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

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.

The transcriptional regulation of pluripotency

“…Together, FGF2-signalling in hESCs not only resulted in phosphorylation of proteins of various signalling cascades such as that of PI3K, MAPK, Wnt, but could also lead to phosphorylation of pluripotency-associated transcription regulators like OCT4, SOX2, SALL4 and DPPA4 [95,96]. These studies while informative in revealing a possible phospho-interactome downstream of FGF2-FGFR, unfortunately do not factor in signalling 'crosstalk' by other receptor kinases onto FGFRassociated pathways [86,87].…”

“…There have already been several large-scale attempts at profiling the global hESC phosphoproteome via mass spectrometry techniques [91][92][93][94], with two studies seeking to specifically address the dynamics of FGF2-dependent tyrosine and serine/threonine phosphorylation [95,96]. Together, FGF2-signalling in hESCs not only resulted in phosphorylation of proteins of various signalling cascades such as that of PI3K, MAPK, Wnt, but could also lead to phosphorylation of pluripotency-associated transcription regulators like OCT4, SOX2, SALL4 and DPPA4 [95,96].…”

Calcium signaling in human pluripotent stem cells

“…PLC-␥, MAPK, PI3-K). In addition, a large number of pY-peptides, not directly involved in the canonical FGF pathway (e.g., Src kinase substrates, additional receptor tyrosine kinases and others), were observed following FGF-2 stimulation [60]. This quantitative phosphoproteomic analysis suggests that intracellular calcium modulation may become important through the activation of the PLC-␥ pathway (Fig.…”

Mechanobiology of Human Pluripotent Stem Cells

“…hPSCs treated with Src kinase inhibitors rapidly differentiate, indicating that high Src family kinase activity is important for the self-renewal of the hPSCs. 64 Increased Src activation could help explain the improved self-renewal some groups report for PSCs grown on softer substrates.…”