Research Papers:

Quantitative phosphoproteome analysis of embryonic stem cell differentiation toward blood

Manuela Piazzi _, Andrew Williamson, Chia-Fang Lee, Stella Pearson, Georges Lacaud, Valerie Kouskoff, James A. McCubrey, Lucio Cocco and Anthony D. Whetton

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Oncotarget. 2015; 6:10924-10939. https://doi.org/10.18632/oncotarget.3454

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Manuela Piazzi1, Andrew Williamson2, Chia-Fang Lee2, Stella Pearson3, Georges Lacaud4, Valerie Kouskoff3, James A. McCubrey5, Lucio Cocco1, Anthony D. Whetton2

1Cell Signaling Laboratory, Department of Biomedical Science (DIBINEM), University of Bologna, Italy

2Stem Cell and Leukaemia Proteomics Laboratory, Manchester Academic Health Science Centre, The University of Manchester, Manchester, UK

3Stem Cell Research Group, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK

4Stem Cell Biology Group Paterson Institute for Cancer Research, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK

5Department of Microbiology and Immunology, Brody School of Medicine at East Carolina University, Greenville, NC, USA

Correspondence to:

Manuela Piazzi, e-mail: [email protected]

Keywords: hemangioblast, iTRAQ, phosphoproteomic, nucleus

Received: January 27, 2015     Accepted: February 24, 2015     Published: March 26, 2015


Murine embryonic stem (ES) cells can differentiate in vitro into three germ layers (endodermic, mesodermic, ectodermic). Studies on the differentiation of these cells to specific early differentiation stages has been aided by an ES cell line carrying the Green Fluorescent Protein (GFP) targeted to the Brachyury (Bry) locus which marks mesoderm commitment. Furthermore, expression of the Vascular Endothelial Growth Factor receptor 2 (Flk1) along with Bry defines hemangioblast commitment. Isobaric-tag for relative and absolute quantification (iTRAQTM) and phosphopeptide enrichment coupled to liquid chromatography separation and mass spectrometry allow the study of phosphorylation changes occurring at different stages of ES cell development using Bry and Flk1 expression respectively. We identified and relatively quantified 37 phosphoentities which are modulated during mesoderm-induced ES cells differentiation, comparing epiblast-like, early mesoderm and hemangioblast-enriched cells. Among the proteins differentially phosphorylated toward mesoderm differentiation were: the epigenetic regulator Dnmt3b, the protein kinase GSK3b, the chromatin remodeling factor Smarcc1, the transcription factor Utf1; as well as protein specifically related to stem cell differentiation, as Eomes, Hmga2, Ints1 and Rif1. As most key factors regulating early hematopoietic development have also been implicated in various types of leukemia, understanding the post-translational modifications driving their regulation during normal development could result in a better comprehension of their roles during abnormal hematopoiesis in leukemia.

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