Wiskott-Aldrich Syndrome Protein Is a Key Regulator of the Phagocytic Cup Formation in Macrophages

Abstract

Phagocytosis is a vital first-line host defense mechanism against infection involving the ingestion and digestion of foreign materials such as bacteria by specialized cells, phagocytes. For phagocytes to ingest the foreign materials, they form an actin-based membrane structure called phagocytic cup at the plasma membranes. Formation of the phagocytic cup is impaired in phagocytes from patients with a genetic immunodeficiency disorder, Wiskott-Aldrich syndrome (WAS). The gene defective in WAS encodes Wiskott-Aldrich syndrome protein (WASP). Mutation or deletion of WASP causes impaired formation of the phagocytic cup, suggesting that WASP plays an important role in the phagocytic cup formation. However, the molecular details of its formation remain unknown. We have shown that the WASP C-terminal activity is critical for the phagocytic cup formation in macrophages. We demonstrated that WASP is phosphorylated on tyrosine 291 in macrophages, and the WASP phosphorylation is important for the phagocytic cup formation. In addition, we showed that WASP and WASPinteracting protein (WIP) form a complex at the phagocytic cup and that the WASP⅐WIP complex plays a critical role in the phagocytic cup formation. Our results indicate that the phosphorylation of WASP and the complex formation of WASP with WIP are the essential molecular steps for the efficient formation of the phagocytic cup in macrophages, suggesting a possible disease mechanism underlying phagocytic defects and recurrent infections in WAS patients. The Wiskott-Aldrich syndrome (WAS)2 is an X chromosome-linked immunodeficiency disorder. Patients with WAS suffer from severe bleeding, eczema, recurrent infections, autoimmune diseases, and an increased risk of lymphoreticular malignancy (1-3). The causative gene underlying WAS encodes Wikott-Aldrich syndrome protein (WASP) (4). WASP is a 62-kDa cytosolic protein comprising distinct domains that interact with other cellular factors to regulate, mediate, and target the many functions of WASP (Fig. 3A) (5, 6). WASP is expressed predominantly in hematopoietic cells and functions in the assembly of the actin cytoskeleton (7, 8), signal transduction (9 -12), apoptosis (13,14), and the regulation of gene expression (15,16). Furthermore, WASP translocates to lipid rafts after T-cell receptor ligation, localizing to immune synapses, the junctions between T cells, and antigen-presenting cells (17,18), and recent investigations of T-cell profiles from WASP-deficient mice have revealed a critical role for WASP in regulatory T-cell function (19 -21).The WASP C-terminal region (residues 178 -502) regulates the organization of the actin cytoskeleton. Phosphatidylinositol 4,5-bisphosphate binds to the Basic Region, whereas Cdc42 binds the GTPase binding domain. Binding of these cellular factors activates the WASP C-terminal VCA (verprolin/cofilin/ acidic) domain, which in turn activates the actin-related protein complex (Arp2/3 complex), stimulating actin polymerization through the formation of F-actin branch junctions (8,22,23). ...

Rapid platelet turnover in WASP(−) mice correlates with increased ex vivo phagocytosis of opsonized WASP(−) platelets

“…Our finding of normal phagocytosis of antibody opsonized WT platelets by WASP(−) macrophages contrasts with reports of impaired phagocytosis of opsonized particles, opsonized bacteria, or apoptotic cells by human WASP(−) macrophages [56], impaired phagocytic cup formation [57], and impaired uptake of opsonized sheep red blood cells and apoptotic Jurkat cells by WASP-deficient murine macrophages [58]. Possible reasons for our divergent results include the substrates themselves (platelets), and several additional differences between our studies and those cited, such as the genetic background of the donor mice, the sources of opsonizing antibodies, and the use of flow cytometric vs microscopic readouts.…”

Phosphorylation of WASp is a key regulator of activity and stability in vivo

“…However, the GBD mutants used for these experiments could have resulted in a disturbance of the autoinhibited confirmation. Phosphorylation of Y291 has also been implicated in formation of filopodia and phagocytic cups in macrophages, and osteoclast sealing zones (17,18). However, the physiological significance and importance for normal activation in vivo is uncertain.…”

“…In some circumstances tyrosine phosphorylation may occur independently of binding to GTP-bound Cdc42 (14,16). In model systems, phosphorylation at Y291 has been reported to be necessary for WASp effector functions downstream of the T cell receptor including efficient actin polymerization, immunological synapse (IS) formation, and T cell activation, as well as for phagocytic cup formation and generation of osteoclast sealing zones (16)(17)(18).…”

Cdc42 Regulates FcγReceptor-mediated Phagocytosis through the Activation and Phosphorylation of Wiskott-Aldrich Syndrome Protein (WASP) and Neural-WASP

“…Phosphorylation of WASP on tyrosine 291 has been shown to enhance the ability of WASP to stimulate actin polymerization in vitro (Cory et al, 2002) and to potentiate phagocytic cup formation (Tsuboi and Meerloo, 2007). Thus, the importance of the WASP phosphorylation downstream of Cdc42-mediated conformation change in WASP was evaluated.…”

“…Fc ␥ R-mediated phagocytosis is impaired in peripheral blood monocytes for Wiskott-Aldrich syndrome patients in which formation of the actin cup is also markedly attenuated (Lorenzi et al, 2000;Dewey et al, 2006). Furthermore, the VCA region of WASP was demonstrated to be critical for the phagocytic cup formation (Tsuboi and Meerloo, 2007). However, the mechanism regulating WASP activation during phagocytosis has not been examined.…”