Serine Phosphorylation of L-Selectin Regulates ERM Binding, Clustering, and Monocyte Protrusion in Transendothelial Migration.

Abigail Newe,Melanie König,Justin Joachim,Angela Rey-Gallardo,Carsten F E Schroer,Karolina Rzeniewicz,Aleksandar Ivetic,Siewert J Marrink,Maddy Parsons,Jürgen Deka

doi:10.3389/fimmu.2019.02227

Abigail Newe, Melanie König + Show 8 more

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https://doi.org/10.3389/fimmu.2019.02227

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Abstract

The migration of circulating leukocytes toward damaged tissue is absolutely fundamental to the inflammatory response, and transendothelial migration (TEM) describes the first cellular barrier that is breached in this process. Human CD14+ inflammatory monocytes express L-selectin, bestowing a non-canonical role in invasion during TEM. In vivo evidence supports a role for L-selectin in regulating TEM and chemotaxis, but the intracellular mechanism is poorly understood. The ezrin-radixin-moesin (ERM) proteins anchor transmembrane proteins to the cortical actin-based cytoskeleton and additionally act as signaling adaptors. During TEM, the L-selectin tail within transmigrating pseudopods interacts first with ezrin to transduce signals for protrusion, followed by moesin to drive ectodomain shedding of L-selectin to limit protrusion. Collectively, interaction of L-selectin with ezrin and moesin fine-tunes monocyte protrusive behavior in TEM. Using FLIM/FRET approaches, we show that ERM binding is absolutely required for outside-in L-selectin clustering. The cytoplasmic tail of human L-selectin contains two serine (S) residues at positions 364 and 367, and here we show that they play divergent roles in regulating ERM binding. Phospho-S364 blocks direct interaction with ERM, whereas molecular modeling suggests phospho-S367 likely drives desorption of the L-selectin tail from the inner leaflet of the plasma membrane to potentiate ERM binding. Serine-to-alanine mutagenesis of S367, but not S364, significantly reduced monocyte protrusive behavior in TEM under flow conditions. Our data propose a model whereby L-selectin tail desorption from the inner leaflet of the plasma membrane and ERM binding are two separable steps that collectively regulate protrusive behavior in TEM.

Highlights

The migration of circulating leukocytes toward extravascular sites of damage or infection is absolutely fundamental to the inflammatory response, and transendothelial migration (TEM) describes the first physical barrier that is breached in this process [1]
We found that L-selectin/ERM binding is absolutely required for outside-in clustering, and biochemical interactions further showed that phospho-Ser364, but not phospho-Ser367, directly blocked ERM binding
To test the impact of blocking ectodomain shedding on Antibody-Mediated Clustering (AMC) of L-selectin, THP-1 cells expressing M-N L-selectin-GFP/RFP revealed no significant increase in Förster resonance energy transfer (FRET) efficiency when cells were at rest, again suggesting that blocking ectodomain shedding of L-selectin did not lead to clustering

Summary

Introduction

The migration of circulating leukocytes toward extravascular sites of damage or infection is absolutely fundamental to the inflammatory response, and transendothelial migration (TEM) describes the first physical barrier that is breached in this process [1]. L-selectin is constitutively expressed in most circulating leukocytes, and is rapidly cleaved (shed) from the plasma membrane following challenge with formyl peptides, TNF-α, lipopolysaccharide, the complement-derived fragment C5a, or phorbol myristate acetate (PMA)—a potent PKC agonist [3,4,5]. L-selectin shedding occurs at a defined extracellular location, nine amino acids above the plasma membrane [6, 7]. Most shedding assays are conducted in vitro, using isolated leukocyte subsets (typically monocytes, neutrophils, and naive Tcells). L-selectin shedding in primary human CD14+ monocytes has been recently shown to be triggered exclusively during TEM, and not before [8]. The shedding event is restricted to transmigrating pseudopods in cells captured in mid-TEM (see later)

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