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

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.

ERM Proteins at the Crossroad of Leukocyte Polarization, Migration and Intercellular Adhesion

“…In addition to this consensus motif, specific Ser in the cytoplasmic tail of adhesion molecules can regulate their binding to ERMs through phosphorylation-dependent mechanisms. Interactions between Ser and the FERM domain have been reported in ICAM-3, PSGL-1, N-CAM-L1 and L-selectin; whereas phosphomimetic mutations of key Ser residues susceptible to phosphorylation by PKC in the cytoplasmic tail of ICAM-3 (Ser6), CD43 (Ser76), CD44 (Ser2) and L-selectin (Ser9) interfere with their binding to the FERM domain, likely by reducing the net positive charge of their FERM-binding motifs [17,[26][27][28][29][30]. The FERM domain can also bind indirectly to ion transporters and other transmembrane receptors (e.g., the β 2 -adrenergic receptor, Na + /H + exchangers [NHE3], and the cystic fibrosis transmembrane conductance regulator, CFTR) through two PDZ domains in the scaffolding ERM-binding phosphoprotein 50 (EBP50, also called NHERF1) and NHE3 kinase A regulatory proteins (E3KARP, also called NHERF-2) (reviewed in [31]).…”

“…Knowing whether regulation of ERM dephosphorylation can facilitate this passage by fostering PM deformation and activating Cdc42-dependent actin polymerization for formation of the invasive pseudopod would thus provide new insights on how TEM is regulated. Interestingly, recent findings from the study of inflammatory monocytes suggest that ERMs can work differentially in TEM, since ezrin binds first to L-selectin and promotes formation of a main pseudopod, whereas moesin interacts later on, fostering shedding of the L-selectin ectodomain and restricting the appearance of additional pseudopods that would disturb directional transmigration [30,101].…”

L-selectin regulates human neutrophil transendothelial migration

“…Given that monocyte and neutrophil PECAM-1 have a well-established role in TEM ( Muller et al, 1993 ; Thompson et al, 2001 ), and to determine whether L-selectin plays a direct role in regulating TEM, we focused exclusively on the relationship between L-selectin and PECAM-1. Moreover, studying the contribution of PECAM-1 to driving the co-clustering of L-selectin aligned with our recent interests in understanding how L-selectin contributes to monocyte protrusive behaviour in TEM ( Newe et al, 2019 ; Rey-Gallardo et al, 2018 ; Rzeniewicz et al, 2015 ). To further determine whether antibody-mediated clustering of PECAM-1 caused L-selectin to cluster locally or distally to PECAM-1 clusters, laser scanning confocal microscopy was used to quantify the extent of overlap in fluorescence signals corresponding to PECAM-1 and L-selectin.…”

“…Based on previous work in mice, where a cytokine-specific role for PECAM-1-dependent migration through venular walls has been documented ( Thompson et al, 2001 ), we were motivated to explore whether the cytokines TNF or IL-1β could play divergent roles in driving the co-clustering of L-selectin with PECAM-1 – specifically during TEM. We have successfully used THP-1 cells to investigate mechanisms regulating L-selectin clustering in TEM, and its interaction with calmodulin or ERM proteins during TEM ( Newe et al, 2019 ; Rey-Gallardo et al, 2018 ; Rzeniewicz et al, 2015 ). Owing to their size, the majority of THP-1 cells are trapped in mid-TEM and rarely complete TEM (i.e.…”

“…The intracellular tails of L-selectin and PECAM-1 can both interact with ERM proteins ( Gamulescu et al, 2003 ; Ivetic et al, 2002 ). With respect to L-selectin, ERM binding is absolutely fundamental to antibody mediated (outside-in) clustering ( Newe et al, 2019 ). PECAM-1 also harbours an immunoreceptor tyrosine-based inhibition motif (ITIM) within its relatively longer cytoplasmic domain.…”