Quantitative Phosphoproteomic Study Reveals that Protein Kinase A Regulates Neural Stem Cell Differentiation Through Phosphorylation of Catenin Beta-1 and Glycogen Synthase Kinase 3β.
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 TiO2 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. Stem Cells 2016;34:2090-2101.
State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Beijing, Peoples' Republic of China.
Shuxin
S
Wang
State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Beijing, Peoples' Republic of China.
Zheyi
Z
Li
State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Beijing, Peoples' Republic of China.
Hongyan
H
Shen
State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Beijing, Peoples' Republic of China.
Zhong
Z
Zhang
Institute of Systems Biomedicine, Peking University Health Science Center, Peking University, Beijing, Peoples' Republic of China.
Yuxin
Y
Yin
State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Beijing, Peoples' Republic of China.
Qingsong
Q
Wang
Institute of Systems Biomedicine, Peking University Health Science Center, Peking University, Beijing, Peoples' Republic of China.
Xuyang
X
Zhao
State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Beijing, Peoples' Republic of China.
Jianguo
J
Ji
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D005333:Fetus / Q000166:cytology; D063990:Gene Ontology; D000071679:Glycogen Synthase Kinase 3 beta / Q000378:metabolism*; D051379:Mice; D058977:Molecular Sequence Annotation; D058953:Neural Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D040901:Proteomics / Q000379:methods*; D051381:Rats; D014212:Tretinoin / Q000494:pharmacology; D060449:Wnt Signaling Pathway / Q000187:drug effects; D051176:beta Catenin / Q000378:metabolism*
D010750:Phosphoproteins; D051176:beta Catenin; D014212:Tretinoin; D000071679:Glycogen Synthase Kinase 3 beta; D017868:Cyclic AMP-Dependent Protein Kinases
Neural stem cells; Phosphoproteomics; Protein kinase A; TiO2; Wnt signaling pathway