Quantitative Phosphoproteomic Study Reveals that Protein Kinase A Regulates Neural Stem Cell Differentiation Through Phosphorylation of Catenin Beta-1 and Glycogen Synthase Kinase 3β

Abstract

Protein phosphorylation is central to the understanding of multiple cellular signaling pathways responsible for regulating the self-renewal and differentiation of neural stem cells (NSCs). Here we performed a large-scale phosphoproteomic analysis of rat fetal NSCs using strong cation exchange chromatography prefractionation and citric acid-assisted two-step enrichment with TiO 2 strategy followed by nanoLC-MS/MS analysis. Totally we identified 32,546 phosphosites on 5,091 phosphoproteins, among which 23,945 were class I phosphosites, and quantified 16,000 sites during NSC differentiation. More than 65% of class I phosphosites were novel when compared with PhosphoSitePlus database. Quantification results showed that the early and late stage of NSC differentiation differ greatly. We mapped 69 changed phosphosites on 20 proteins involved in Wnt signaling pathway, including S552 on catenin beta-1 (Ctnnb1) and S9 on glycogen synthase kinase 3b (Gsk3b). Western blotting and real-time PCR results proved that Wnt signaling pathway plays critical roles in NSC fate determination. Furthermore, inhibition and activation of PKA dramatically affected the phosphorylation state of Ctnnb1 and Gsk3b, which regulates the differentiation of NSCs. Our data provides a valuable resource for studying the self-renewal and differentiation of NSCs. STEM CELLS 2016;34:2090-2101 SIGNIFICANCE STATEMENTAlthough the self-renewal and differentiation of neural stem cells are very important to the development and impairment of nervous system, the underlying mechanism is still unclear. Here we report the largest comprehensive phosphoproteome of primarily cultured rat neural stem cells and quantified them during differentiation. Many phosphorylation sites on key proteins of some signaling pathways were quantified, including catenin b-1 and glycogen synthase kinase 3b. Finally we proved that PKA regulates neural stem cell differentiation through phosphorylation of catenin b-1 and glycogen synthase kinase 3b. We expect that our data will provide a valuable resource to help understand the mechanism of NSC differentiation more comprehensively.

Maresin-1 and Resolvin E1 Promote Regenerative Properties of Periodontal Ligament Stem Cells Under Inflammatory Conditions

“…Protein phosphorylation is central to the understanding of downstream signaling pathways responsible for regulating stem cell functions. Quantitative phosphoproteomics has been used to study global signaling events in pluripotent, and cancer stem cells and new therapeutic targets have accordingly been proposed ( 59 – 62 ). To our knowledge, however, it has not been used before to assess the impact of inflammation on stem cell biology.…”

Andrographolide Promotes Neural Differentiation of Rat Adipose Tissue-Derived Stromal Cells through Wnt/β-Catenin Signaling Pathway

“…We suggest that same mechanism underlies the effects of ANDRO on the neural differentiation of ADSCs. By quantitative phosphoproteomic analysis, Wang et al [ 16 ] demonstrated that the phosphorylation of GSK-3 β and β -catenin is involved in the regulation of NSC self-renewal and differentiation. Their results showed that S552 on β -catenin and S9 on GSK-3 β play critical roles in activating the Wnt canonical pathway for determining the fate of NSCs.…”

“…The Wnt signaling pathway plays an important role in controlling neuronal differentiation, dendritic development, axonal outgrowth and guidance, neuronal plasticity, and the synaptic function in the adult nervous system [ 15 ]. Recently, a quantitative phosphoproteomic study has reported that the phosphorylation of catenin β -1 (CTNNB1) and GSK3 β regulates the differentiation of neural stem cells (NSCs) [ 16 ].…”

Protein Kinase A Signaling Inhibits Iridophore Differentiation in Zebrafish

“…Zebrafish iridophores are specified and then differentiate beginning at 20 h post fertilization (hpf), as detected by purine nucleoside phosphorylase ( pnp4a ) expression [10]. As PKA signaling functions during neuron differentiation [22], we hypothesized that PKA signaling regulates differentiation of the iridophore precursors, iridoblasts. To test this hypothesis, we examined pnp4a expression in forskolin and H89-treated larvae.…”

“…Embryos were fixed in 4% paraformaldehyde in phosphate-buffered saline at pH 7.2, and processed using standard protocols [21]. Digoxigenin-labeled probes for mitfa [7], foxd3 [22,23], and pnp4a [10] have been previously described. All imaging was done using a Nikon SMZ-1500 stereomicroscope equipped with a Digital Sight DS-Ri1 Digital Camera.…”