The adaptor protein Nck has been shown to link receptor ligation to actin-based signalling in a diverse range of cellular events, such as changes in cell morphology and motility. It has also been implicated in phagocytosis. However, its molecular role in controlling actin remodelling associated with phagocytic uptake remains to be clarified. Here, we show that Nck, which is recruited to phagocytic cups, is required for Fcγ receptor (FcγR)- but not complement receptor 3 (CR3)-induced phagocytosis. Nck recruitment in response to FcγR ligation is mediated by the phosphorylation of tyrosine 282 and 298 in the ITAM motif in the cytoplasmic tail of the receptor. In the absence of FcγR phosphorylation, there is also no recruitment of N-WASP or Cdc42 to phagocytic cups. Nck promotes FcγR-mediated phagocytosis by recruiting N-WASP to phagocytic cups. Efficient phagocytosis, however, only occurs, if the CRIB domain of N-WASP can also interact with Cdc42. Our observations demonstrate that Nck and Cdc42 collaborate to stimulate N-WASP-dependent FcγR-mediated phagocytosis.

Phagocytosis is a multi-step process, initiated by receptor–ligand interactions, that triggers a temporal recruitment of a signalling network that culminates in the local reorganisation of the actin cytoskeleton and the internalisation of a range of foreign particles and pathogens, as well as apoptotic and necrotic cells (Aderem and Underhill, 1999). Two of the best-characterised opsonic phagocytic receptors are the Fcγ receptor (FcγR) and the complement receptor 3 (CR3), which bind the Fc region of immunoglobulins and surface-deposited C3bi, respectively (Groves et al., 2008). Ultra-structural analysis has shown that during CR3-mediated phagocytosis, complement-opsonised particles ‘sink’ into cells with minimal membrane protrusions. In contrast, when the FcγR binds IgG-coated targets, engulfment is associated with extensive actin-rich membrane ruffles (Allen and Aderem, 1996). Consistent with this morphological distinction, there are key differences in the signalling networks downstream of the two receptors (Groves et al., 2008). The activities of Rac and Cdc42 are crucial for engulfment downstream of the FcγR, whereas RhoA is essential for CR3-mediated phagocytosis (Caron and Hall, 1998; Cox et al., 1997; Hoppe and Swanson, 2004; Massol et al., 1998; Park and Cox, 2009; Yamauchi et al., 2004).

During phagocytosis, the active GTP-bound Rho GTPases exert their effects on the actin cytoskeleton by binding several downstream effectors that ultimately lead to the activation of the actin filament nucleating capacity of the actin-related protein 2/3 complex (Arp2/3) (Groves et al., 2008). The stimulation of actin filament assembly downstream of Cdc42 is triggered by the interaction of the C-terminal WA domain of the haematopoietic cell-specific Wiskott–Aldrich syndrome protein (WASP) and the ubiquitously expressed neural WASP (N-WASP) with the Arp2/3 complex, stimulating its activation (Blanchoin et al., 2000; Goley and Welch, 2006; Machesky and Insall, 1998). Macrophages derived from patients with Wiskott–Aldrich syndrome, which lack functional WASP, are defective in uptake of IgG-opsonised particles, as they are impaired in actin cup formation (Lorenzi et al., 2000).

WASP proteins exist in the cell in an inactive auto-inhibitory conformation through intra-molecular interactions (Kim et al., 2000). WASP is converted from an auto-inhibited state into an active form by synergistically binding to GTP-bound Cdc42 and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] (Higgs and Pollard, 2000; Prehoda et al., 2000; Rohatgi et al., 2000). The local recruitment and activation of Cdc42 and the transient enrichment of PtdIns(4,5)P2 at the phagocytic cup after receptor ligation represents a potent activation signal for WASP (Botelho et al., 2000). WASP is also activated by phosphorylation during FcγR-mediated phagocytosis (Park and Cox, 2009). Phosphorylation of tyrosine 291 of WASP enhances the ability of WASP to promote Arp2/3-mediated actin polymerisation in vitro (Cory et al., 2002). Moreover, Cdc42 binding to WASP is thought to be necessary for the tyrosine phosphorylation of WASP both in vitro and in vivo (Cory et al., 2002; Park and Cox, 2009; Torres and Rosen, 2006).

WASP/N-WASP activation occurs downstream of FcγR ligation (Lorenzi et al., 2000; Park and Cox, 2009). Binding of IgG to the FcγR triggers clustering and subsequently, tyrosine kinase phosphorylation, a signalling event not triggered during CR3-mediated phagocytosis. Tyrosine residues 282 and 298, located in a conserved ITAM motif in the cytoplasmic domain of the receptor are phosphorylated by Src family kinases (Ghazizadeh et al., 1994; Greenberg et al., 1993; Greenberg et al., 1994). One of the unanswered questions in the field is how this earliest detectable signalling event brings about the activation of WASP. A potential link could be the SH2/SH3 adaptor protein Nck, which couples activated cell surface receptors to various intracellular effectors that regulate actin dynamics (Blasutig et al., 2008; Buday et al., 2002; Li et al., 2001). Indeed, artificial clustering of all three Nck SH3 domains at the plasma membrane is sufficient to induce localised actin polymerisation (Rivera et al., 2004). Nck exerts its effects on the actin cytoskeleton by activating the ability of WASP/N-WASP to stimulate Arp2/3-dependent actin polymerisation (Rohatgi et al., 2001; Tomasevic et al., 2007).

Previous studies have pointed towards a role for Nck as a regulator of FcγR-mediated phagocytosis (Coppolino et al., 2001; Izadi et al., 1998). Upon FcγRII crosslinking in myeloid cells, Nck becomes tyrosine phosphorylated by Src family kinases (Izadi et al., 1998). Nck is also complexed with WASP in RAW 264.7 macrophages after Fc receptor crosslinking (Coppolino et al., 2001). However, despite these data, the exact role of Nck during phagocytosis remains to be established. Given this, we set out to investigate the function of Nck in FcγR- and CR3-mediated phagocytosis.

Nck is required for FcγR- but not CR3-mediated phagocytosis

In order to extend earlier findings and define the role of Nck in FcγR- or CR3-dependent phagocytic cascades, we examined the distribution of Nck in macrophages after challenge with red blood cells (RBCs) coated with IgG or C3bi, respectively. FcγR-mediated uptake resulted in the recruitment of Nck to phagocytic cups (Fig. 1A,B). In contrast, engulfment of C3bi–RBC did not induce a similar enrichment of Nck at sites of particle attachment, although actin was still present (Fig. 1A,B).

Fig. 1.

Nck is required for FcγR- but not CR3-mediated phagocytosis. (A) Immunofluorescence images of J774A.1 macrophages phagocytosing IgG- or C3bi-opsonised RBCs (blue) reveals Nck (green) is recruited to F-actin-positive phagocytic cups (red) during FcγR- but not CR3-mediated uptake. Dashed boxes indicate enlarged areas (ZOOM). Scale bar: 10 µm. (B) Quantification of Nck recruitment to FcγR- or CR3-induced phagocytic cups. (C) Immunoblot analysis of Nck and actin expression in control (Mock and siCtrl) and Nck siRNA-transfected J774A.1 macrophages. (D) Quantification of the number of attached (Association index; Ai; black bars) and internalised (Phagocytic index; Pi; white bars) IgG- or C3bi-opsonised RBCs in control (Mock and siControl) and Nck siRNA-transfected J774A.1 macrophages. (E) Quantification of the number of attached (Ai; black bars) and internalised (Pi; white bars) IgG- or C3bi-opsonised RBCs in Nck+/+ and Nck−/− MEFs expressing FcγRIIA or CR3 together with Nck1–GFP. Data are means±s.e.m. (n = 3). *P<0.05, **P<0.01, ***P<0.001.

Fig. 1.

Nck is required for FcγR- but not CR3-mediated phagocytosis. (A) Immunofluorescence images of J774A.1 macrophages phagocytosing IgG- or C3bi-opsonised RBCs (blue) reveals Nck (green) is recruited to F-actin-positive phagocytic cups (red) during FcγR- but not CR3-mediated uptake. Dashed boxes indicate enlarged areas (ZOOM). Scale bar: 10 µm. (B) Quantification of Nck recruitment to FcγR- or CR3-induced phagocytic cups. (C) Immunoblot analysis of Nck and actin expression in control (Mock and siCtrl) and Nck siRNA-transfected J774A.1 macrophages. (D) Quantification of the number of attached (Association index; Ai; black bars) and internalised (Phagocytic index; Pi; white bars) IgG- or C3bi-opsonised RBCs in control (Mock and siControl) and Nck siRNA-transfected J774A.1 macrophages. (E) Quantification of the number of attached (Ai; black bars) and internalised (Pi; white bars) IgG- or C3bi-opsonised RBCs in Nck+/+ and Nck−/− MEFs expressing FcγRIIA or CR3 together with Nck1–GFP. Data are means±s.e.m. (n = 3). *P<0.05, **P<0.01, ***P<0.001.

To test whether Nck is required for FcγR-induced phagocytosis, we examined the consequences of its RNAi-mediated depletion. RT-PCR reveals J774A.1 macrophages express both Nck1 and Nck2 (data not shown). As the two Nck isoforms are functionally redundant (Lettau et al., 2009), we ablated Nck1 and Nck2 expression using two independent sets of siRNA duplexes. After 24 hours of siRNA treatment the levels of Nck1 and Nck2 were dramatically reduced by 77.0±11.25% (n = 3) resulting in an ∼50% reduction in the ability of J774A.1 macrophages to internalise IgG-opsonised RBCs as compared with the control (Fig. 1C,D). Importantly, the binding of RBCs to Nck-depleted macrophages was higher than with controls, indicating that its loss led to a defect in FcγR phagocytic signalling and not the ability to bind IgG–RBC (Fig. 1D). In contrast, J774A.1 macrophages with reduced Nck1/2 expression were found to be as competent as controls in binding and internalising C3bi–RBC (Fig. 1D).

To corroborate our RNAi data, we assessed the ability of Nck1/Nck2 double knockout (Nck−/−) and wild-type mouse embryonic fibroblasts (MEFs) expressing FcγR or CR3 to internalise RBCs. Although there was no difference in the ability of the different MEFs to bind RBCs, a distinction could be seen in the FcγR phagocytic capacity of Nck−/− cells. FcγR-dependent phagocytosis was reduced by 66% in the absence of Nck (Fig. 1E). This decrease in uptake could be rescued to near wild-type levels by expression of GFP–Nck1 (Fig. 1E). In contrast, the capacity to bind and engulf RBCs via CR3 was equivalent in the presence or absence of Nck. Furthermore, expression of GFP–Nck1 in Nck−/− cells did not change the levels of associated or CR3-internalised RBCs (Fig. 1E). Our data show that phagocytosis downstream of FcγR but not CR3 involves Nck.

Nck recruitment requires phosphorylation of tyrosine residues 282 and 298 in the FcγR ITAM motif

The phosphorylation of tyrosine residues 282 and 298 in the ITAM motif in the FcγR cytoplasmic tail is essential for actin polymerisation during phagocytosis (Greenberg et al., 1993). To investigate whether Nck recruitment depends on phosphorylation of these two residues, we examined the consequences of replacing tyrosine 282 and 298 with phenylalanine on FcγR-mediated phagocytosis. We found that replacement of these two tyrosine residues had no effect on IgG–RBC binding (Fig. 2A, black bars), indicating that they are not required for ligand binding, in line with previous results (Mitchell et al., 1994). In contrast, the phagocytic index of the single tyrosine mutants (Y282F and Y298F) was decreased to ∼40% of the level observed with the wild-type FcγR (Fig. 2A, white bars). The level of uptake was further reduced to ∼5% in cells expressing the Y282F/Y298F double mutant.

Fig. 2.

Nck recruitment is dependent on tyrosine phosphorylation of the FcγR ITAM motif. (A) Quantification of the ability of COS-7 cells expressing the indicated FcγR mutant to bind (Ai; black bars) and phagocytose (Pi; white bars) IgG-opsonised RBCs. (B) Immunofluorescence images showing the localisation of GFP-tagged Nck1 (green) in COS-7 cells expressing FcγR ITAM wild type and tyrosine mutants. Arrows and insets present examples of the typical cup morphology in all conditions. Scale bar: 10 µm. (C) Percentage of total RBCs demonstrating Nck enrichment as measured from 20 cells for each mutant and wild-type FcγR-expressing cell population, corresponding to ≥100 phagocytic cups analysed in three experiments. (D) Coomassie-stained gel from an in vitro peptide-binding assay using phosphorylated and non-phosphorylated peptides containing the FcγR ITAM motif tyrosine residues. The extracts containing His-tagged Nck1 or Nck2, and peptides, where P represents the phosphopeptide, are indicated. *P<0.05, **P<0.01, ***P<0.001.

Fig. 2.

Nck recruitment is dependent on tyrosine phosphorylation of the FcγR ITAM motif. (A) Quantification of the ability of COS-7 cells expressing the indicated FcγR mutant to bind (Ai; black bars) and phagocytose (Pi; white bars) IgG-opsonised RBCs. (B) Immunofluorescence images showing the localisation of GFP-tagged Nck1 (green) in COS-7 cells expressing FcγR ITAM wild type and tyrosine mutants. Arrows and insets present examples of the typical cup morphology in all conditions. Scale bar: 10 µm. (C) Percentage of total RBCs demonstrating Nck enrichment as measured from 20 cells for each mutant and wild-type FcγR-expressing cell population, corresponding to ≥100 phagocytic cups analysed in three experiments. (D) Coomassie-stained gel from an in vitro peptide-binding assay using phosphorylated and non-phosphorylated peptides containing the FcγR ITAM motif tyrosine residues. The extracts containing His-tagged Nck1 or Nck2, and peptides, where P represents the phosphopeptide, are indicated. *P<0.05, **P<0.01, ***P<0.001.

Concomitant with the loss of RBC uptake, there was also a corresponding reduction in the recruitment of GFP–Nck1 to FcγR-induced phagocytic cups (Fig. 2B,C). Mutation of the individual tyrosine residues resulted in a significant decrease in Nck1 recruitment to bound RBCs (wild type, 66±3%; Y282F, 30±2%; and Y298F, 13±2%). Moreover, when tyrosine 282 and 298 were replaced by phenylalanine, only 2% of phagocytic cups were GFP–Nck1 positive. We reasoned that, when phosphorylated, the tyrosine residues in the FcγR ITAM motifs might recruit Nck by interacting directly with its SH2 domain. To explore this possibility, we carried out in vitro peptide-binding assays on Nck1 and Nck2 produced in E. coli. We found that Nck1 and Nck2 were able to bind to tyrosine 282 and 298 when these residues were phosphorylated (Fig. 2D). The binding of Nck1 to the phosphorylated ITAM tyrosine residues is considerably weaker than that of Nck2, which also has a stronger preference for phospho-tyrosine 298 (Fig. 2D).

The recruitment of Cdc42 but not Rac1 to the FcγR is Nck-dependent

Activation of Cdc42 and Rac directs the actin restructuring at the phagocytic cup (Caron and Hall, 1998; Cougoule et al., 2006; Cox et al., 1997; Massol et al., 1998; Patel et al., 2002). To examine whether Nck is involved in recruiting these Rho GTPases, we challenged Nck+/+ and Nck−/− cells expressing GFP-tagged Cdc42 or Rac1 and the FcγR with IgG-opsonised RBCs. The accumulation of GFP–Cdc42 at phagocytic cups was reduced by 41% in Nck−/− as compared with Nck+/+ cells (Fig. 3A,B). However, this level of recruitment was similar to that with GFP alone (Fig. 3A,B). An absence of Nck did not result in a reduction in Rac1 recruitment to FcγR phagocytic cups (Fig. 3A,B). The presence of Rac would explain why there are no apparent differences in the number of phagocytic cups inducing actin polymerisation in the absence of Nck or Cdc42 (supplementary material Fig. S1). In agreement with this suggestion, dominant-negative Cdc42 or Rac reduce uptake but do not abrogate actin accumulation at phagocytic cups (Cougoule et al., 2006). Only when both Cdc42 and Rac signalling is inhibited is there no actin accumulation at sites of particle binding or uptake (Cougoule et al., 2006). A similar inhibition of phagocytosis occurs in the absence of FcγR phosphorylation because Cdc42, Rac, Nck and N-WASP are not recruited to phagocytic cups (Fig. 3C). The recruitment of Rac downstream of FcγR phosphorylation in the absence of Nck accounts for the phagocytotic uptake seen in Nck−/− cells (Fig. 1E).

Fig. 3.

Nck promotes recruitment of Cdc42 and N-WASP but not Rac1 to the FcγR. (A) Representative immunofluorescence images illustrating GFP–Cdc42 and GFP–Rac1 localisation in Nck+/+ and Nck−/− MEFs. Enrichment at sites of particle binding is indicated by arrows and enlargements in insets. Scale bar: 10 µm. (B) Recruitment of GFP-tagged proteins to phagocytic cups was quantified as the percentage of positive phagocytic cups. Data are means±s.e.m. (n = 3), each with ≥20 transfected cells analysed per experiment. (C) Percentage of total RBCs with GFP-tagged protein enrichment as measured from 20 cells for each wild-type (WT) FcγR and mutant FcγR Y282F/Y298F-expressing cell population, corresponding to ≥100 phagocytic cups analysed in three experiments. **P<0.01, ***P<0.001.

Fig. 3.

Nck promotes recruitment of Cdc42 and N-WASP but not Rac1 to the FcγR. (A) Representative immunofluorescence images illustrating GFP–Cdc42 and GFP–Rac1 localisation in Nck+/+ and Nck−/− MEFs. Enrichment at sites of particle binding is indicated by arrows and enlargements in insets. Scale bar: 10 µm. (B) Recruitment of GFP-tagged proteins to phagocytic cups was quantified as the percentage of positive phagocytic cups. Data are means±s.e.m. (n = 3), each with ≥20 transfected cells analysed per experiment. (C) Percentage of total RBCs with GFP-tagged protein enrichment as measured from 20 cells for each wild-type (WT) FcγR and mutant FcγR Y282F/Y298F-expressing cell population, corresponding to ≥100 phagocytic cups analysed in three experiments. **P<0.01, ***P<0.001.

Nck and Cdc42 collaborate to stimulate N-WASP-dependent FcγR-mediated phagocytosis

To examine whether Nck recruits N-WASP during engulfment, we co-expressed GFP–N-WASP and the FcγR in Nck+/+ and Nck−/− cells. We found that in the absence of Nck, GFP–N-WASP is recruited to phagocytic cups to a similar degree to that of GFP (Fig. 3B). This confirms that both FcγR phosphorylation and Nck are required to recruit N-WASP. They are also required to recruit Cdc42 (Fig. 3B,C). This immediately raises the question as to what is the role of Cdc42 during phagocytosis. Previous experiments using dominant-negative Cdc42 have revealed it is involved in FcγR-induced phagocytosis (Caron and Hall, 1998). Subsequent, RNAi-based approaches have shown that Cdc42 is required to activate N-WASP/WASP during FcγR-induced phagocytosis (Park and Cox, 2009). To extend these studies, we assessed the ability of Cdc42−/− and Cdc42fl/− fibroblastoid cell lines expressing FcγR to bind and internalise IgG-coated RBCs. As expected, Cdc42fl/− cells were competent in internalising RBCs (Fig. 4A). In contrast, cells lacking Cdc42 exhibited a marked decrease in their phagocytic potential (Fig. 4A). This level of uptake is similar to that seen in cells expressing dominant-negative Cdc42 (Caron and Hall, 1998) or after treatment with shRNA against Cdc42 (Park and Cox, 2009). Cells treated with siRNA against Nck1/2 and MEFs lacking Nck also had a similar phagocytic index (Fig. 1D,E). The level of GFP–Nck1 recruitment to phagocytic cups was, however, similar in the presence or absence of Cdc42 (Fig. 4B). This confirms that Nck is upstream of Cdc42 in the FcγR phagocytic signalling cascade (Fig. 3B). In contrast, the recruitment of GFP–N-WASP to phagocytic cups in Cdc42−/− cells was significantly lower than that of the wild-type cells (Fig. 4B). These data suggest that the localisation of N-WASP to the phagocytic cup is enhanced but not totally dependent on Cdc42. To confirm whether this is the case, we examined the accumulation of GFP-tagged Nck1, Cdc42 and N-WASP(H208D) to FcγR-induced phagocytic cups in the presence and absence of N-WASP (Fig. 4C,D). The N-WASP(H208D) is deficient in Cdc42 binding (Miki et al., 1998). As expected from the above observations, we found that the recruitment of GFP–Nck1 was not influenced by the presence or absence of N-WASP. In contrast, the recruitment of GFP–N-WASP(H208D) was significantly reduced in N-WASP−/− cells (Fig. 4C,D). This confirms that the recruitment of N-WASP to phagocytic cups is partially dependent on Cdc42. Consistent with this, we found that a loss in the ability of N-WASP to bind Cdc42 led to a similar reduction in the phagocytic index as the absence of the complete protein (Fig. 4E). Unexpectedly, GFP–Cdc42 enrichment at phagocytic cups was also significantly reduced in N-WASP−/− cells (Fig. 4C,D).

Fig. 4.

Nck and Cdc42 co-operate to recruit N-WASP to promote FcγR-mediated phagocytosis. (A) Quantification of the binding (Ai; black bars) and phagocytosis (Pi; white bars) of IgG-opsonised RBCs in Cdc42fl/− and Cdc42−/− cells, expressing FcγR+/− GFP–Cdc42. (B) Quantification of the recruitment of GFP-tagged Nck and N-WASP to phagocytic cups in Cdc42fl/− and Cdc42−/− cells. (C) Representative images showing the recruitment of Nck1, Cdc42 and N-WASP(H208D) (green) to RBCs (red) in N-WASP+/+ and N-WASP−/− cells. Scale bar: 10 µm. (D) Quantification of the number of phagocytic cups showing enrichment of GFP tagged protein. Data are means±s.e.m. (n = 3). (E) Quantification of the binding (Ai; black bars) and phagocytosis (Pi; white bars) of IgG-opsonised RBCs in N-WASP+/+ and N-WASP−/− MEFs expressing FcγR together with GFP, GFP–N-WASP or GFP–N-WASP(H208D). *P<0.05, **P<0.01, ***P<0.001.

Fig. 4.

Nck and Cdc42 co-operate to recruit N-WASP to promote FcγR-mediated phagocytosis. (A) Quantification of the binding (Ai; black bars) and phagocytosis (Pi; white bars) of IgG-opsonised RBCs in Cdc42fl/− and Cdc42−/− cells, expressing FcγR+/− GFP–Cdc42. (B) Quantification of the recruitment of GFP-tagged Nck and N-WASP to phagocytic cups in Cdc42fl/− and Cdc42−/− cells. (C) Representative images showing the recruitment of Nck1, Cdc42 and N-WASP(H208D) (green) to RBCs (red) in N-WASP+/+ and N-WASP−/− cells. Scale bar: 10 µm. (D) Quantification of the number of phagocytic cups showing enrichment of GFP tagged protein. Data are means±s.e.m. (n = 3). (E) Quantification of the binding (Ai; black bars) and phagocytosis (Pi; white bars) of IgG-opsonised RBCs in N-WASP+/+ and N-WASP−/− MEFs expressing FcγR together with GFP, GFP–N-WASP or GFP–N-WASP(H208D). *P<0.05, **P<0.01, ***P<0.001.

Taken together our observations clearly demonstrate that the combined action of Nck and Cdc42 are required to recruit and/or activate N-WASP to promote efficient FcγR-mediated phagocytosis (supplementary material Fig. S2). Nevertheless, it is clear that tyrosine phosphorylation of the FcγR, and the subsequent recruitment of Nck, are the most upstream events in the signalling network leading to Fc-mediated phagocytosis. We envisage that FcγR phosphorylation is the initial trigger for Nck-dependent recruitment and activation of N-WASP. Efficient phagocytosis, however, will only occur if the CRIB domain of N-WASP also interacts with Cdc42. We envisage that the role of Cdc42 during FcγR-mediated phagocytosis is largely one of stabilising rather than activating N-WASP during Arp2/3-mediated actin polymerisation.

DNA constructs and antibodies

Expression vectors for FcγRIIA and CD11b, CD18 (integrin chains of the CR3), GFP-tagged Rac1, Cdc42, Nck1 and N-WASP are as described previously (Caron and Hall, 1998; Moreau et al., 2000). Tyrosine 282 and 298 phenylalanine mutants of the FcγR are as described previously (Cougoule et al., 2006). GFP–N-WASP(H208D) (Lommel et al., 2001) was a kind gift of Jürgen Wehland (The Helmholtz Centre for Infection Research, Braunschweig, Germany). Rabbit antibodies against Nck (Upstate) and actin (Sigma) as well as mouse anti-Nck (BD Biosciences) and HRP-donkey anti-rabbit (Santa Cruz Biotechnology) antibodies were used.

Cell culture and transfection

COS-7 and J774A.1 cell lines were obtained from the ATCC. Murine embryonic fibroblast (MEF) cell lines Nck+/+ and Nck−/− (Bladt et al., 2003), N-WASP+/+ and N-WASP−/− (Snapper et al., 2001), and fibroblastoid cell lines Cdc42fl/− and Cdc42−/− (Czuchra et al., 2005) were kind gifts of Tony Pawson (Samuel Lunenfeld Research Institute, Toronto, Canada), Scott Snapper (Dept. of Medicine and Immunology, Massachusetts General Hospital, Boston, USA) and Cord Brakebusch (Biotech Research & Innovation Centre, University of Copenhagen, Denmark), respectively. COS-7, Nck+/+, Nck−/−, N-WASP+/+ and N-WASP−/− MEFs were transfected using Lipofectamine 2000 (Invitrogen). Cdc42fl/− and Cdc42−/− MEFs were transfected using the Amaxa system (Amaxa Inc. program A-023). Transfected cells were grown for 18–24 hours prior to experimentation to allow for expression of the corresponding proteins.

siRNA transfection, phagocytosis assays and analysis

Reduction of endogenous Nck expression was achieved using siRNA duplexes from Dharmacon RNA Technologies: siGENOME SMARTpool targeting mouse Nck1 (M-042187-00) and Nck2 (M-059051-01) or a non-targeting siRNA pool (siControl; D-001206-13-05). siRNA oligonucleotides were transfected into cells using Lipofectamine RNAiMAX (Invitrogen) in accordance with the manufacturer's instructions. Cells were grown for 24 hours prior to western blotting and phagocytic assays, which were performed as previously described (Caron and Hall, 1998). Immunofluorescence analysis and the quantification of RBC binding (Association Index; Ai) and internalisation (Phagocytic Index; Pi) were performed as described previously (Cougoule et al., 2006). Student's t-test was performed to determine statistical significance of the results. Data sets were considered different for P<0.05.

FcγR phosphopeptide pulldowns

Phosphorylated and non-phosphorylated peptides (TADGGYMTLNP and DDKNIYLTLPP), corresponding to 5 residues either side of the tyrosine residues 282 and 293 in the ITAM motifs of the FcγR, were coupled via an additional N-terminal CGG to SulfoLink resin (Pierce Biotechnology). Pulldown assays using these peptide-coupled resins were then performed with E.coli BL21(DE3) soluble extracts containing His-tagged Nck1 and 2 as described previously (Scaplehorn et al., 2002).

This manuscript is dedicated to the memory of Emmanuelle Caron who sadly passed away from cancer on 8 July 2009. She was an enthusiastic and inspiring supervisor with an unending passion for science who is greatly missed.

We thank Jürgen Wehland for providing GFP–N-WASP(H208D) as well as Tony Pawson, Scott Snapper and Cord Brakebusch for Nck, N-WASP and Cdc42 null cell lines, respectively. We also thank Gareth Jones and Claire Wells (King's College, London) and Jasmine Abella (London Research Institute, Cancer Research UK) for comments on the manuscript.

Funding

This work was supported by a Biotechnology and Biological Sciences Research Council studentship to A.E.D.; and a Cancer Research studentship to S.K.D.

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