicative of functional signaling pathways. The analysis indicates that receptor tyrosine kinases are functionally compartmentalized into distinct collaborative groups distinguished by activation and intracellular localization of SRC-family kinases, especially FYN and LYN. Changes in intracellular localization of activated FYN and LYN were observed in response to stimulation of the receptor tyrosine kinases, ALK and KIT. The results suggest a mechanism to distinguish signaling responses to activation of different receptors, or combinations of receptors, that govern the behavior of the neural crest, which gives rise to neuroblastoma.
Highlights
Neuroblastoma arises from cells of the neural crest, a population of multipotent, migrating cells that differentiate into neurons in the peripheral nervous system, melanocytes, and structural cells [1]
To identify patterns in tyrosine phosphorylation in neuroblastoma, we analyzed tyrosine phosphoproteomic data acquired from 21 neuroblastoma cell lines using immunoprecipitation of tyrosine phosphorylated peptides as previously described [41,42]
For the first analysis described below, phosphopeptide amounts were summed for each protein in each sample, with the exception of the SRC-family kinases (SFKs), where the C-terminal inhibitory phosphorylation was summed separately and given the names SRC_i; LYN phosphorylated on the C-terminal inhibitory site (LYN_i); FYN_i; and YES1_i
Summary
Neuroblastoma arises from cells of the neural crest, a population of multipotent, migrating cells that differentiate into neurons in the peripheral nervous system, melanocytes, and structural cells [1]. Neural crest cells appear to restrict their range of cell fate choices in sequential steps [7,8], and the profound heterogeneity in neuroblastoma is caused by a failure to differentiate at different stages. Neuroblastoma tumors and cell lines represent a snapshot of failed differentiation at different stages in the neural crest sympathoadrenal lineage [2,4,7,8]. Anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase (RTK), is frequently mutated and activated in both familial and spontaneous neuroblastomas, suggesting that this receptor can prevent a key differentiation step in neural crest cells [9,10,11,12,13,14,15]. The tragic outcome is too often a metastatic cancer with poor prognosis
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