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

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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.