Phosphorylation Dynamics during Early Differentiation of Human Embryonic Stem Cells

Developmental Biology and Stem Cell Research, Hubrecht Institute, Utrecht, The Netherlands.
Cell stem cell (Impact Factor: 22.27). 09/2009; 5(2):214-26. DOI: 10.1016/j.stem.2009.05.021
Source: PubMed


Pluripotent stem cells self-renew indefinitely and possess characteristic protein-protein networks that remodel during differentiation. How this occurs is poorly understood. Using quantitative mass spectrometry, we analyzed the (phospho)proteome of human embryonic stem cells (hESCs) during differentiation induced by bone morphogenetic protein (BMP) and removal of hESC growth factors. Of 5222 proteins identified, 1399 were phosphorylated on 3067 residues. Approximately 50% of these phosphosites were regulated within 1 hr of differentiation induction, revealing a complex interplay of phosphorylation networks spanning different signaling pathways and kinase activities. Among the phosphorylated proteins was the pluripotency-associated protein SOX2, which was SUMOylated as a result of phosphorylation. Using the data to predict kinase-substrate relationships, we reconstructed the hESC kinome; CDK1/2 emerged as central in controlling self-renewal and lineage specification. The findings provide new insights into how hESCs exit the pluripotent state and present the hESC (phospho)proteome resource as a complement to existing pluripotency network databases.

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Available from: Albert J R Heck, Oct 09, 2015
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    • "Consistently, we detected a significant loss of ACINUS protein expression (Figures S2F–S2H) and consequently serine-1004 phosphorylation-dependent activation, in Medalist +C cells (Figures S2F, S2G, and S2I). Failure to activate expression of ACINUS, which is necessary for early differentiation of embryonic stem cells (ESCs) (Rigbolt et al., 2011; Van Hoof et al., 2009) may, in part, underlie the impairment in differentiation observed in Medalist +C patients. It is possible that exposure of fibroblasts to prolonged periods of hyperglycemia in the Medalist patients, and consequent hyperosmotic stress, would have impacted their ability to reprogramming as well as the subsequent potential for proliferation and/or differentiation (Madonna et al., 2013) and requires further investigation. "
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    ABSTRACT: The mechanisms underlying the development of complications in type 1 diabetes (T1D) are poorly understood. Disease modeling of induced pluripotent stem cells (iPSCs) from patients with longstanding T1D (disease duration ≥ 50 years) with severe (Medalist +C) or absent to mild complications (Medalist -C) revealed impaired growth, reprogramming, and differentiation in Medalist +C. Genomics and proteomics analyses suggested differential regulation of DNA damage checkpoint proteins favoring protection from cellular apoptosis in Medalist -C. In silico analyses showed altered expression patterns of DNA damage checkpoint factors among the Medalist groups to be targets of miR200, whose expression was significantly elevated in Medalist +C serum. Notably, neurons differentiated from Medalist +C iPSCs exhibited enhanced susceptibility to genotoxic stress that worsened upon miR200 overexpression. Furthermore, knockdown of miR200 in Medalist +C fibroblasts and iPSCs rescued checkpoint protein expression and reduced DNA damage. We propose miR200-regulated DNA damage checkpoint pathway as a potential therapeutic target for treating complications of diabetes.
    Cell Metabolism 08/2015; 22(2). DOI:10.1016/j.cmet.2015.07.015 · 17.57 Impact Factor
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    • "We selected strictly binary interactions between proteins for which we gathered evidence of expression at the protein level in human ESCs. We derived the human ESC proteome from quantitative mass spectrometry-based proteomics studies (Phanstiel et al., 2011; van Hoof et al., 2009). The relationship between the PER and DEX sets was estimated by calculating the following quantities: the count of direct interactions between the proteins encoded by PER and DEX genes respectively, the count of topological first-order interaction neighbours common to the PER and DEX sets, the count of significant interface (INT) proteins in the Inweb network. "
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    ABSTRACT: The shortage of molecular information on cell cycle changes along embryonic stem cell (ESC) differentiation prompts an in silico approach, which may provide a novel way to identify candidate genes or mechanisms acting in coordinating the two programs. We analyzed germ layer specific gene expression changes during the cell cycle and ESC differentiation by combining four human cell cycle transcriptome profiles with thirteen in vitro human ESC differentiation studies. To detect cross-talk mechanisms we then integrated the transcriptome data that displayed differential regulation with protein interaction data. A new class of non-transcriptionally regulated genes was identified, encoding proteins which interact systematically with proteins corresponding to genes regulated during the cell cycle or cell differentiation, and which therefore can be seen as interface proteins coordinating the two programs. Functional analysis gathered insights in fate-specific candidates of interface functionalities. The non-transcriptionally regulated interface proteins were found to be highly regulated by post-translational ubiquitylation modification, which may synchronize the transition between cell proliferation and differentiation in ESCs.
    Stem Cell Research 09/2014; 13(2). DOI:10.1016/j.scr.2014.07.008 · 3.69 Impact Factor
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    • "Recent studies indicate that post-translational modifications of iPS factors regulate their activity. For example, phosphorylation of human Sox2 at Ser249, Ser250 and Ser 251 (Van Hoof et al., 2009) inhibits Sox2 DNA binding activity (Tsuruzoe et al., 2006). Acetylation of mouse Sox2 at Lys75 by p300/CBP enhances nuclear export and degradation of Sox2 through an ubiquitin-mediated degradation pathway (Baltus et al., 2009). "
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    ABSTRACT: Nanog regulates human and mouse embryonic stem (ES) cell self-renewal activity. Activation of ERKs signaling negatively regulates ES cell self-renewal and induces differentiation, but the mechanisms are not understood. We found that ERK1 binds and phosphorylates Nanog. Activation of MEK/ERKs signaling and phosphorylation of Nanog inhibit Nanog transactivation, inducing ES cell differentiation. Conversely, suppression of MEK/ERKs signaling enhances Nanog transactivation to inhibit ES cell differentiation. We observed that phosphorylation of Nanog by ERK1 decreases Nanog stability through ubiquitination-mediated protein degradation. Further, we found that this phosphorylation induces binding of FBXW8 with Nanog to reduce Nanog protein stability. Overall, our results demonstrated that ERKs-mediated Nanog phosphorylation plays an important role in self-renewal of ES cells through FBXW8-mediated Nanog protein stability.
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