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

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.

SRC Homology 2 Domain-containing Leukocyte Phosphoprotein of 76 kDa (SLP-76) N-terminal Tyrosine Residues Regulate a Dynamic Signaling Equilibrium Involving Feedback of Proximal T-cell Receptor (TCR) Signaling

“…1, label-free heatmap). We recently showed in Jurkat T cells (46), as others have shown in primary T cells (47), that inhibition of Erk activation leads to inhibition of positive feedback path-ways through decreased phosphorylation of Ser 59 on Lck (48,49). Our data support the hypothesis that N-terminal tyrosine residues of SLP-76 regulate competing negative feedback through Csk and positive feedback through Erk.…”

Vav1 Regulates T-Cell Activation through a Feedback Mechanism and Crosstalk between the T-Cell Receptor and CD28

“…The J.Vav1.WT cell line medium additionally contained 0.5mg/ml Geneticin (Thermo Fisher Scientific, Rockford, IL). TCR stimulation and lysis was performed as described (32). Briefly, cells were reconstituted at a concentration of 1×10 8 cells/ml in PBS.…”

Adaptation of HepG2 cells to a steady-state reduction in the content of protein phosphatase 6 (PP6) catalytic subunit

“…Label-free comparison of phosphopeptide abundance was accomplished as described previously [34], as was retention time alignment of individual replicate analyses [29]. Peak areas were calculated by inspection of select ion chromatograms (SICs) using in-house software programmed in Microsoft Visual Basic 6.0 based on Xcalibur Development kit 2.1 (Thermo Fisher Scientific).…”