Open AccessQuantitative Phosphoproteomic Study Reveals that Protein Kinase A Regulates Neural Stem Cell Differentiation Through Phosphorylation of Catenin Beta‐1 and Glycogen Synthase Kinase 3β
Author(s) -
Wang Shuxin,
Li Zheyi,
Shen Hongyan,
Zhang Zhong,
Yin Yuxin,
Wang Qingsong,
Zhao Xuyang,
Ji Jianguo
Publication year - 2016
Publication title -
stem cells
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.159
H-Index - 229
eISSN - 1549-4918
pISSN - 1066-5099
DOI - 10.1002/stem.2387
Subject(s) - biology , wnt signaling pathway , gsk 3 , phosphorylation , neural stem cell , microbiology and biotechnology , kinase , signal transduction , cellular differentiation , protein kinase a , glycogen synthase , stem cell , biochemistry , gene
A bstract 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 3β (Gsk3β). 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 Gsk3β, which regulates the differentiation of NSCs. Our data provides a valuable resource for studying the self‐renewal and differentiation of NSCs. S tem C ells 2016;34:2090–2101