Phosphoproteomic analysis of thrombin- and p38 MAPK-regulated signaling networks in endothelial cells

Olivia Molinar-Inglis,Jacob M Wozniak,Neil J Grimsey,Lennis B Orduña-Castillo,Norton Cheng,Ying Lin,Monica L Gonzalez Ramirez,Cierra A Birch,John D Lapek,David J Gonzalez,Joann Trejo

doi:10.1016/j.jbc.2022.101801

Abstract

Endothelial dysfunction is a hallmark of inflammation and is mediated by inflammatory factors that signal through G protein–coupled receptors including protease-activated receptor-1 (PAR1). PAR1, a receptor for thrombin, signals via the small GTPase RhoA and myosin light chain intermediates to facilitate endothelial barrier permeability. PAR1 also induces endothelial barrier disruption through a p38 mitogen-activated protein kinase–dependent pathway, which does not integrate into the RhoA/MLC pathway; however, the PAR1-p38 signaling pathways that promote endothelial dysfunction remain poorly defined. To identify effectors of this pathway, we performed a global phosphoproteome analysis of thrombin signaling regulated by p38 in human cultured endothelial cells using multiplexed quantitative mass spectrometry. We identified 5491 unique phosphopeptides and 2317 phosphoproteins, four distinct dynamic phosphoproteome profiles of thrombin-p38 signaling, and an enrichment of biological functions associated with endothelial dysfunction, including modulators of endothelial barrier disruption and a subset of kinases predicted to regulate p38-dependent thrombin signaling. Using available antibodies to detect identified phosphosites of key p38-regulated proteins, we discovered that inhibition of p38 activity and siRNA-targeted depletion of the p38α isoform increased basal phosphorylation of extracellular signal–regulated protein kinase 1/2, resulting in amplified thrombin-stimulated extracellular signal–regulated protein kinase 1/2 phosphorylation that was dependent on PAR1. We also discovered a role for p38 in the phosphorylation of α-catenin, a component of adherens junctions, suggesting that this phosphorylation may function as an important regulatory process. Taken together, these studies define a rich array of thrombin- and p38-regulated candidate proteins that may serve important roles in endothelial dysfunction.

Highlights

We show that p38 negatively regulates basal phosphorylation of extracellular signal–regulated protein kinase 1/ 2 (ERK1/2) in endothelial cells resulting in amplified ERK1/2 phosphorylation after thrombin stimulation
Cell lysates from endothelial cells treated with or without SB203580 stimulated with thrombin showed agonist-induced p38 phosphorylation in DMSO control cells that was markedly reduced in cells pretreated with the p38 inhibitor SB203580 (Fig. 1E), confirming thrombin-stimulated p38 signaling is inhibited by SB203580 pretreatment in EA.hy926 as we previously reported [4, 6]
Our study further identified several thrombin- and p38regulated key proteins associated with endothelial dysfunction and important biological processes including microtubules, focal adhesions, stress fiber, Rho, small GTPases, cell–cell adherens junction proteins, and a subset of kinases

Summary

Introduction

To identify p38 MAPK targets that mediate thrombin-induced proinflammatory responses, human cultured endothelial EA.hy296 cells were pretreated with SB203580, a p38α and p38β selective inhibitor, or dimethylsulfoxide (DMSO) vehicle control and stimulated with thrombin to determine changes in protein phosphorylation linked to p38 signaling. No change in phosphorylation was detected in C4 peptides in SB203580-treated cells following thrombin stimulation compared to DMSO control (Fig. 2, A and B).

Results

Conclusion