Phosphorylation of FcγRIIA is required for the receptor-induced actin rearrangement and capping: the role of membrane rafts

Abstract

Activation of Fcγ receptor II (FcγRII) induces rearrangement of the actin-based cytoskeleton that serves as a driving force for FcγRII-mediated phagocytosis and FcγRII capping. To get insight into the signaling events that lead to the actin reorganization we investigated the role of raft-associated Src family tyrosine kinases in capping of FcγRII in U937 cells. After crosslinking, FcγRII was found to be recruited to detergent-resistant membrane domains (DRMs), rafts,where it coexisted with Lyn kinase and underwent tyrosine phosphorylation. Lyn was displaced from DRMs under the influence of DL-α-hydroxymyristic acid and 2-bromopalmitic acid, agents blocking N-terminal myristoylation and palmitoylation of proteins, respectively, and after disruption of DRM integrity by depletion of plasma membrane cholesterol withβ-cyclodextrin. Under these conditions, phosphorylation of the crosslinked FcγRII was diminished and assembly of FcγRII caps was blocked. The similar reduction of FcγRII cap formation correlated with inhibition of receptor phosphorylation was achieved with the use of PP1 and herbimycin A, specific inhibitors of Src family tyrosine kinases. Phosphorylation of FcγRIIA expressed in BHK cells, lacking endogenous FcγRs, was abolished by substitution of tyrosine 298 by phenylalanine in the ITAM of the receptor. The mutant receptor did not undergo translocation towards cap-like structures and failed to promote the receptor-mediated spreading of the cells, as compared to BHK cells transfected with the wild-type FcγRIIA. On the basis of these data, we suggest that tyrosine phosphorylation of activated FcγRIIA by raft-residing tyrosine kinases of the Src family triggers signaling pathways that control the rearrangement of the actin cytoskeleton required for FcγRII-mediated motility.

The coordination of signaling during Fc receptor-mediated phagocytosis

“…DRM have been implicated in early stages of FcR activation. In particular, ligation or cross-linking FcR facilitates its localization to DRM [76,77], possibly through regulated generation of ceramide [78]. The consequences of FcR recruitment to DRM are not yet clear, but they appears to facilitate interaction of the receptor with early signaling kinases such as Lyn [76].…”

Cholesterol-Binding Peptides and Phagocytosis

“…Syk activation in turn initiates a signaling cascade, including activation of phosphatidylinositol 3-kinase (PI 3-kinase) and of the small GTPases Rac and Cdc42, which coordinate actin remodeling (Henry et al, 2004). As was reported (Kwiatkowska & Sobota, 2001;Katsumata et al, 2001;Kono et al, 2002;Kwiatkowska et al, 2003), one of the earliest signal events after the cross-binding of Fcγ receptors is lateral raft assembly that occurs before the activation of kinases of the Src family and independent of their activity. To trigger the lateral assembly of rafts, sufficient was the expression of ligand-binding monomer FcγR, without signal subunits carrying the activating fragment with tyrosine.…”

“…Moreover, expression of the ligandbinding fragment of the receptor triggered fast mobilization of calcium. The authors (Kono et al, 2002;Kwiatkowska et al, 2003) suggested that lateral assembly of rafts is caused by ligand-binding subunits of the Fcγ-receptor and that it is the raft coalescence that triggers the signaling cascade of the biochemical reactions leading to rearrangements of membrane and cytoskeleton and eventually, to the formation of a membrane vesicle containing the particle. The importance of the integrity of the plasma membrane detergent-resistant microdomains for IgG-dependent phagocytosis was also shown by Marois et al, 2011.…”

“…Binding of cholesterol at the level of the transmembrane domain of the receptor may not only account for segregation of the receptors in the lipid rafts at the early stages of phagocytosis (Kono et al, 2002;Kwiatkowska et al, 2003) but also favour an optimal FcγR configuration required for further interactions with kinases. As regards the channel proteins, binding of cholesterol may regulate the state of the ion-conducting pore.…”

Cell Surface Ceramide Generation Precedes and Controls FcγRII Clustering and Phosphorylation in Rafts

“…Within the rafts, protein-tyrosine kinases of the Src family, most likely Lyn, phosphorylate tyrosine residues of ITAM. The receptor phosphorylation triggers a cascade of tyrosine phosphorylation of several downstream proteins, which is required for a local reorganization of the actin cytoskeleton (2)(3)(4). During Fc␥RII-mediated phagocytosis, the actin cytoskeleton drives uptake of IgG-coated particles, e.g.…”