Quantitative global phosphoproteomics of human umbilical vein endothelial cells after activation of the Rap signaling pathway

Abstract

The small GTPase Rap1 is required for proper cell-cell junction formation and also plays a key role in mediating cAMP-induced tightening of adherens junctions and subsequent increased barrier function of endothelial cells. To further study how Rap1 controls barrier function, we performed quantitative global phosphoproteomics in human umbilical vein endothelial cells (HUVECs) prior to and after Rap1 activation by the Epac-selective cAMP analog 8-pCPT-2 0 -O-Me-cAMP-AM (007-AM). Tryptic digests were labeled using stable isotope dimethyl labeling, enriched with phosphopeptides by strong cation exchange (SCX), followed by titanium(IV) immobilized metal affinity chromatography (Ti 4+ -IMAC) and analyzed by high resolution mass spectrometry. We identified 19 859 unique phosphopeptides containing 17 278 unique phosphosites on 4594 phosphoproteins, providing the largest HUVEC phosphoproteome to date. Of all identified phosphosites, 220 (B1%) were more than 1.5-fold up-or downregulated upon Rap activation, in two independent experiments. Compatible with the function of Rap1, these alterations were found predominantly in proteins regulating the actin cytoskeleton, cell-cell junctions and cell adhesion.

cAMP signalling in the vasculature: the role of Epac (exchange protein directly activated by cAMP)

“…Rap1 is an important regulator of cell-cell contacts in a variety of cells, and Rap1 activation can be mediated by several Rap1 GEFs [67], which raises the possibility that other Rap1 GEFs, in addition to Epac and PDZ-GEF1/2, may mediate endothelial barrier stabilization via Rap1. Whereas the mechanisms by which Epac mediates VEC barrier function appear to be diverse, it is noteworthy that a recent comprehensive phosphoproteomics study has shown that the majority of proteins that undergo phosphorylation in response to selective activation of Epac in HUVECs are those involved in facilitating cell-cell junction formation, adhesion and actin reorganization [68]. This further supports the notion that Epac is a central regulator of these processes.…”

Strong anion exchange‐mediated phosphoproteomics reveals extensive human non‐canonical phosphorylation

“…TiO 2 or IMAC, we hypothesise that this is because UPAX separates, rather than specifically enriches, phosphopeptides from non-phosphopeptides. Based on a careful comparison of the number of phosphosites identified using our "all-pX" search strategy with the number of phosphorylation sites identified for the theoretical non-phosphorylatable residue Ala, we show that the commonly applied ptmRS score of 0.75 for "class I" phosphosite localisation (Taus et al, 2011;Meijer et al, 2013;Giansanti et al, 2015;Roitinger et al, 2015;Lombardi et al, 2017) is not acceptable for broad-scale analysis of canonical and non-canonical phosphorylation, since it yields an unacceptably high FLR (Fig 2 and Appendix Tables S5 and S7). To improve confidence in our phosphopeptide datasets, facilitating motif and functional characterisation of the sites and proteins identified, we increased the acceptable site localisation stringency to ptmRS ≥ 0.90, further filtering the data to a ptmRS value ≥ 0.99 for improved confidence when interrogating pX consensus motifs.…”

Quantitative phosphoproteomics unveils temporal dynamics of thrombin signaling in human endothelial cells

“…[15][16][17] Despite the fact that robust workflows have been developed to perform quantitative MS proteomic analysis and extensively used to study phosphorylation dynamics in cell cultures, global phosphoproteomics has only very recently been successfully applied to primary ECs. [18][19][20][21] In this study, we have performed a system-wide and time-resolved characterization of thrombin-induced signaling in primary human blood outgrowth ECs (BOECs). BOECs are ECs derived from human peripheral blood and are a bona fide EC culture model with superior expansion capacity over traditional EC culture models.…”