In-depth qualitative and quantitative profiling of tyrosine phosphorylation using a combination of phosphopeptide immunoaffinity purification and stable isotope dimethyl labeling.

Several mass spectrometry-based assays have emerged for the quantitative profiling of cellular tyrosine phosphorylation. Ideally, these methods should reveal the exact sites of tyrosine phosphorylation, be quantitative, and not be cost-prohibitive. The latter is often an issue as typically several milligrams of (stable isotope-labeled) starting protein material are required to enable the detection of low abundance phosphotyrosine peptides. Here, we adopted and refined a peptidecentric immunoaffinity purification approach for the quantitative analysis of tyrosine phosphorylation by combining it with a cost-effective stable isotope dimethyl labeling method. We were able to identify by mass spectrometry, using just two LC-MS/MS runs, more than 1100 unique non-redundant phosphopeptides in HeLa cells from about 4 mg of starting material without requiring any further affinity enrichment as close to 80% of the identified peptides were tyrosine phosphorylated peptides. Stable isotope dimethyl labeling could be incorporated prior to the immunoaffinity purification, even for the large quantities (mg) of peptide material used, enabling the quantification of differences in tyrosine phosphorylation upon pervanadate treatment or epidermal growth factor stimulation. Analysis of the epidermal growth factor-stimulated HeLa cells, a frequently used model system for tyrosine phosphorylation, resulted in the quantification of 73 regulated unique phosphotyrosine peptides. The quantitative data were found to be exceptionally consistent with the literature, evidencing that such a targeted quantitative phosphoproteomics approach can provide reproducible results. In general, the combination of immunoaffinity purification of tyrosine phosphorylated peptides with large scale stable isotope dimethyl labeling provides a cost-effective approach that can alleviate variation in sample preparation and analysis as samples can be combined early on. Using this approach, a rather complete qualitative and quantitative picture of tyrosine phosphorylation signaling events can be generated.

FGF-2 modulates Wnt signaling in undifferentiated hESC and iPS cells through activated PI3-K/GSK3beta signaling.

Fibroblast growth factor-2 (FGF-2) is widely used to culture human embryonic stem cells (hESC) and induced pluripotent stem (iPS) cells. Despite its importance in maintaining undifferentiated hESC phenotype, a lack of understanding in the role of FGF-2 still exists. Here, we investigate the signaling events in hESC following the addition of exogenous FGF-2. In this study, we show that hESC express all forms of fibroblast growth factor receptors (FGFRs) which co-localize on Oct3/4 positive cells. Furthermore, downregulation of Oct3/4 in hESC occurs following treatment with an FGFR inhibitor, suggesting that FGF signaling may regulate Oct3/4 expression. This is also observed in iPS cells. Also, downstream of FGF signaling, both mitogen activated protein kinase (MAPK) and phosphoinositide 3-kinase pathways (PI3-K) are activated following FGF-2 stimulation. Notably, inhibition of MAPK and PI3-K signaling using specific kinase inhibitors revealed that activated PI3-K, rather than MAPK, can mediate pluripotent marker expression. To understand the importance of PI3-K activation, activation of Wnt/beta-catenin by FGF-2 was investigated. Wnt signaling had been implicated to have a role in maintaining of pluripotent hESC. We found that upon FGF-2 stimulation, GSK3beta is phosphorylated following which nuclear translocation of beta-catenin and TCF/LEF activation occurs. Interestingly, inhibition of the Wnt pathway with Dikkopf-1 (DKK-1) resulted in only partial suppression of the FGF-2 induced TCF/LEF activity. Prolonged culture of hESC with DKK-1 did not affect pluripotent marker expression. These results suggest that FGF-2 mediated PI3-K signaling may have a direct role in modulating the downstream of Wnt pathway to maintain undifferentiated hESC.

Tyrosine phosphorylation profiling in FGF-2 stimulated human embryonic stem cells.

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.