ERK Positive Feedback Regulates a Widespread Network of Tyrosine Phosphorylation Sites across Canonical T Cell Signaling and Actin Cytoskeletal Proteins in Jurkat T Cells

Ynes A Helou,Samantha P Beik,Arthur R Salomon,Vinh Nguyen,Emilio Hirsch

doi:10.1371/journal.pone.0069641

Ynes A Helou, Samantha P Beik + Show 3 more

Open Access

https://doi.org/10.1371/journal.pone.0069641

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Journal: PLoS ONE	Publication Date: Jul 17, 2013
Citations: 94	License type: CC BY 4.0

Affiliation: Brown University, Providence College

Abstract

Competing positive and negative signaling feedback pathways play a critical role in tuning the sensitivity of T cell receptor activation by creating an ultrasensitive, bistable switch to selectively enhance responses to foreign ligands while suppressing signals from self peptides. In response to T cell receptor agonist engagement, ERK is activated to positively regulate T cell receptor signaling through phosphorylation of Ser59 Lck. To obtain a wide-scale view of the role of ERK in propagating T cell receptor signaling, a quantitative phosphoproteomic analysis of 322 tyrosine phosphorylation sites by mass spectrometry was performed on the human Jurkat T cell line in the presence of U0126, an inhibitor of ERK activation. Relative to controls, U0126-treated cells showed constitutive decreases in phosphorylation through a T cell receptor stimulation time course on tyrosine residues found on upstream signaling proteins (CD3 chains, Lck, ZAP-70), as well as downstream signaling proteins (VAV1, PLCγ1, Itk, NCK1). Additional constitutive decreases in phosphorylation were found on the majority of identified proteins implicated in the regulation of actin cytoskeleton pathway. Although the majority of identified sites on T cell receptor signaling proteins showed decreases in phosphorylation, Tyr598 of ZAP-70 showed elevated phosphorylation in response to U0126 treatment, suggesting differential regulation of this site via ERK feedback. These findings shed new light on ERK’s role in positive feedback in T cell receptor signaling and reveal novel signaling events that are regulated by this kinase, which may fine tune T cell receptor activation.

Highlights

The adaptive immune response relies the T cell receptor (TCR) to discriminate between foreign and self antigen
The immunoreceptor tyrosine-based activation motifs (ITAMs) serve as binding sites for the Syk family protein tyrosine kinase f-chain associated protein of 70 kDa (ZAP-70), which is recruited to the TCR
Using the human Jurkat T cell leukemia cell line, the preferred model system for studying TCR signaling [31], a quantitative mass spectrometry-based phosphoproteomic strategy was undertaken to investigate the systems-wide effects of ERK feedback in TCR signaling (Figure 2)

Summary

Introduction

The adaptive immune response relies the T cell receptor (TCR) to discriminate between foreign and self antigen. In canonical T cell activation, signaling events induced by the interaction between a TCR and peptide-major histocompatibility complex (MHC) agonist generates a set of cellular physiological changes that culminate in T cell proliferation, differentiation, and cytokine secretion. Upon activation of the TCR, the Src family protein tyrosine kinases Lck and Fyn phosphorylate the TCR CD3 chain immunoreceptor tyrosine-based activation motifs (ITAMs). The ITAMs serve as binding sites for the Syk family protein tyrosine kinase f-chain associated protein of 70 kDa (ZAP-70), which is recruited to the TCR. There, ZAP-70 is phosphorylated and activated by the Src kinase Lck. A number of signaling proteins, including the scaffolding proteins linker for activation of T cells (LAT) and SH2 domain-containing leukocyte protein of 76kDa (SLP-76) are subsequently phosphorylated by active ZAP-70. LAT and SLP-76 form a signalosome complex essential for the assembly and activation of downstream signaling proteins. LAT and SLP-76 form a signalosome complex essential for the assembly and activation of downstream signaling proteins. [1,2,3]

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