Distinct Mechanisms Regulate Lck Spatial Organization in Activated T Cells.

Natasha Kapoor-Kaushik,Jieqiong Lou,Sophie V Pageon,Jérémie Rossy,Ewoud B Compeer,Katharina Gaus,Elizabeth Hinde,Yui Yamamoto,Zhengmin Yang,Thibault Tabarin,Felix Kraus

doi:10.3389/fimmu.2016.00083

Natasha Kapoor-Kaushik, Jieqiong Lou + Show 9 more

Open Access

https://doi.org/10.3389/fimmu.2016.00083

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Abstract

Phosphorylation of the T cell receptor (TCR) by the kinase Lck is the first detectable signaling event upon antigen engagement. The distribution of Lck within the plasma membrane, its conformational state, kinase activity, and protein–protein interactions all contribute to determine how efficiently Lck phosphorylates the engaged TCR. Here, we used cross-correlation raster image correlation spectroscopy and photoactivated localization microscopy to identify two mechanisms of Lck clustering: an intrinsic mechanism of Lck clustering induced by locking Lck in its open conformation and an extrinsic mechanism of clustering controlled by the phosphorylation of tyrosine 192, which regulates the affinity of Lck SH2 domain. Both mechanisms of clustering were differently affected by the absence of the kinase Zap70 or the adaptor Lat. We further observed that the adaptor TSAd bound to and promoted the diffusion of Lck when it is phosphorylated on tyrosine 192. Our data suggest that while Lck open conformation drives aggregation and clustering, the spatial organization of Lck is further controlled by signaling events downstream of TCR phosphorylation.

Highlights

T lymphocytes participate in an immune response when they become activated through the T cell receptor (TCR)
We have demonstrated previously that TCR activation leads to an increase in Lck clustering and that this clustering is driven by the open/active conformation of Lck [16]
Our data further indicated that signaling proteins downstream of TCR contributed to regulating Lck distribution in the plasma membrane of activated T cells, through mechanisms that were not related to conformation-induced clustering

Summary

Introduction

T lymphocytes participate in an immune response when they become activated through the T cell receptor (TCR). Despite the identification of the major players and sequences of events involved in T cell signaling pathways, the question of “How does T cell receptor signaling begin?” remains poorly understood [1, 2]. The first detectable signaling event is the phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) on TCR/CD3 subunits by the Src kinase Lck. Lck is attached to the plasma membrane through the myristoylation and palmitoylation of residues at its amino terminus. To the membrane anchor are a Src homology 3 (SH3) and a SH2 domains, followed by a catalytic tyrosine kinase domain and a short carboxy-terminal tail. Phosphorylation and dephosphorylation of a carboxy-terminal inhibitory tyrosine (Y505) and an activating tyrosine (Y394) in the catalytic domain regulate Lck kinase activity

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