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Quantitative phosphoproteome analysis of embryonic stem cell differentiation toward blood.

Piazzi M, Williamson A, Lee CF, Pearson S, Lacaud G, Kouskoff V, McCubrey JA, Cocco L, Whetton AD - Oncotarget (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Cell Signaling Laboratory, Department of Biomedical Science (DIBINEM), University of Bologna, Italy.

ABSTRACT
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 (iTRAQ(TM)) 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.

No MeSH data available.


Related in: MedlinePlus

Comparison between phosphopeptides and protein expression levelsPanels show changes in identified phosphopeptides compared to the expression profile of the referring proteins previously identified [4]. Blue histograms represent phosphorylation and orange histograms refer to protein expression level changes in Bry−Flk1− (Epiblast-like cells), Bry+Flk1− (Early mesoderm) and Bry+Flk1+ (Hemangioblast).
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Figure 3: Comparison between phosphopeptides and protein expression levelsPanels show changes in identified phosphopeptides compared to the expression profile of the referring proteins previously identified [4]. Blue histograms represent phosphorylation and orange histograms refer to protein expression level changes in Bry−Flk1− (Epiblast-like cells), Bry+Flk1− (Early mesoderm) and Bry+Flk1+ (Hemangioblast).

Mentions: Twenty-one phosphopeptides within this dataset were found to emanate from proteins analyzed in the nuclear proteome expression analysis associated with Bry and Flk1 expression previously published [4] (Figure 3). Although the expression of the proteins did not change during hematopoietic differentiation, 9 phosphopeptides displayed differential regulation: Hmga1, Sec61b, Ctnnd1, Gsk3b, Dnajc5, Nucks1, Rif1, Ssrp1, Raver1. Among these, phosphorylation on Hmga1 and Raver1 only decreased in the progression from Bry+Flk1− to Bry+Flk1+; while Raver1 is involved in the regulation of alternative splicing [21], Hmga1 plays a role in modulation of gene expression during development and embryogenesis, and its expression is known to be markedly diminished in differentiated cells [22]. Interestingly, Ctnnd1, Gsk3 and Rif1, are involved either in the regulation of cell differentiation and developmental processes, but also in the regulation of the cellular response to signaling pathways, such as the Wnt receptor signaling pathway. Moreover, GSK3 was found to play a pivotal role in controlling the decision fate of ES cells between self-renewal and differentiation; in fact inhibition of GSK3 has been found to promote and maintain mESC self-renewal [23]. As shown in Figure 3, in the majority of the cases normalization against protein level revealed no protein level change modulating stoichiometry of phosphorylation. For example, Hmga2 expression was augmented as ES cells differentiate and it remains high in the Bry+Flk1+: we found a peptide whose phosphorylation increased as mES cells differentiate, and this phosphorylation is maintained in the Bry+Flk1+ population. Hmga2 can exert a negative regulation on the Jak-Stat signaling cascade [24]. The Jak/Stat pathway promotes ESCs self-renewal [25] and is required for self-renewal of Drosophila sperm stem cells [26]. Conversely, only few phosphopeptides did not exhibit a correspondence between phosphorylation and protein expression. Among these, is the phosphopeptide emanating from the chromatin remodeling factor Smarcc1/Baf155, whose phosphorylation increased as mESCs differentiated toward mesoderm lineage commitment. Interestingly, Schaniel et co-workers [27], showed that Smarcc1 plays a balance between gene repression, which maintains ESCs in the self-renewal state and chromatin rearrangements, that led to the expression of genes involved in differentiation.


Quantitative phosphoproteome analysis of embryonic stem cell differentiation toward blood.

Piazzi M, Williamson A, Lee CF, Pearson S, Lacaud G, Kouskoff V, McCubrey JA, Cocco L, Whetton AD - Oncotarget (2015)

Comparison between phosphopeptides and protein expression levelsPanels show changes in identified phosphopeptides compared to the expression profile of the referring proteins previously identified [4]. Blue histograms represent phosphorylation and orange histograms refer to protein expression level changes in Bry−Flk1− (Epiblast-like cells), Bry+Flk1− (Early mesoderm) and Bry+Flk1+ (Hemangioblast).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4484429&req=5

Figure 3: Comparison between phosphopeptides and protein expression levelsPanels show changes in identified phosphopeptides compared to the expression profile of the referring proteins previously identified [4]. Blue histograms represent phosphorylation and orange histograms refer to protein expression level changes in Bry−Flk1− (Epiblast-like cells), Bry+Flk1− (Early mesoderm) and Bry+Flk1+ (Hemangioblast).
Mentions: Twenty-one phosphopeptides within this dataset were found to emanate from proteins analyzed in the nuclear proteome expression analysis associated with Bry and Flk1 expression previously published [4] (Figure 3). Although the expression of the proteins did not change during hematopoietic differentiation, 9 phosphopeptides displayed differential regulation: Hmga1, Sec61b, Ctnnd1, Gsk3b, Dnajc5, Nucks1, Rif1, Ssrp1, Raver1. Among these, phosphorylation on Hmga1 and Raver1 only decreased in the progression from Bry+Flk1− to Bry+Flk1+; while Raver1 is involved in the regulation of alternative splicing [21], Hmga1 plays a role in modulation of gene expression during development and embryogenesis, and its expression is known to be markedly diminished in differentiated cells [22]. Interestingly, Ctnnd1, Gsk3 and Rif1, are involved either in the regulation of cell differentiation and developmental processes, but also in the regulation of the cellular response to signaling pathways, such as the Wnt receptor signaling pathway. Moreover, GSK3 was found to play a pivotal role in controlling the decision fate of ES cells between self-renewal and differentiation; in fact inhibition of GSK3 has been found to promote and maintain mESC self-renewal [23]. As shown in Figure 3, in the majority of the cases normalization against protein level revealed no protein level change modulating stoichiometry of phosphorylation. For example, Hmga2 expression was augmented as ES cells differentiate and it remains high in the Bry+Flk1+: we found a peptide whose phosphorylation increased as mES cells differentiate, and this phosphorylation is maintained in the Bry+Flk1+ population. Hmga2 can exert a negative regulation on the Jak-Stat signaling cascade [24]. The Jak/Stat pathway promotes ESCs self-renewal [25] and is required for self-renewal of Drosophila sperm stem cells [26]. Conversely, only few phosphopeptides did not exhibit a correspondence between phosphorylation and protein expression. Among these, is the phosphopeptide emanating from the chromatin remodeling factor Smarcc1/Baf155, whose phosphorylation increased as mESCs differentiated toward mesoderm lineage commitment. Interestingly, Schaniel et co-workers [27], showed that Smarcc1 plays a balance between gene repression, which maintains ESCs in the self-renewal state and chromatin rearrangements, that led to the expression of genes involved in differentiation.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Cell Signaling Laboratory, Department of Biomedical Science (DIBINEM), University of Bologna, Italy.

ABSTRACT
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 (iTRAQ(TM)) 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.

No MeSH data available.


Related in: MedlinePlus