Phagocytosis is one of the important innate immune responses that function to eliminate invading infectious agents. Monocytes, macrophages, and neutrophils are the professional phagocytic cells. Phagocytosis is a complex process involving the recognition of invading foreign particles by specific types of phagocytic receptors and the subsequent internalization of the particles. Fc gamma receptors (FCGRs) are among the best studied phagocytic receptors that bind to Fc portion of immunoglobulin G (IgG). Through their antigen binding F(ab) end, antibodies bind to specific antigen while their constant (Fc) region binds to FCGRs on phagocytes. The clustering of FCGRs by IgG antibodies on the phagocyte initiates a variety of signals, which lead, through the reorganisation of actin cytoskeleton and membrane remodelling, to the formation of pseudopod and phagosome. Fc gamma receptors are classified into three classes: FCGRI, FCGRII and FCGRIII. Each class of these FCGRs consists of several individual isoforms. Among all these isoforms FCGRI, FCGRIIA and FCGRIIIA, are able to mediate phagocytosis (Joshi et al. 2006, Garcia Garcia & Rosales 2002, Nimmerjahn & Ravetch 2006).
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Inositol 1,4,5-triphosphate (IP3) is a second messenger produced by phospholipase C (PLC) metabolism of phosphoinositol 4,5-bisphosphate (PIP2) (Canossa et al. 2001).
The IP3 receptor (IP3R) is an IP3-gated calcium channel. It is a large, homotetrameric protein, similar to other calcium channel proteins such as ryanodine. The four subunits form a 'four-leafed clover' structure arranged around the central calcium channel. Binding of ligands such as IP3 results in conformational changes in the receptor's structure that leads to channel opening.
Myosin-X (Myosin 10) is one of the downstream effectors of PI3K in FCGR-phagocytosis and is involved in pseudopod extension and closure of phagocytic cups. It is recruited to the forming phagosome by binding, through its second PH domain to membrane PIP3, a major product of PI3-kinase (Cox et al. 2002). Myosin-X may act as a motor to transport membrane cargo molecules to the forming pseudopods, influencing actin dynamics. It is not understood with certainty how myosin X contributes to the mechanism of pseudopod extension. It selectively binds to actin bundle such that each head may bind, in an ATP-sensitive manner, to two adjacent actin filaments within the actin bundle. Myosin X hydrolyze ATP and converts this chemical energy to mechanical energy moving toward the plus end/barbed end of the actin filament facing towards the tip of the growing pseudopods (Araki 2006, Chavrier 2003, Watanabe et al 2010).
FCGRII (CD32) is a low-affinity receptor encoded by three different genes (A, B and C) (Brooks et al. 1989). FCGRIIA functions as a single-chain transmembrane receptor containing both the ligand-binding extracellular domain and a signal transducing cytoplasmic domain that contains distinct immunoreceptor tyrosine-based activation motif (ITAM). This ITAM-like domain in FCGRIIA contains two YXXL motifs with a spacer sequence of 12 amino acids instead of the usual 7. Isoform FCGRIIB is expressed in various leukocytes, including human monocytes and, as opposed to the activating Fc receptors it has an immunoreceptor tyrosine-based inhibitory motif (ITIM) and negatively regulates phagocytosis (Van den Herik-Oudijk IE et al. 1995, Mitchell et al. 1994, Tridandapani et al. 2002). The first step in Fc-gamma receptor (FCGR) phagocytosis is binding and clustering of FCGRs by IgG-coated foreign particles. FCGR are clustered at the cell surface by multivalent antigen-antibody complexes and recruited to lipid raft micro domains; monovalent ligand binding is insufficient to generate a signal. This cross-linking results in the localization of FCGRs into lipid rafts and this may aid in recruiting and complexing with additional signalling proteins associated with lipid rafts (Bournazos et al. 2009, Kwiatkowska & Sobota 2001, Kono et al. 2002). This is followed by phosphorylation of the tyrosine residues within the ITAM located on the cytoplasmic portion of FCGRIIA by membrane-associated tyrosine kinases of the Src family (Mitchell et al. 1994).
Diacylglycerol (DAG) positively regulates the autophosphorylation of protein kinase C-delta (PKC-delta), which stimulates ERK1/2 and triggers neurite outgrowth. DAG also stimulates the translocation of PKC from the cytosol to the plasma membrane. PKC-delta contributes to growth factor specificity and response to neuronal cells by promoting cell-type-specific differences in growth factor signalling. DAG can also activate PKC-epsilon in the same manner.
PLCG is tyrosine phosphorylated by either SYK or Src kinases on three tyrosine residues and this phosphorylation enhances the activity of PLCG. Although maximal activation requires binding of PLCG to PIP3 with its plecstrin homology (PH) domain.
PLCgamma (PLCG) is recruited to FCGR and the phosphorylated Y342 and Y346 in SYK have been reported to be involved in the interaction of PLCG (Law et al. 1996). PLCG accumulates at the phagocytic cup during FCGR, but the exact role of PLCG in the regulation of phagocytosis is not clear. It may be involved in FCGR signaling by activating PKC through DAG production (Garcia-Garcia & Rosales 2002 )
Activated PI3K phosphorylates phosphatidylinositol (PI) 4-phosphate and PI 4,5-bisphosphate (PIP2) to generate PI 3,4-bisphosphate and PI 3,4,5-triphosphate (PIP3) and these second messengers recruit other signaling proteins containing plecstrin homology (PH) domain. PIP3 rapidly accumulates at sites of phagocytosis and disappears after the phagosome has been sealed off from the plasma membrane.
Activated PLCG translocates to the plasmamembrane and interacts with the inositol ring of the membrane bound phosphatidylinositol 4,5-bisphosphate (PIP2) with its PH domain. The active enzyme promotes intracelllular signaling by catalysing the hydrolysis of PIP2 to generate the second messengers IP3 and DAG.
PI3K is one of the downstream effector of activated SYK. The p85 alpha regulatory subunit of PI3K have been shown to interact with SYK and FCGRs, and transient and restricted accumulation of lipid products of the kinase have been observed at sites of the phagosomal cup (Yanagi et al. 1994, Marshall et al. 2001). Leverrier at al. has demonstrated that p110beta is the major class I catalytic isoform required for FCGR-mediated phagocytosis by primary macrophages (Leverrier at al. 2003). p85 alpha subunit interacts with phosphorylated SYK at tyrosine 317 with its C-terminal SH2 domain, whereas other tyrosine residues like 342 and 346 in the linker region may contribute to the interaction with N-terminal SH2 domain (Moon et al, 2005). The main role of PI3K in phagocytosis appears to be the regulation of pseudopod extension necessary for particle internalization and may also regulate phagocytosis through activation of ERK (Garcia-Garcia & Rosales. 2002).
FCGR mediated phagocytosis requires CDC42 to stimulate actin polymerization, generating the force for phagocytic cup protrusion or pseudopod extension. CDC42 activation is restricted at the advancing edge of the phagocytic cup, where actin is concentrated, and is deactivated at the base of the phagocytic cup (Beemiller et al 2010). The mechanism behind the recruitment and activation of CDC42 during FCGR phagocytosis is unknown. VAV regulates the activation of RAC1 but not CDC42 and the GEF responsible for CDC42 activation during FCGR-mediated phagocytosis remains unidentified (Adam et al 2004, Patel et al 2002).
Multiple sites of phosphorylation are known to exist in SYK, which both regulate its activity and also serve as docking sites for other proteins. Some of these sites include Y131 of interdomain A, Y323, Y348, and Y352 of interdomain B, and Y525 and Y526 within the activation loop of the kinase domain and Y630 in the C-terminus (Zhang et al. 2002, Lupher et al. 1998, Furlong et al. 1997). Phosphorylation of these tyrosine residues disrupts autoinhibitory interactions and results in kinase activation even in the absence of phosphorylated ITAM tyrosines (Tsang et al. 2008). SYK is primarily phosphorylated by Src family kinases and this acts as an initiating trigger by generating few molecules of activated SYK which are then involved in major SYK autophosphorylation (Hillal et al. 1997).
The organized movements of membranes and the actin cytoskeleton are coordinated in phagocytosis by small GTPases of the Rho family. Specifically, RAC1 and CDC42 are known to be stimulated upon engagement of FCGR and are essential for the extension of the pseudopods that surround and engulf the phagocytic particle (Scott et al 2005). RAC1 is known to regulate actin dynamics. It is active throughout the phagocytic cup and activated RAC1 is necessary to assemble F actin. However, closing the phagocytic cup requires RAC1 to be deactivated (Naakaya et al 2007). Deletion of RAC1 prevents FCGR mediated phagocytosis (Hall et al 2006). RAC1 activation involves transition from an inactive GDP bound to an active GTP bound state catalysed by guanine exchanges factors (GEFs). VAV has been implicated in the activation of RAC1 (Patel et al 2002).
SYK is a tyrosine kinase related to ZAP70 that is expressed in all hematopoietic cells and coimmunoprecipitates with the gamma chain associated with FCGRIIIA in macrophages and with FCERI in mast cells. SYK is very important for FCGR phagocytosis and is recruited to these phosphorylated ITAM residues through its two SRC homology 2 (SH2) domains (Agarwal et al. 1993). When SYK kinase expression is inhibited with antisense oligonucleotides both in vitro and in vivo, phagocytosis and inflammation are abolished (Matsuda et al. 1997). The domain structure of SYK comprises a regulatory region at the N-terminus consisting of a pair of SH2 domains separated by an inter-SH2 linker called interdomain A, an SH2-domain-kinase linker termed interdomain B, and a C-terminal kinase domain (Arias-Palomo et al. 2009). In resting state SYK exists in an auto-inhibited conformation by the interactions between the SH2-SH2 regulatory region and the inter-SH2 linker and the catalytic domain. This interdomain interaction reduces the conformational flexibility required by the kinase domain for catalysis (Arias-Palomo et al. 2007). Changes in the orientation of the SH2 domains could control the disruption of the auto inhibitory interactions and the activation of SYK. These movements could be totally or partially induced by the binding to phosphorylated ITAMs and/or phosphorylation of tyrosine residues in interdomain A or B (Arias-Palomo et al. 2009). Tsang et al. suggested that SYK functions as an OR-gate switch with respect to phosphorylation and ITAM binding, as either one stimulus OR the other is sufficient to cause full activation (Tsang et al. 2008).
VAV proteins exist in an auto-inhibitory state folded in such a way as to inhibit the GEF activity of its DH domain. This folding is mediated through binding of tyrosines in the acidic domain to the DH domain and through binding of the CH domain to the C1 region. Activation of VAV may involve at least three different events to relieve this auto-inhibition. Phosphorylation of the tyrosines in the acidic domain causes them to be displaced from the DH domain, binding of a ligand to the CH domain may cause it to release the C1 domain and binding of PIP3 to PH domain may alter its conformation. VAV1 is phosphorylated on Y174 in the acidic domain, and this is mediated by Syk and Src-family tyrosine kinases. Once activated, VAV1 is then involved in the activation of RAC and CDC42 downstream of FCGR.
PAK1 needs autophosphorylation for complete activation. PAK1 is autophosphorylated at several sites, but S144 flanking the kinase inhibitor region and T423 within the catalytic domain are the two conserved sites that regulate the catalytic activity (Chong et al. 2001, Parrini et al. 2001).
FCGRI is coded by three different genes (A, B, and C) and is expressed on most myeloid cells including monocytes, macrophages and dendritic cells (Allen & Seed 1988). FCGRI is a high affinity IgG receptor capable of binding monomeric IgG. FCGRI exists as a complex containing ligand (IgG) binding extracellular alpha-chain and homodimer of signal transducing FcR gamma (CD3G) chains, or a heterodimer of signal transducing FcR gamma and zeta chains (Ernst et al. 1993, Scholl & Geha 1993, van Vugt et al. 1996). The cytoplasmic domain of FCGRI does not have signaling motifs, however it is suggested that the gamma-subunit might be required for generating the phagocytic signal (Duchemin et al. 1994, Indik et al. 1995). The first step in Fc-gamma receptor (FCGR) phagocytosis is binding and clustering of FCGRs by IgG-coated foreign particles. FCGR are clustered at the cell surface by multivalent antigen-antibody complexes and recruited to lipid raft micro domains; monovalent ligand binding is insufficient to generate a signal. This cross-linking results in the localization of FCGRs into lipid rafts and this may aid in recruiting and complexing with additional signalling proteins associated with lipid rafts (Bournazos et al. 2009, Kwiatkowska & Sobota 2001, Kono et al. 2002). This is followed by phosphorylation of the tyrosine residues with in the immuno tyrosine activation motif (ITAM) located on the cytoplasmic portion of accessory gamma/zeta chains by membrane-associated tyrosine kinases of the Src family (Duchemin et al. 1994, van Vugt et al. 1996).
PAK1, a downstream effector of CDC42 and RAC1, is found localized in phagosomes. Upon activation, PAK1 phosphorylates LIMK, which directly phosphorylates and inactivates cofilin, a protein that mediates depolymerization of actin filaments. Thus, RAC and CDC42 coordinate actin dynamics by inducing actin polymerization via ARP2/3 on one hand, and inhibiting actin depolyerization via LIMK and cofilin on the other (Garcia-Garcia & Rosales 2002). PAK1 exists as homodimer in a trans-inhibited conformation. The kinase inhibitory (KI) domain of one PAK1 molecule binds to the C-terminal catalytic domain of the other and inhibits catalytic activity. GTPases RAC1/CDC42 bind the GBD domain of PAK1 thereby altering the conformation of the KI domain, relieving inhibition of its catalytic domain, and allowing PAK1 autophosphorylation that is required for full kinase activity (Parrini et al. 2002, Zhao & Manser 2005).
FCGRIII (CD16) is a low affinity Fc gamma receptor and is encoded by two genes (A and B), the transmembrane form FCGRIIIA and the GPI anchored FCGRIIIB (Edberg et al. 1989). Between the two isoforms FCGRIIIA is involved in phagocytosis whereas FCGRIIIB is capable of inducing calcium signalling and actin polymerisation, but its role in phagocytosis is still not clear (Garcia Garcia & Rosales 2002). FCGRIIIA is expressed in macrophages and natural killer cells as a multi chain complex consisting of a single alpha chain containing IgG binding domains and a signal transducing gamma and/or zeta dimer (Wirthmuller et al. 1992, Lanier et al. 1989). Both gamma and zeta chains contain a conserved immunoreceptor tyrosine based activation motif (ITAM), which has 2 copies of the YXXL sequence (Isakov 1997). However, the gamma chain of FCGRIIIA is approximately sixfold more efficient in mediating phagocytosis than the zeta subunit (Park & Schreiber 1995). Phosphorylation of the conserved tyrosine residues of the ITAM in these accessory proteins is required for the phagocytic signal mediated by FCRGIIIA. The first step in Fc-gamma receptor (FCGR) phagocytosis is binding and clustering of FCGRs by IgG-coated foreign particles. FCGR are clustered at the cell surface by multivalent antigen-antibody complexes and recruited to lipid raft micro domains; monovalent ligand binding is insufficient to generate a signal. This cross linking results in the localisation of FCGRs into lipid rafts and this may aid in their recruiting and complexing with additional signalling proteins associated with lipid rafts (Kono et al. 2002). This is followed by phosphorylation of the tyrosine residues within the ITAM located on the cytoplasmic portion of accessory gamma/zeta chains by membrane associated tyrosine kinases of the Src family (Park et al. 1993).
VAV family members are cytoplasmic guanine nucleotide exchange factors (GEFs) for Rho-family GTPases (RAC, RHO and CDC42). VAV1 is found predominantly in hematopoietic cells, whereas VAV2 and VAV3 are more broadly expressed. VAV proteins link the cell surface receptors like FCGR to the intracellular Rho GTPases and the actin cytoskeleton during phagocytosis (Hall et al 2006). Experiments using two-hybrid system suggest that VAV1 with its SH2 domain directly binds to the phosphorylated Y342 of SYK (Deckert et al. 1996). VAV proteins are also recruited to membrane through their PH domain by binding PI(3,4,5)P3 produced by PI3K.
After cross linking, Fc gamma receptors are sequestered to lipid rafts where they are complexed with some of the tyrosine kinases of Src family and undergo phosphorylation on the tyrosine residues contained in conserved ITAM sequences. At least six out of nine members of the Src family kinases (SRC, FYN, FGR, HCK, YES and LYN ) have been identified in the phagocytic cells and are implicated in the initiation of Fc gamma mediated signaling. (Suzuki et al. 2000, Majeed et al. 2001, Kwiatkowska et al. 2003). Some of these kinases have been found associated with specific receptors. In monocytes HCK and LYN have been found associated with FCGRI (Durden et al. 1995), whereas only HCK with FCGRIIA (Ghazizadeh et al. 1994) while FGR in neutrophils (Hamada et al. 1993) and LCK in NK cells with FCGRIIIA (Pignata et al. 1993) The implication of Src kinases in phosphorylation was first supported by pharmacological findings that herbimycin A, a tyrosine kinase inhibitor relatively specific for Src-family kinases, potently suppressed Fc receptor mediated functions (Greenberg et al. 1993, Suzuki et al. 2000). However, their particular involvement in phagocytosis remains unclear, as targeted disruption of single or multiple Src family genes did not result in significant alterations in phagocytosis (Hunter et al. 1993, Fitzer Attas et al. 2000, Suzuki et al. 2000). HCK, FGR and LYN triple-deficient (-/-) macrophages have shown significant delays in FCGR mediated phagocytosis, but these deficiencies do not completly disrupt the process (Fitzer Attas et al. 2000). Tyrosine residues Y288 and Y304 (Y282 and Y298 according to the literature reference, it is 6 residues shorter compared to uniprot entry due to an alternate initiation codon usage), within ITAM sequence in the cytoplasmic domain of FCGRIIA are the key target sites that are phosphorylated by Src family kinases (Mitchell et al, 1994). In case of FCGRIA and FCGRIIIA the specific tyrosine residues within ITAMs of the associated gamma/zeta chains are phosphorylated by activated Src family kinases (SFKs) (Park et al. 1993).
LIM kinases are serine protein kinases with a unique combination of two N-terminal LIM motifs, a central PDZ domain, and a C-terminal protein kinase domain. LIMK1 is one of the downstream targets of PAK1 and is activated through phosphorylation by PAK1 on T508 within its activation loop (Edwards et al. 1999, Aizawa et al. 2001). LIM-kinase is responsible for the tight regulation of the activity of cofilin (a protein that depolymerizes actin filaments) and thus maintains the balance between actin assembly and disassembly. Phosphorylated cofilin is inactive, resulting in stabilization of the actin cytoskeleton.
WASP family verprolin-homologous proteins (WAVEs) function downstream of RAC1 and are involved in activation of the ARP2/3 complex. The resulting actin polymerization mediates the projection of the plasma membrane in lamellipodia and membrane ruffles. WAVEs exist as a pentameric hetero-complex called WAVE Regulatory Complex (WRC). The WRC consists of a WAVE family protein (WASF1, WASF2 or WASF3 - commonly known as WAVE1, WAVE2 or WAVE3), ABI (Abelson-interacting protein), NCKAP1 (NAP1, p125NAP1), CYFIP1 (SRA1) or the closely related CYFIP2 (PIR121), and BRK1 (HSPC300, BRICK). Of the three structurally conserved WAVEs in mammals, the importance of WAVE2 in activation of the ARP2/3 complex and the consequent formation of branched actin filaments is best established. WAVEs in the WRC are intrinsically inactive and are stimulated by RAC1 GTPase and phosphatidylinositols (PIP3). The C-terminal VCA domain of WAVE2 (and likely WAVE1 and WAVE3) which can bind both the ARP2/3 complex and actin monomers (G-actin) is masked in the inactive state. After PIP3 binds to the polybasic region of WAVE2 (and likely WAVE1 and WAVE3) and RAC1:GTP binds to the CYFIP1 (or CYFIP2) subunit of the WRC, allosteric changes most likely occur which allow WAVEs to interact with the ARP2/3 complex. The interactions between WAVEs and RAC1 are indirect. BAIAP2/IRSp53, an insulin receptor substrate, acts as a linker, binding both activated RAC1 and the proline-rich region of WAVE2 (and likely WAVE1 and WAVE3) and forming a trimolecular complex. CYFIP1 (or CYFIP2) in the WAVE regulatory complex binds directly to RAC1:GTP and links it to WAVE2 (and likely WAVE1 and WAVE3) (Derivery et al. 2009, Yamazaki et al. 2006, Takenawa & Suetsugu 2007, Chen et al. 2010, Pollard 2007, Lebensohn & Kirschner 2009).
Once activated, the ARP2/3 complex nucleates new actin filaments that extend from the sides of pre-existing mother actin filaments at a 70-degree angle to form Y-branched networks (Firat-Karalar & Welch 2010). These branched actin filaments push the cell membrane forward to form a pseudopod. The ARP2/3 complex is composed of two Arps (actin-related proteins), ARP2 and ARP3, and five unique proteins ARPC1, ARPC2, ARPC3, ARPC4 and ARPC5 (Gournier et al. 2001). Both ARP2 and ARP3 subunits bind ATP. There are two proposed models to explain the process of actin nucleation by ARP2/3 complex: the barbed-end branching model and the dendritic nucleation/side branching model (Le Clainche & Carlier 2008). In barbed-end branching model, the branching/ternary complex (G-actin-WASP/WAVE-Arp2/3 complex) binds to the barbed end of the mother filament. G-actin bound to VCA domain or one of the Arp subunits incorporates into the mother filament at the barbed end, thus positioning ARP2/3 complex to initiate the daughter branch on the side of the mother filament. ARP2/3 nucleates the formation of new actin filament branches, which elongate at the barbed ends (Le Clainche & Carlier 2008, Pantaloni et al 2000, Le Clainche et al. 2003, Egile et al. 2005). In side branching model, the branching complex binds to the side of the mother actin filament mimicking an actin nucleus and initiates a lateral branch (Le Clainche & Carlier 2008, Amann & Pollard 2001).
Phosphatidic acid phosphatase (PAP) bound to the plasma membrane catalyzes the dephosphorylation of phosphatidic acid (PA), yielding diacylglycerol (DAG) and inorganic phosphate. In humans, Diacylglycerol pyrophosphate (DPPL1 and DPPL2) perform this reaction.
The ARP2/3 complex shows higher affinity for the phosphorylated VCA domain of WAVE2 than for the unphosphorylated VCA domain. WAVE proteins can be phosphorylated by various kinases. Active ERK (Mitogen activated protein kinase 3) phosphorylates the WAVE regulatory complex (WRC) on multiple serine/threonine sites within the proline-rich domains (PRDs) of WAVE2 and ABI1. Phosphorylation of the PRDs would disrupt their interaction with SH3 and PLP binding domains, potentially altering WRC activation. ERK phosphorylates both S343 and T346 in WAVE2 and S183, S216, S225, S392, and S410 in ABI1. Cumulatively, the phosphorylation of both WAVE2 and ABI in the WAVE regulatory complex (WRC) contributes to the RAC-induced WRC conformational change that exposes the VCA domain, leading to binding and activation of ARP2/3 (Mendoza et al. 2011, Nakanishi et al. 2007). ERK phosphorylation sites in WAVE2 are not strictly conserved in WAVE1 and WAVE3 but, based on the amino acid sequence, other potential ERK phosphorylation sites exist.
Phospholipase D (PLD) catalyses the hydrolysis of the membrane phospholipid, phosphatidylcholine (PC) to generate choline and metabolically active phosphatidic acid (PA) (Lennartz 1999). Pharmacological inhibition studies show that PLD participates in FCGR-mediated phagocytosis (Kusner et al. 1996). There is an increase in the activity of PLD following the activation of phagocytosis via FCGR (Kusner et al. 1999). Following activation of FCGR, PLD translocates to the plasma membrane at the phagocytic cup and generate PA. This PA can be converted to DAG through the action of phosphatidic acid phosphatase-1 (PAP-1). Thus activation of PLD may be an additional pathway leading to PKC activation. The two isoforms PLD1 and PLD2 are both shown to be essential for the formation of phagosome at different stages. PLD1 is localized on the endosomal/lysosomal compartment and PLD2 is localized at the plasma membrane. PLD2 may be linked to phagosome formation whereas PLD1 may be involved in the focal exocytosis at the plasma membrane and also in the maturation process (Carrotte et al. 2006).
ATP bound G-actin monomers are added to the fast growing barbed ends of both mother and daughter filaments and the polymerization of these filaments drives membrane protrusion. In the process of phagocytosis, pseudopodia extend around the antibody-bound particle to form the phagocytic cup. This elongation continues until the filament reaches steady state equilibrium with free G-actin monomers (Millard et al. 2004, Le Clainche et al. 2008).
Several members of phospholipase A (PLA) are involved in phagocytosis. Macrophages express three classes of PLA2: secreted Ca-dependent (sPLA2), cytosolic Ca-dependent (cPLA2) and cytosolic Ca-independent (iPLA2) of which iPLA2 is involved in FCGR mediated arachidonic acid (AA) production. Aggregation of FCGR triggers phosphorylation and membrane translocation of iPLA2. Protein kinase C (PKC), ERK and p38MAPK seems to regulate iPLA2 in monocytes, neutrophils and macrophages. Phosphorylated iPLA2 then mediates production of AA and lysoophospholipids from phosphatidylcholine. iPLA2 inhibitors (bromoenol lactone) block AA release and phagocytosis which can be restored upon addition of exogenous AA, suggesting a critical role for iPLA2 in FCGR phagocytosis (Lennartz et al. 1993, Tay & Melendez). Release of AA by activated iPLA2 changes the physical characteristic of the membrane which may facilitate pseudopod extension.
In addition to the membrane remodeling for pseudopod extension, particle internalization requires a contractility force pulling the forming phagosome into the cytoplasm. Myosin motor proteins are the actin-binding proteins, with ATPase activity move along actin fibers, and produce the driving force for phagosome formation and transport. Several myosin motors including myosins IC, II, V, IXb are involved in FCGR-mediated phagocytosis as force generators and actin-based transport motors (Swanson et al. 1999). Nonmuscle myosin II, is a motor protein known to generate intracellular contractile forces and tension by associating with F-actin. It has been observed to localize around forming phagosomes and suggested a role in phagocytic-cup squeezing during FCGR-mediated phagocytosis. Each myosin II motor protein exists as a complex consisting of two copies each of myosin II heavy chain (MHC), essential light chains (ELC), and myosin regulatory light chain (MRLC). Selective inhibition of myosin II by ML-7, a myosin light-chain kinase (MLCK) inhibitor, prevents phagocytic cup closure, but not pseudopod extension for the formation of phagocytic cups in FCGR-mediated phagocytosis (Grooves et al. 2008, Araki 2006). Tight ring of actin filaments within the elongating pseudopodia squeezes the deformable particles. In the classical zipper model for phagocytosis, the pseudopod extends over the IgG-coated particles, in which FCGRs in the phagocyte plasma membrane interact sequentially with Fc portions of IgG molecules zippering the membrane along the particle. This sequential IgG-FCGR binding might not occur by itself, but requires forced zipper closure, where myosin-II contractile activity may promote the binding between the FCGR and its ligands, to facilitate the efficient extension and subsequent closure of phagocytic cups (Araki 2006, ). Myosin IC mediates the purse-string-like contraction that closes phagosomes. Myosin-V has been implicated in membrane trafficking events (Swanson et al. 1999).
Abelson interactor-1 (ABL) tyrosine kinase phosphorylates the strictly conserved tyrosine 150 in WAVE2 (Y151 in WAVE1 and WAVE3) (Leng et al. 2003, Chen et al. 2010).
After incorporation at the branch, the actin bound to VCA domain of WASP/WAVE undergoes ATP hydrolysis and this destabilizes its interaction with WASP/WAVE. This dissociates the branched junction from the membrane-bound WASP/WAVE (Kovar 2006).
WASP interacting proteins (WIP) family includes WIPF1 (WIP), WIPF2 (WIRE,WICH) and WIPF3 (CR16, corticosteroids and regional expression-16). WIPs share a specific proline rich sequence that interacts with the WH1 domain of WASP and N-WASP (WASL). WIPs form heterocomplexes with WASPs and may contribute to the WASP protein stability (Aspenstrom 2002, Kato et al. 2002, Ho et al. 2001, Moreau et al. 2000). SH3 domain containing adaptor proteins like GRB2 (Carlier et al. 2000), NCK (Rohatgi et al. 2001) and WISH (DIP/SPIN90) (Fukuoka et al. 2001) bind to the proline rich domain in WASPs and activate the ARP2/3 complex. By binding simultaneously to N-WASP and the ARP2/3 complex, GRB2 works synergistically with CDC42 in the activation of ARP2/3 complex-mediated actin assembly (Carlier et al. 2000).
Macrophages lacking all the three isoforms of VAV did not affect FCGR-mediated phagocytosis suggesting that RAC1 is regulated by GEFs other than VAV downstream of the FCGR (Hall et al 2006). DOCK180, a member of GEFs, is found to be involved in the activation of RAC1. DOCK180 associates with the adaptor protein CRKII and the complex is found to accumulate at the phagocytic cup. DOCK180 is recruited to the sites of phagocytosis by binding to SH3 domain of CRKII through its proline-rich motif (Hasegawa et al 1996). CRKII is likely recruited to the activated FCGR complex by binding phosphorylated ITAM tyrosines on the receptor or through other phosphotyrosines on ancillary proteins that are recruited to the receptor complex (Lee et al 2007). Unlike the usual GEFs, DOCK180 does not contain the conserved Dbl homology (DH) domain. Instead, it has a DHR-2 or DOCKER domain capable of loading RAC with GTP (Brugnera et al 2002). Binding of DOCK180 to RAC alone is insufficient for GTP loading, and a DOCK180-ELMO interaction is required. ELMO1, as well as ELMO2, form a complex with DOCK180 and they function together as a bipartite GEF to optimally activate RAC (Gumienny et al 2001, Brugnera et al 2002).
WASP is phosphorylated on Tyr291 (Cory et al. 2002) and N-WASP (WASL) on Tyr256 (Wu et al. 2004) by Src family of tyrosine kinases and this phosphorylation may release the autoinhibitory intramolecular interactions. The phosphorylation seems to be enhanced by the activation of CDC42. WASP phosphorylation and binding of CDC42 have a synergistic effect on the activation of the ARP2/3 complex (Takenawa & Suetsugu 2007). In N-WASP, the phosphorylation may reduce its nuclear translocation and may sustain it in its functional site in the cytoplasm (Wu et al. 2004).
The EPHB2-FAK pathway partially promotes dendritic spine stability through LIMK-mediated cofilin (CFL1) phosphorylation (Shi et al. 2009). CFL1 is a member of the ADF (actin-depolymerizing factor) protein family that is involved in regulating actin dynamics in the growth cone. It binds to actin in a one-to-one molar ratio, and stimulates both the severing of actin filaments and depolymerization of actin subunits from the actin filament end. Activated LIMK phosphorylates CFL1 on the conserved serine 3 residue located near the actin-binding site. After phosphorylation, CFL1 is inactive, loses its affinity for actin and dissociates from G-actin monomers. Once freed, ADP-actin monomers can exchange ADP with cytoplasmic ATP, ready for reincorporation at the barbed end of a growing filament (Gungabissoon & Bamburg 2003).
Binding of Hsp90 to the LIMK proteins protects them from degradation and promotes their dimer formation and transphosphorylation. It is estimated that LIMK1 contains at least 5 phospho-amino acids primarily phospho-serines, in its kinase domain. The positions of these serine residues are not known. Transphosphorylation of these serine residues in LIMK1 increases its stability.
After phosphorylation on Thr 508, LIMK undergoes homodimerization. Homodimer formation is promoted by the binding of heat shock protein 90 (Hsp90) to a short sequence in the kinase domain of LIMKs. LIMKs are further phosphorylated after homodimer formation and transphosphorylation of the kinase domain.
Wiskott-Aldrich syndrome protein (WASP) and Neural-WASP (N-WASP, WASL) proteins are scaffolds that transduce signals from cell surface receptors to the activation of the ARP2/3 complex and actin polymerization. WASP and N-WASP possess a central GTPase binding domain (GBD) and an NH2-terminal WASP homology domain 1 (WH1) followed by a basic region (B), and a C-terminal VCA region that contains: a V domain (verprolin homology/WASP homology 2), a C domain (connecting), and an A motif (acidic). The VCA region is responsible for binding to and activating the ARP2/3 complex (Bompard & Caron 2004, Callebaut et al 1998). Under resting conditions, WASP and N-WASP are maintained in an autoinhibited state via interaction of the GBD and the VCA domains. This prevents access of the ARP2/3 complex and G-actin to the VCA region. Activated CDC42 binds to the GBD region of WASPs and this interaction releases the VCA region from autoinhibition, enabling binding of the ARP2/3 complex and stimulating actin polymerization (Kim et al 2000, Park & Cox 2009). Phosphoinositides (PtdIns(4,5)P2) interact with the basic (B) region in WASPs and this interaction is important for activation of the WASPs and the ARP2/3 complex (Higgs & Pollard 2000).
Once WASPs (WASP and N-WASP) and WAVEs (WAVE2 and probably WAVE1 and WAVE3) are activated, their VCA region becomes available for binding to the ARP2/3 complex and actin monomer (G-actin). The actin monomer binds to the V domain and ARP2/3 complex binds to the CA domain. The simultaneous binding of G-actin and the ARP2/3 complex to the VCA region contributes to the activation of the ARP2/3-complex-mediated actin polymerization. The VCA module acts as a platform on which an actin monomer binds to the ARP2/3 complex to trigger actin polymerization (Takenawa & Suetsugu 2007).
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WASP/N-WASP:ARP2/3
complex:G-actincomplex recruited
to phagocytic cupfilament:branching complex:daughter
filamentfilament:branching
complexAnnotated Interactions
WASP/N-WASP:ARP2/3
complex:G-actinWASP/N-WASP:ARP2/3
complex:G-actincomplex recruited
to phagocytic cupcomplex recruited
to phagocytic cupfilament:branching complex:daughter
filamentfilament:branching complex:daughter
filamentfilament:branching complex:daughter
filamentfilament:branching
complexfilament:branching
complexThe first step in Fc-gamma receptor (FCGR) phagocytosis is binding and clustering of FCGRs by IgG-coated foreign particles. FCGR are clustered at the cell surface by multivalent antigen-antibody complexes and recruited to lipid raft micro domains; monovalent ligand binding is insufficient to generate a signal. This cross-linking results in the localization of FCGRs into lipid rafts and this may aid in recruiting and complexing with additional signalling proteins associated with lipid rafts (Bournazos et al. 2009, Kwiatkowska & Sobota 2001, Kono et al. 2002). This is followed by phosphorylation of the tyrosine residues within the ITAM located on the cytoplasmic portion of FCGRIIA by membrane-associated tyrosine kinases of the Src family (Mitchell et al. 1994).
The first step in Fc-gamma receptor (FCGR) phagocytosis is binding and clustering of FCGRs by IgG-coated foreign particles. FCGR are clustered at the cell surface by multivalent antigen-antibody complexes and recruited to lipid raft micro domains; monovalent ligand binding is insufficient to generate a signal. This cross-linking results in the localization of FCGRs into lipid rafts and this may aid in recruiting and complexing with additional signalling proteins associated with lipid rafts (Bournazos et al. 2009, Kwiatkowska & Sobota 2001, Kono et al. 2002). This is followed by phosphorylation of the tyrosine residues with in the immuno tyrosine activation motif (ITAM) located on the cytoplasmic portion of accessory gamma/zeta chains by membrane-associated tyrosine kinases of the Src family (Duchemin et al. 1994, van Vugt et al. 1996).
PAK1 exists as homodimer in a trans-inhibited conformation. The kinase inhibitory (KI) domain of one PAK1 molecule binds to the C-terminal catalytic domain of the other and inhibits catalytic activity. GTPases RAC1/CDC42 bind the GBD domain of PAK1 thereby altering the conformation of the KI domain, relieving inhibition of its catalytic domain, and allowing PAK1 autophosphorylation that is required for full kinase activity (Parrini et al. 2002, Zhao & Manser 2005).
The first step in Fc-gamma receptor (FCGR) phagocytosis is binding and clustering of FCGRs by IgG-coated foreign particles. FCGR are clustered at the cell surface by multivalent antigen-antibody complexes and recruited to lipid raft micro domains; monovalent ligand binding is insufficient to generate a signal.
This cross linking results in the localisation of FCGRs into lipid rafts and this may aid in their recruiting and complexing with additional signalling proteins associated with lipid rafts (Kono et al. 2002). This is followed by phosphorylation of the tyrosine residues within the ITAM located on the cytoplasmic portion of accessory gamma/zeta chains by membrane associated tyrosine kinases of the Src family (Park et al. 1993).
The implication of Src kinases in phosphorylation was first supported by pharmacological findings that herbimycin A, a tyrosine kinase inhibitor relatively specific for Src-family kinases, potently suppressed Fc receptor mediated functions (Greenberg et al. 1993, Suzuki et al. 2000). However, their particular involvement in phagocytosis remains unclear, as targeted disruption of single or multiple Src family genes did not result in significant alterations in phagocytosis (Hunter et al. 1993, Fitzer Attas et al. 2000, Suzuki et al. 2000). HCK, FGR and LYN triple-deficient (-/-) macrophages have shown significant delays in FCGR mediated phagocytosis, but these deficiencies do not completly disrupt the process (Fitzer Attas et al. 2000).
Tyrosine residues Y288 and Y304 (Y282 and Y298 according to the literature reference, it is 6 residues shorter compared to uniprot entry due to an alternate initiation codon usage), within ITAM sequence in the cytoplasmic domain of FCGRIIA are the key target sites that are phosphorylated by Src family kinases (Mitchell et al, 1994). In case of FCGRIA and FCGRIIIA the specific tyrosine residues within ITAMs of the associated gamma/zeta chains are phosphorylated by activated Src family kinases (SFKs) (Park et al. 1993).
In barbed-end branching model, the branching/ternary complex (G-actin-WASP/WAVE-Arp2/3 complex) binds to the barbed end of the mother filament. G-actin bound to VCA domain or one of the Arp subunits incorporates into the mother filament at the barbed end, thus positioning ARP2/3 complex to initiate the daughter branch on the side of the mother filament. ARP2/3 nucleates the formation of new actin filament branches, which elongate at the barbed ends (Le Clainche & Carlier 2008, Pantaloni et al 2000, Le Clainche et al. 2003, Egile et al. 2005). In side branching model, the branching complex binds to the side of the mother actin filament mimicking an actin nucleus and initiates a lateral branch (Le Clainche & Carlier 2008, Amann & Pollard 2001).
The two isoforms PLD1 and PLD2 are both shown to be essential for the formation of phagosome at different stages. PLD1 is localized on the endosomal/lysosomal compartment and PLD2 is localized at the plasma membrane. PLD2 may be linked to phagosome formation whereas PLD1 may be involved in the focal exocytosis at the plasma membrane and also in the maturation process (Carrotte et al. 2006).
SH3 domain containing adaptor proteins like GRB2 (Carlier et al. 2000), NCK (Rohatgi et al. 2001) and WISH (DIP/SPIN90) (Fukuoka et al. 2001) bind to the proline rich domain in WASPs and activate the ARP2/3 complex. By binding simultaneously to N-WASP and the ARP2/3 complex, GRB2 works synergistically with CDC42 in the activation of ARP2/3 complex-mediated actin assembly (Carlier et al. 2000).