Phosphoinositides regulate the TCR/CD3 complex membrane dynamics and activation

Nassima Chouaki Benmansour,Nassima Chouaki Benmansour,Nassima Chouaki Benmansour,Ambroise Wu,Ambroise Wu,Ambroise Wu,Gaëtan Chicanne,Gaëtan Chicanne,Anne-Marie Sartre,Didier Marguet,Didier Marguet,Didier Marguet,Anne-Marie Sartre,Elise Bergot,Mathieu Fallet,Anthony Formisano,Mathieu Fallet,Sébastien Mailfert,Elise Bergot,Yannick Hamon,Kilian Ruminski,Anthony Formisano,Anthony Formisano,Cyrille Billaudeau,Cyrille Billaudeau,Cyrille Billaudeau,Cyrille Billaudeau,Cyrille Billaudeau,Cyrille Billaudeau,Cyrille Billaudeau,Yannick Hamon,Bernard Payrastre,Bernard Payrastre,Bernard Payrastre,Sophie Brasselet,Sophie Brasselet,Sophie Brasselet,Yannick Hamon,Elise Bergot,Anne-Marie Sartre,Sébastien Mailfert,Mathieu Fallet,Hai-Tao He,Hai-Tao He,Hai-Tao He,Audrey Salles,Audrey Salles,Audrey Salles,Audrey Salles,Kilian Ruminski,Sébastien Mailfert,Marie-Claire Phelipot,Sophie Brustlein,Sophie Brustlein,Sophie Brustlein,Kilian Ruminski

doi:10.1038/s41598-018-23109-8

Abstract

Phosphoinositides (PIs) play important roles in numerous membrane-based cellular activities. However, their involvement in the mechanism of T cell receptor (TCR) signal transduction across the plasma membrane (PM) is poorly defined. Here, we investigate their role, and in particular that of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] in TCR PM dynamics and activity in a mouse T-cell hybridoma upon ectopic expression of a PM-localized inositol polyphosphate-5-phosphatase (Inp54p). We observed that dephosphorylation of PI(4,5)P2 by the phosphatase increased the TCR/CD3 complex PM lateral mobility prior stimulation. The constitutive and antigen-elicited CD3 phosphorylation as well as the antigen-stimulated early signaling pathways were all found to be significantly augmented in cells expressing the phosphatase. Using state-of-the-art biophotonic approaches, we further showed that PI(4,5)P2 dephosphorylation strongly promoted the CD3ε cytoplasmic domain unbinding from the PM inner leaflet in living cells, thus resulting in an increased CD3 availability for interactions with Lck kinase. This could significantly account for the observed effects of PI(4,5)P2 dephosphorylation on the CD3 phosphorylation. Our data thus suggest that PIs play a key role in the regulation of the TCR/CD3 complex dynamics and activation at the PM.

Highlights

To cite this version: Nassima Chouaki-Benmansour, Kilian Ruminski, Anne-Marie Sartre, Marie-Claire Phelipot, Audrey Salles, et al
One major hole has remained in this picture despite extensive studies carried out over the last three decades, which is the lack of understanding on the mechanism by which the ligand engagement of T cell receptor (TCR)/CD3 at the cell surface brings about the phosphorylation of CD3 immunoreceptor tyrosine-based activation motifs (ITAMs) by Lck located at the inner leaflet of the plasma membrane (PM), a process that is commonly called TCR triggering[1]
Using a combination of state-of-the-art biophotonic approaches including excitation-polarization-resolved fluorescence microscopy and spot-variaton fluorescence correlation spectroscopy, we provide evidence that PI(4,5) P2 dephosphorylation strongly altered the CD3ε cytoplasmic domain binding to the PM inner surface in live cells, and this event could significantly account in the observed modification on the TCR lateral dynamics and activation at the PM

Summary

Introduction

To cite this version: Nassima Chouaki-Benmansour, Kilian Ruminski, Anne-Marie Sartre, Marie-Claire Phelipot, Audrey Salles, et al. Phosphoinositides (PIs) play important roles in numerous membrane-based cellular activities Their involvement in the mechanism of T cell receptor (TCR) signal transduction across the plasma membrane (PM) is poorly defined. Using state-of-the-art biophotonic approaches, we further showed that PI(4,5)P2 dephosphorylation strongly promoted the CD3ε cytoplasmic domain unbinding from the PM inner leaflet in living cells, resulting in an increased CD3 availability for interactions with Lck kinase This could significantly account for the observed effects of PI(4,5)P2 dephosphorylation on the CD3 phosphorylation. Upon TCR binding to its peptide-major histocompatibility complex (pMHC) ligand, one of the earliest and essential signaling is phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs), located in the cytoplasmic domain of the CD3 subunits, by Lck kinase. Since Lck is known to be constitutively activated in the PM of resting T cells[5], we could ask why are the CD3 ITAMs only phosphorylated upon TCR engagement by ligand, and not in the resting state

Methods

Results

Conclusion