Abstract
Embryonic stem cells are pluripotent and capable of unlimited self-renewal. Elucidation of the underlying molecular mechanism may contribute to the advancement of cell-based regenerative medicine. In the present work, we performed a large scale analysis of the phosphoproteome in mouse embryonic stem (mES) cells. Using multiplex strategies, we detected 4581 proteins and 3970 high confidence distinct phosphosites in 1642 phosphoproteins. Notably, 22 prominent phosphorylated stem cell marker proteins with 39 novel phosphosites were identified for the first time by mass spectrometry, including phosphorylation sites in NANOG (Ser-65) and RE1 silencing transcription factor (Ser-950 and Thr-953). Quantitative profiles of NANOG peptides obtained during the differentiation of mES cells revealed that the abundance of phosphopeptides and non-phosphopeptides decreased with different trends. To our knowledge, this study presents the largest global characterization of phosphorylation in mES cells. Compared with a study of ultimately differentiated tissue cells, a bioinformatics analysis of the phosphorylation data set revealed a consistent phosphorylation motif in human and mouse ES cells. Moreover, investigations into phosphorylation conservation suggested that phosphoproteins were more conserved in the undifferentiated ES cell state than in the ultimately differentiated tissue cell state. However, the opposite conclusion was drawn from this conservation comparison with phosphosites. Overall, this work provides an overview of phosphorylation in mES cells and is a valuable resource for the future understanding of basic biology in mES cells.
Highlights
Embryonic stem (ES) cells, which are derived from the inner cell mass of early blastocysts [1], are pluripotent and capable of self-renewal [2]
The cells were examined under a microscope to ensure that the mouse embryonic stem (mES) cells were more than 90% confluent and had a high nucleus/cytosol ratio
Immunostaining of cells cultured under the same conditions for the murine-specific stem cell surface markers alkaline phosphatase and SSEA-1 and for the expression of OCT4, SOX2, and NANOG, which are master regulators of mES cells, strongly demonstrates the undifferentiated state of the mES cells used in the assays
Summary
Embryonic stem (ES) cells, which are derived from the inner cell mass of early blastocysts [1], are pluripotent and capable of self-renewal [2]. Significant progress has been made toward understanding the characteristics of ES cells, including uncovering some notable transcription factors, such as OCT4 [4, 5], SOX2 [6], and NANOG [7, 8] These key regulators are thought to be critical for the differentiation of ES cells because of their unique expression profiles and their essential roles in early development. We developed a “StemCell Project” web site to retrieve and analyze the data from the study This information provides an overview of phosphorylation in mES cells and should be a valuable resource for the future understanding of the basic biology in mES cells
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