Tyrosine Phosphorylation Profiling in FGF-2 Stimulated Human Embryonic Stem Cells

Vanessa M Y Ding,Christian Preisinger,Paul J Boersema,Albert J R Heck,Subaashini Natarajan,Simone Lemeer,Geoffrey Koh,Dong-Yup Lee,Berend Snel,Leong Yan Foong,Jos Boekhorst,Andre Choo,Martin Pera

doi:10.1371/journal.pone.0017538

Abstract

The role of fibroblast growth factor-2 (FGF-2) in maintaining undifferentiated human embryonic stem cells (hESC) was investigated using a targeted phosphoproteomics approach to specifically profile tyrosine phosphorylation events following FGF-2 stimulation. A cumulative total number of 735 unique tyrosine phosphorylation sites on 430 proteins were identified, by far the largest inventory to date for hESC. Early signaling events in FGF-2 stimulated hESC were quantitatively monitored using stable isotope dimethyl labeling, resulting in temporal tyrosine phosphorylation profiles of 316 unique phosphotyrosine peptides originating from 188 proteins. Apart from the rapid activation of all four FGF receptors, trans-activation of several other receptor tyrosine kinases (RTKs) was observed as well as induced tyrosine phosphorylation of downstream proteins such as PI3-K, MAPK and several Src family members. Both PI3-K and MAPK have been linked to hESC maintenance through FGF-2 mediated signaling. The observed activation of the Src kinase family members by FGF-2 and loss of pluripotent marker expression post Src kinase inhibition may point to the regulation of cytoskeletal and actin depending processes to maintain undifferentiated hESC.

Highlights

Human ESCs are a powerful tool for drug screening, studying early lineage differentiation in vitro, and generating specific cell phenotypes for therapeutic applications
Several pathways have been implicated in human embryonic stem cells (hESC) self-renewal, including transforming growth factor-b/Activin-A/Nodal [1], sphingosine-1-phosphate/plateletderived growth factor (S1P/PDGF) [2], insulin growth factor (IGF)/insulin [3] and fibroblast growth factor-2 (FGF-2) [4]
Activation of the hESC was validated by Western blotting using two antibodies specific for the phosphorylation of proteins downstream of the fibroblast growth factor receptors (FGFRs)

Summary

Introduction

Human ESCs are a powerful tool for drug screening, studying early lineage differentiation in vitro, and generating specific cell phenotypes for therapeutic applications. Optimizing the efficient expansion and differentiation of these cells still requires further understanding, especially of the signaling pathways responsible for regulating hESC. The process of self-renewal appears to be regulated synergistically through various pathways via growth factor or cytokine supplementation. FGF2 signaling appears indispensible to hESC self-renewal just as leukemia inhibitory factor is to mESC [6]. FGF-2 is widely used for sustained long-term culture of human embryonic stem cells (hESC) and induced pluripotent stem (iPS) cells under both feeder and feeder–free culture conditions [7,8,9]

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Discussion

Conclusion