Retinoic acid induced specific changes in the phosphoproteome of C17.2 neural stem cells

Retinoic acid (RA), a vitamin A derivative, is an effective cell differentiating factor which plays critical roles in neuronal differentiation induction and the production of neurotransmitters in neurons. However, the specific changes in phosphorylation levels and downstream signalling pathways associated with RA remain unclear. This study employed qualitative and quantitative phosphoproteomics approaches based on mass spectrometry to investigate the phosphorylation changes induced by RA in C17.2 neural stem cells (NSCs). Dimethyl labelling, in conjunction with TiO₂ phosphopeptide enrichment, was utilized to profile the phosphoproteome of self-renewing and RA-induced differentiated cells in C17.2 NSCs. The results of our study revealed that, qualitatively, 230 and 14 phosphoproteins were exclusively identified in the self-renewal and RA-induced groups respectively. Quantitatively, we successfully identified and quantified 177 unique phosphoproteins, among which 70 exhibited differential phosphorylation levels. Analysis of conserved phosphorylation motifs demonstrated enrichment of motifs corresponding to cyclin-dependent kinase and MAPK in the RA-induced group. Additionally, through a comprehensive literature and database survey, we found that the differentially expressed proteins were associated with the Wnt/β-catenin and Hippo signalling pathways. This work sheds light on the changes in phosphorylation levels induced by RA in C17.2 NSCs, thereby expanding our understanding of the molecular mechanisms underlying RA-induced neuronal differentiation.