Role of CrkII in Fcγ Receptor-mediated Phagocytosis

Warren L Lee,Gabriela Cosio,Keith Ireton,Sergio Grinstein

doi:10.1074/jbc.m700823200

Abstract

Phagocytosis of IgG-opsonized pathogens by Fcgamma receptors requires extensive remodeling of the actin cytoskeleton, a process regulated by the small GTPase Rac. Vav was thought to be the guanine nucleotide exchange factor responsible for the activation of Rac, but recent evidence indicates that Fcgamma receptor-mediated phagocytosis is unaffected in macrophages lacking all three isoforms of Vav. We therefore tested whether another GEF, DOCK180, participates in Fcgamma receptor-initiated phagocytosis. DOCK180 associates with the adaptor protein Crk, which mediates recruitment of the GEF to sites of tyrosine phosphorylation. CrkII and DOCK180 were found to accumulate at the phagocytic cup. Knockdown of Crk or DOCK180 in murine macrophages using small interfering RNA inhibited phagocytosis of IgG-opsonized particles. Moreover, transfection of dominant negative CrkII prevented both recruitment of DOCK180 and the activation of Rac at the phagocytic cup. This is the first report of a role for either Crk or DOCK180 in Fcgamma receptor-mediated phagocytosis. The Crk-DOCK180 complex is involved in the clearance of apoptotic cells, which unlike the ingestion of IgG-opsonized particles, is an anti-inflammatory process. The finding that CrkII-DOCK180 is also responsible, at least in part, for the effects of Fcgamma receptors implies that additional, parallel pathways must account for the associated pro-inflammatory effect.

Highlights

Actin restructuring during Fc-mediated phagocytosis is regulated by the small GTPases Cdc42 and Rac
CrkII Is Recruited to the Phagocytic Cup during Fc␥-mediated Phagocytosis—To confirm that Crk is expressed in macrophages, we analyzed whole cell lysates of human blood monocyte-derived macrophages and murine RAW264.7 cells by immunoblotting using a monoclonal antibody that recognizes the two known splice variants of Crk [10]
As found for endogenous Crk, the recruitment of CrkII-HA was noticeable during the initial stages of phagocytosis (Fig. 1E), but despite the improved detectability conferred by the epitope tag, the adaptor protein was virtually absent from sealed phagosomes (Fig. 1F)

Summary

Introduction

Actin restructuring during Fc-mediated phagocytosis is regulated by the small GTPases Cdc42 and Rac. We identified a critical role for CrkII in the phagocytosis of IgG-opsonized particles and provided evidence that this adaptor serves to recruit the Rac GEF DOCK180 to the site of ingestion. We exposed siRNA-transfected cells inant negative constructs, these results indicate that Crk has an to IgG-opsonized beads and determined the phagocytic index essential function in Fc␥-mediated phagocytosis.

Results

Conclusion