Syk-mediated translocation of PI3Kdelta to the leading edge controls lamellipodium formation and migration of leukocytes.

Jürgen Schymeinsky,Barbara Walzog,Zoltán Jakus,Ronald Gerstl,Cornelia Then,Victor L J Tybulewicz,Anca Sindrilaru,Karin Scharffetter-Kochanek,Nick Gay

doi:10.1371/journal.pone.0001132

Abstract

The non-receptor tyrosine kinase Syk is mainly expressed in the hematopoietic system and plays an essential role in β2 integrin-mediated leukocyte activation. To elucidate the signaling pathway downstream of Syk during β2 integrin (CD11/CD18)-mediated migration and extravasation of polymorphonuclear neutrophils (PMN), we generated neutrophil-like differentiated HL-60 (dHL-60) cells expressing a fluorescently tagged Syk mutant lacking the tyrosine residue at the position 323 (Syk-Tyr323) that is known to be required for the binding of the regulatory subunit p85 of the phosphatidylinositol 3-kinase (PI3K) class IA. Syk-Tyr323 was found to be critical for the enrichment of the catalytic subunit p110δ of PI3K class IA as well as for the generation of PI3K products at the leading edge of the majority of polarized cells. In accordance, the translocation of PI3K p110δ to the leading edge was diminished in Syk deficient murine PMN. Moreover, the expression of EGFP-Syk Y323F interfered with proper cell polarization and it impaired efficient migration of dHL-60 cells. In agreement with a major role of β2 integrins in the recruitment of phagocytic cells to sites of lesion, mice with a Syk-deficient hematopoietic system demonstrated impaired PMN infiltration into the wounded tissue that was associated with prolonged cutaneous wound healing. These data imply a novel role of Syk via PI3K p110δ signaling for β2 integrin-mediated migration which is a prerequisite for efficient PMN recruitment in vivo.

Highlights

The efficient recruitment of polymorphonuclear neutrophils (PMN) to sites of lesion is critical for host defense, inflammation and tissue repair [1,2]
Downstream signaling of CD18 via specific Immunoreceptor Tyrosine-based Activation Motif (ITAM)-bearing adapter proteins (i.e. DAP12 and the Fc receptor (FcR) c-chain) leads to the activation of the non-receptor tyrosine kinase Syk [7,8,9] which has been shown to be critical for migration of neutrophil-like differentiated HL-60 cells and primary murine PMN [10,11]
Whereas 87.4% of the EGFP-Syk transfectants performed efficient migration on immobilized fibrinogen along a gradient of 10 nM fMLP (Figure 1E and F), this percentage was significantly reduced to 39.5% in cells expressing the Syk mutant which again formed multiple and unstable lamellipodia (Please see Movie S1 and Movie S2 presented as supplemental information). These results indicate that Syk-Tyr323 supported the maintenance of cell polarity, a prerequisite for efficient cell migration suggesting that the phosphatidylinositol 3-kinases (PI3K) class IA may act downstream of Syk

Summary

Introduction

The efficient recruitment of polymorphonuclear neutrophils (PMN) to sites of lesion is critical for host defense, inflammation and tissue repair [1,2]. A recent in vivo study demonstrated an impaired adhesion of LFA-12/2 PMN to the wall of postcapillary venules of the murine cremaster muscle upon stimulation with macrophage inflammatory protein-2 (Mip-2) and a decreased intraluminal crawling of Mac-1-/- PNM accompanied by a delayed emigration through nonoptimal sites [6]. Downstream signaling of CD18 via specific Immunoreceptor Tyrosine-based Activation Motif (ITAM)-bearing adapter proteins (i.e. DAP12 and the Fc receptor (FcR) c-chain) leads to the activation of the non-receptor tyrosine kinase Syk [7,8,9] which has been shown to be critical for migration of neutrophil-like differentiated HL-60 cells (dHL-60) and primary murine PMN [10,11]. A direct interaction between Syk and the regulatory subunit p85 of the phosphatidylinositol 3-kinases (PI3K) class IA has been demonstrated [13,18]

Methods

Results

Conclusion