Src phosphorylation of cortactin enhances actin assembly

Abstract

Src kinase mediates growth factor signaling and causes oncogenic transformation, which includes dramatic changes in the actin cytoskeleton, cell shape, and motility. Cortactin was discovered as a substrate for Src. How phosphorylation of cortactin can enhance actin assembly is unknown. Here, using an actin assembly system reconstituted from purified components, we demonstrate for the first time a biochemical mechanism by which Src phosphorylation of cortactin affects actin assembly. The adaptor Nck is an important component of the system, linking phosphorylated cortactin with neuronal WASp (N-WASp) and WASp-interacting protein (WIP) to activate Arp2/3 complex.

The actin cytoskeleton is necessary for cellular migration (1), endocytosis (2), and cancer cell invasion (3). Actin cytoskeletal reorganization downstream of cellular Src (c-Src) plays an important role in growth factor and integrin signaling. c-Src mediates EGF-induced cell migration, cell shape, and actin stress fiber rearrangement (4, 5). Platelet-derived growth factor receptor activation results in actin-rich, circular dorsal ruffle formation, also downstream of Src activity (6).

Activating mutations in c-Src or infections with the Src-encoding Rous sarcoma virus lead to oncogenic transformation accompanied by dramatic changes in the actin cytoskeleton (7). A key feature of Src-transformed fibroblasts is the formation of ventral actin-rich adhesive protrusions (8), known as invadopodia or podosomes. Invadopodia secrete proteases that degrade local extracellular matrix to facilitate invasion (9). Elevated c-Src activity increases invadopodia formation in carcinoma cells (10), and elevation in phosphotyrosine levels at invadopodia specifically correlate with increased proteolytic activity (11).

Invadopodia are composed of actin filaments and a variety of actin assembly regulators (10, 12, 13). Cortactin is required for the assembly of invadopodia in carcinoma cells (10), and it is important for invasion and cell motility in fibrosarcoma cells (14). In metastatic carcinoma cells, invadopodia formation also depends on the Arp2/3 complex, its activator neuronal WASp (N-WASp), and the adaptor protein Nck1 (15). WASp-interacting protein (WIP), an important binding partner of WASp family proteins, is also crucial for invadopodia formation (15).

Studies of dynamic membrane protrusions in a variety of cell types reveal a crucial role for N-WASp, Nck, cortactin, and WIP in Arp2/3 complex-based actin assembly. For example, in breast cancer cells, active N-WASp localizes to the actin nucleation zone of the dynamic leading edge (16), and knockdown of N-WASp or Nck2 leads to a decrease in actin polymerization at the leading edge (17). Mouse embryonic fibroblasts lacking Nck1/2 fail to form dorsal ruffles upon platelet-derived growth factor stimulation (18). In fibrosarcoma cells, cortactin knockdown leads to a defect in the persistence of lamellipodial protrusions (14), and in epithelial cells, the combined expression of cortactin and WIP promotes membrane protrusions (19).

Biochemical studies of Arp2/3-induced actin assembly reveal a number of functional and physical interactions among cortactin, Nck1, N-WASp, and WIP. Alone, cortactin promotes actin polymerization by simultaneously binding to Arp2/3 complex and F-actin, which has the dual effect of activating Arp2/3 complex for actin nucleation and stabilizing filament branches created by Arp2/3 complex (20). N-WASp is a potent activator of Arp2/3 complex, and its effect is synergistic with that of cortactin (21). In addition, cortactin and Nck bind directly to, and thereby activate, N-WASp (22, 23). Furthermore, WIP binds directly to cortactin, which enhances cortactin's ability to activate Arp2/3 complex (19). WIP's proline-rich region also binds Nck's SH3 domain (24, 25).

In cells, tyrosine phosphorylation of cortactin is essential for several actin-based processes. Cortactin is tyrosine-phosphorylated downstream of growth factor signaling and Src activity (26). In osteoclasts, tyrosine phosphorylation of cortactin is essential for actin-based podosome formation (27). In transmigrating leukocytes, cortactin phosphorylation downstream of Src is required for endothelial cell ICAM-1 clustering and actin remodeling (28). In human breast cancer cell lines, cortactin phosphorylation after EGF stimulation enhances cortactin binding to CD2AP, which may facilitate trafficking of endocytosed EGF receptors (29). Cortactin phosphorylation is necessary for optimal cadherin-mediated intercellular adhesion strength (30). Breast cancer cell lines expressing the cortactin mutant deficient in tyrosine phosphorylation induce 74% fewer osteolytic metastases as compared with cell lines expressing WT cortactin (31).

Here, we have sought a biochemical mechanism to account for the importance of cortactin phosphorylation downstream of Src in cells. Reconstituting actin assembly with a set of purified components, we find that the phosphorylation of cortactin by Src greatly enhanced Arp2/3 complex-mediated actin polymerization. This effect required Nck, and polymerization is enhanced by the addition of N-WASp and WIP. We explored the molecular interactions among the components with mutational analysis. Physical biochemical studies supported the functional assays, and cell biology studies supported the feasibility of this mechanism in cells.

Results

Our actin assembly reconstitution system included combinations of cortactin Nck1, N-WASp, and WIP in addition to Arp2/3 complex and actin. Supporting information (SI) Fig. 4A illustrates the domain structures of these proteins and their known interactions (19, 22, 24, 25, 32). We viewed the inclusion of WIP as potentially critical, because in cells, WASp family proteins are often in a complex with WIP (33–35), and the stability of WASp/N-WASp and WIP appears to depend on their association (36, 37). In addition, the majority of mutations in WASp that result in the Wiskott–Aldrich syndrome occur in the WIP-binding EVH1 domain of WASp (32). The adaptor protein Nck1 was included because its SH2 domain binds phosphocortactin strongly (38), in addition to the known links of Nck with actin assembly discussed in the Introduction.

To study the functional and physical interactions between cortactin, Nck1, N-WASp, and WIP in vitro, full-length recombinant proteins were expressed and purified from bacterial, insect, and human cells, as described in Materials and Methods. Protein purity was assessed by SDS/PAGE with silver staining (SI Fig. 4B). Recombinant N-WASp was the limiting reagent in most of our studies, because it was purified from human 293 cells.

Dissection of Cortactin Interaction with N-WASp-WIP.

We first considered the role of nonphosphorylated cortactin in actin assembly in the presence of N-WASp, WIP, and Nck1 (SI Fig. 5A). The assay included real-time observation of actin polymerization based on the fluorescence of pyrene-labeled actin. Here, the degree of Arp2/3 complex activation is proportional to the rate of actin polymerization, and the number of actin filament barbed ends nucleated by Arp2/3 complex can be calculated from the slope of the fluorescence versus time plot (39). The addition of cortactin to the combination of N-WASp and WIP produced high levels of activation of Arp2/3 complex, resulting in 5-fold higher barbed end generation (SI Fig. 5B, lane 8 versus lane 12; SI Fig. 5C).

We then asked which domains of cortactin were important for its effect on N-WASp and WIP. The W525K mutant form of cortactin, with an inactive SH3 domain (19), had no effect when added to the N-WASp-WIP complex (SI Fig. 5D, lane 4 versus lane 7). In contrast, the W22A cortactin mutant, which cannot bind Arp2/3 complex (21), was nearly as effective as WT cortactin (SI Fig. 5D, lane 5 versus lane 6). Thus, cortactin appears to interact with N-WASp/WIP via its SH3 domain, and the acidic region of N-WASp is the likely activator of Arp2/3 complex.

We asked how the combination of N-WASp-WIP and cortactin would be affected by the addition of Nck1. Nck1 addition to N-WASp, WIP, and cortactin increased Arp2/3 complex activation (SI Fig. 5E, lane 8 versus 9). This was not observed when W22A or W525K cortactin was substituted for WT cortactin, in combination with N-WASp, WIP, and Nck1 (SI Fig. 5E, lane 9 versus lanes 10 and 11), suggesting an important role for cortactin in binding Arp2/3 complex, N-WASp, and WIP in the presence of Nck1.

Levels of Activation of N-WASp-WIP.

Because N-WASp was the likely activator of Arp2/3 complex in our assays, we wanted to compare the level of N-WASp activation here with other activators of N-WASp observed in previous studies. N-WASp is autoinhibited, and in previous studies, the combination of PIP₂ and active Cdc42 provided high levels of activation of N-WASp (23). Nck1 alone has also been found to activate N-WASp (25). We compared the level of Nck1 activation of N-WASp with that of the combination of PIP₂ and active Cdc42 (SI Fig. 6A). Nck1 activated N-WASp to a higher level than did PIP₂ and Cdc42 (SI Fig. 6B, lanes 3 and 4 versus lane 5; SI Fig. 6C). We asked whether addition of WIP would lead to a further increase in Arp2/3 complex activity. Indeed, the combination of Nck1, N-WASp, and WIP was a more potent activator of Arp2/3 complex than Nck1 and N-WASp (SI Fig. 6D, lane 4 versus lane 5). The combination of Nck1 and WIP, without N-WASp, had similar Arp2/3 complex activity to that of the negative control (unpublished data).

Src-Phosphorylated Cortactin Activates Actin Polymerization in the Presence of Nck1.

We hypothesized that Nck1 may act as an adaptor connecting phosphocortactin with N-WASp or WIP. To test this hypothesis, we included phosphorylation of cortactin as a variable in the reconstitution system. Recombinant cortactin was phosphorylated in vitro with recombinant Src. Immunoblots with a general antiphosphotyrosine antibody and with two antibodies specific for cortactin phosphotyrosines documented that specific tyrosine phosphorylation did occur (SI Fig. 4C). We carried out additional kinase assays, using adenosine 5′-triphosphate [γ-³²P] to assess the stoichiometry of cortactin phosphorylation by Src kinase. Stoichiometric calculations revealed a 2:1 ratio of incorporated [γ-³²P] ATP:cortactin. Assuming that all three of cortactin's tyrosines targeted by Src are phosphorylated, then ≈2/3 of cortactin was fully phosphorylated under these reaction conditions.

We asked how combinations of phosphocortactin, Nck1, N-WASp, and WIP would affect Arp2/3-mediated actin polymerization (Fig. 1A). First, we found that the combination of Src-phosphorylated cortactin, Nck1, N-WASp, and WIP activated Arp2/3 complex better than did the same combination without phosphorylation of cortactin (Fig. 1B, lane 6 versus 7; SI Fig. 7B, curve 6 versus 7). The concentration of barbed ends created was ≈45% higher with cortactin phosphorylation. In addition, the combination of the four proteins produced significantly more Arp2/3 complex activation than any combination of three proteins (Fig. 1B, lane 7 versus lanes 1–6; SI Fig. 7B, curve 7 versus curves 1–6).

Fig. 1.

Cortactin phosphorylation enhances Arp2/3 complex activation and actin filament barbed end generation. (A) Schematic of interactions between phosphocortactin, Nck, N-WASp, and WIP. Point mutations in cortactin's acidic DDW (W22A) and SH3 (W525K) regions disrupt Arp2/3 and N-WASp or WIP binding, respectively. (B) Concentration of actin barbed ends generated by activated Arp2/3 complex. Phosphocortactin, Nck1, N-WASp, and WIP strongly activate Arp2/3 complex in a Src-dependent manner. (C) The function of phosphocortactin's acidic DDW region is dispensable for Arp2/3 activation in combination with Nck1 and N-WASp but necessary for Arp2/3 complex activation in combination with Nck1 and WIP. (D) Phosphocortactin's SH3 domain is dispensable for Arp2/3 activation in combinations of Nck1 with either N-WASp or WIP.

To dissect the roles of the multiple potential interactions among the proteins, we next examined the effects of Src-phosphorylation of cortactin in the presence of Nck1 and either N-WASp or WIP. Phosphorylation of cortactin by Src enhanced actin polymerization in the presence of Nck1 and N-WASp, as indicated by faster actin polymerization and up to a 3-fold increase in barbed end generation (Fig. 1C, lane 7 versus lane 8; SI Fig. 7C, curve 6 versus curve 8). An enhancement of actin polymerization was also observed upon Src phosphorylation of cortactin in combination with Nck1 and WIP, without N-WASp (Fig. 1C, lane 2 versus lane 3; SI Fig. 7C, curve 2 versus curve 4). Thus, cortactin phosphorylation by Src had a strong and positive effect on Arp2/3 complex activation in the presence of Nck1 and either N-WASp or WIP.

Cortactin can bind to Arp2/3 complex via an N-terminal acidic region with a DDW motif (20). To test the importance of this interaction, we used the W22A mutant form of cortactin, which is unable to bind Arp2/3 complex (21). Phosphorylated W22A cortactin activated actin polymerization as well as phospho-WT cortactin did, in combination with Nck1 and N-WASp, (Fig. 1C, lane 8 versus lane 10; SI Fig. 7C, curve 7 versus curve 8). Thus, phosphocortactin's acidic DDW region is not necessary for Arp2/3 complex activation in the presence of Nck1 and N-WASp, suggesting that the C-terminal acidic region of N-WASp plays this role. In addition, the effects of phosphocortactin on actin polymerization in the presence of Nck1 and N-WASp are likely mediated through cortactin domains other than the acidic DDW region.

To test this idea further, we replaced N-WASp with WIP in the assay, keeping phosphocortactin and Nck1. In this context, phosphocortactin's acidic DDW region is the only potential activator of Arp2/3 complex, so one might expect very little actin polymerization upon substituting W22A cortactin for WT cortactin in the assay. Indeed, the W22A mutation essentially eliminated Arp2/3 complex activation in this setting (Fig. 1C, lane 3 versus lane 5; SI Fig. 7C, curve 3 versus curve 4). Thus, the acidic DDW region of phosphocortactin was critical for Arp2/3 activation in the absence of N-WASp and the presence of Nck1 and WIP.

Cortactin's SH3 domain is able to bind proline-rich regions of N-WASp or WIP (19, 22). To determine the importance of these potential interactions in the reconstitution, we tested the W525K mutant form of cortactin, which is unable to bind proline-rich sequences (19). Phosphorylated W525K cortactin enhanced actin polymerization as efficiently as phospho-WT cortactin did, in the presence of Nck1 and N-WASp (Fig. 1D, lane 7 versus lane 9; SI Fig. 7D, curve 8 versus curve 9). Next, we substituted WIP for N-WASp in an assay with the same design. Again, the W525K mutant form of phosphocortactin induced actin polymerization as well as did phospho-WT cortactin (Fig. 1D, lane 3 versus lane 5; SI Fig. 7D, curve 4 versus curve 5). Thus, phosphocortactin's SH3 domain function is dispensable in the presence of Nck1 and either N-WASp or WIP.

Effects of Src Phosphorylation on Cortactin Are Mediated by Tyrosines 421, 466, and 482 of Cortactin.

In cells, cortactin is phosphorylated on tyrosines 421, 466, and 482 downstream of Src activity (40) and Nck1's SH2 domain can bind phosphocortactin (38). We asked whether the enhanced actin polymerization observed with phosphocortactin, Nck1 and N-WASp was mediated by these three tyrosines of cortactin. To answer this question, we used a mutant form of cortactin with Tyr 421, 466, and 482 changed to Phe (3YF) (SI Fig. 8A), which cannot be phosphorylated by Src (40). Src had no effect on the 3YF cortactin mutant in the presence of Nck1 and N-WASp, and the activity of this combination was similar to that of WT cortactin, Nck1, and N-WASp without Src (Fig. 2A, lane 7 versus lane 10; SI Fig. 8B, curve 5 versus curve 6). In addition, Src had no effect on the basal activity of Nck1 and N-WASp in the presence of 3YF cortactin (Fig. 2A, lane 9 versus lane 10; SI Fig. 8B, curve 4 versus curve 5).

Fig. 2.

The effects of Src phosphorylation on cortactin depend on tyrosines 421, 466, and 482. (A) Cortactin phosphorylated in vitro, using recombinant Src forms a strong Arp2/3 activator with Nck1 and N-WASp, which depends on cortactin's tyrosines 421, 466, and 482. (B) Cortactin phosphorylated in vitro strongly activates Arp2/3 complex with Nck1 and WIP. The effects of Src phosphorylation on cortactin require tyrosines 421, 466, and 482.

We asked whether cortactin's tyrosines 421, 466, and 482 also mediated enhanced Arp2/3 activation by phosphocortactin, Nck1, and WIP (SI Fig. 8C). Src had no effect on the 3YF cortactin mutant in the presence of Nck1 and WIP, and the activity of this combination was similar to that of WT cortactin, Nck1, and WIP in the absence of Src (Fig. 2B, lane 2 versus lane 5; SI Fig. 8D curve 3 versus curve 4).

Src Phosphorylation of Cortactin Augments Cortactin Binding to Nck1 and WIP.

Next, we investigated the physical basis of how Src phosphorylation of cortactin enhances Arp2/3 activation. In particular, we asked whether phosphorylation increased the amounts of complexes of cortactin, Nck1, and WIP that can form. We tested physical binding and complex formation with surface plasmon resonance (SPR). Nonphosphorylated cortactin was coupled to flow cell A on an SPR chip, and Src-phosphorylated cortactin was coupled to flow cell B. Various analytes were then flowed over the two cells of the chip concurrently. Nck1 bound to phosphocortactin but not cortactin (Fig. 3), consistent with the functional assay results. Conversely, WIP bound cortactin and phosphocortactin equally well. This result was also expected because cortactin's SH3 domain is responsible for binding WIP.

Fig. 3.

Physical binding of phosphocortactin, Nck1, and WIP. (A) SPR sensograms of Nck1 and WIP binding to cortactin and phosphocortactin. Cortactin and phosphocortactin were coupled to different flow cells of one SPR chip. Nck1 and/or WIP were flowed in as analytes. After each association and dissociation cycle, the chip was regenerated (arrows). Vertical lines in sensograms indicate the change from analyte to buffer. The arrowhead indicates the calculated value of the sum of the response units for Nck1 alone and WIP alone, binding to phosphocortactin. The observed value was consistently higher.

We asked whether phosphorylated cortactin could bind WIP and Nck1 simultaneously. Because WIP and Nck1 can also interact with each other, we reasoned that their binding to phosphocortactin might be enhanced. Flowing WIP and Nck1 onto the phosphocortactin flow cell together led to SPR response unit levels greater than the sum of the levels for Nck1 alone and WIP alone (Fig. 3), suggesting enhancement in the binding interactions.

In Vivo Expression, Phosphorylation, and Localization of Cortactin.

We asked whether cortactin, Nck, N-WASp, and WIP are coexpressed and colocalized in a primary cell type. Osteoclasts were examined because Src and cortactin phosphorylation are critical for cellular function (41). RT-PCR with total RNA from cultured primary osteoclasts revealed that cortactin, Nck1/2, WIP, and N-WASp were expressed (SI Fig. 9A). Immunofluorescence staining of osteoclasts revealed colocalization of cortactin, Nck, N-WASp, and WIP to podosomes (SI Fig. 9B), the dynamic actin-rich structures that resemble invadopodia of cancer cells (42, 43).

Next, we asked whether cortactin is phosphorylated on tyrosines in osteoclasts. The three tyrosines known to be phosphorylated downstream of Src in other studies are residues 421, 466, and 482 of mouse cortactin (5). Polyclonal antibodies specific for the phosphotyrosine form of two of these residues have been characterized (40). We found that these antibodies recognized endogenous phosphocortactin in osteoclast cell lysates by immunoblot, as did a general anti-phosphotyrosine antibody (SI Fig. 9C).

Discussion

The effects of Src activity, as part of oncogenic transformation and growth factor signaling, include dramatic increases in dynamic actin assembly. Cortactin is tyrosine-phosphorylated downstream of Src in growth factor signaling (40), transformation (11), and pathogen invasion (44). A key challenge for understanding the biology of Src has been to uncover a biochemical mechanism that might account for the importance of cortactin's tyrosine phosphorylation downstream of Src. Here, we have discovered a biochemical mechanism that is able to account for Src's activation of actin assembly by phosphorylation of cortactin. In reconstituted actin assembly assays, cortactin phosphorylation by Src enhanced Arp2/3-mediated actin polymerization in the presence of Nck1, N-WASp, and WIP.

Cortactin is a multidomain protein with several potential roles in linking signaling molecules with activators of actin assembly. We investigated the functional significance of cortactin's acidic DDW region, which can bind Arp2/3 complex (21), and cortactin's SH3 domain, which can bind N-WASp and WIP, among several proteins (19, 22). We used full-length cortactin containing single amino acid changes, known to have specific defects in these interactions. Both the acidic DDW region and the SH3 domain were important or essential in certain settings but completely dispensable in others (summarized in SI Table 1).

One can generalize the results as follows. For the acidic DDW region, when cortactin was the only Arp2/3 complex activator present (i.e., in the absence of N-WASp), the DDW motif was absolutely necessary for Arp2/3 activation of actin polymerization (SI Table 1, lines 1, 2, 4, and 5). The DDW motif was important for Arp2/3 complex activation when WIP was present, either in the presence or absence of N-WASp (SI Table 1, lines 2, 5, 7, and 8). At the other extreme, the DDW motif was dispensable when WIP was absent and N-WASp was present (SI Table 1, lines 3 and 6).

The SH3 domain was important for optimal Arp2/3 complex activation when Nck1 was absent and N-WASp and WIP were present alone or together (SI Table 1, lines 10, 11, and 15). This was also the case when Nck1 was present along with both N-WASp and WIP (SI Table 1, line 16). On the other hand, when Nck1 was present with either N-WASp alone or WIP alone, the cortactin SH3 domain function was largely dispensable (SI Table 1, lines 13 and 14). Considering these results together and in combination with the Src phosphorylation results, one can see that various combinations of domain interactions linking these membrane adaptors and regulators in different ways may suffice to activate Arp2/3 complex.

In addition to having a positive influence on actin polymerization, cortactin phosphorylation downstream of Src may enhance cortactin's ability to act as an adaptor or negatively regulate cortactin function. In cells, cortactin's ability to bind proline-rich binding partners (including CD2AP and myosin light chain kinase) is enhanced upon phosphorylation of cortactin (29, 45). Biochemically, cortactin's ability to activate N-WASp in vitro is enhanced by Erk phosphorylation of Ser 405 and 418 of cortactin (46). However, the effects of Erk phosphorylation on cortactin are negatively regulated by Src phosphorylation of cortactin (46). The difference between the Erk-Src phosphorylation data and our data may be explained by the absence and presence of Nck, respectively.

Our results provide a biochemical model that may help to explain these and other cases of actin polymerization at membranes. One possible scenario is that Nck, N-WASp, WIP, and cortactin are recruited to a membrane site in response to Src activation, creating a highly potent activator for Arp2/3 complex. Certain subsets of the four proteins may also be sufficient, and the composition of the activating complex may change over time. As actin polymerization proceeds at the membrane, cortactin may stabilize filament branch points and move away from the membrane with the growing actin filament network, whereas N-WASp, WIP, and Nck remain at the membrane.

Materials and Methods

Protein Purification.

Arp2/3 complex was purified from bovine thymus as described in ref. 39. Actin was purified from porcine muscle and gel-filtered as described in ref. 47. Human Cdc42 cDNA [American Type Culture Collection] was used as a template for QuikChange site-directed mutagenesis (Stratagene, La Jolla, CA) to mutate G12 to V. The insert was cloned into pDEST15 vector (Invitrogen, Carlsbad, CA). WT full-length human Nck1 was also cloned into pDEST15. GST-tagged VCA, Cdc42, Nck1, and cortactin were expressed in BL21star E. coli (Invitrogen) and purified by using glutathione-Sepharose 4B beads (Amersham Biosciences, Piscataway, NJ). GST-cortactin was further purified as described (48), including a TEV protease (Invitrogen) cleavage step to remove the GST tag. GST-viral cap antigen was further purified as described in ref. 49.

Recombinant WIP was purified as described in ref. 50, with substitution of a MonoS column for the gel filtration column. Recombinant TAP-tagged N-WASp was produced by using the Freestyle 293 expression system (Invitrogen) according to manufacturer's instructions. Briefly, suspension Freestyle 293-FS cells were grown in defined serum-free media in shaker flasks at 37°C with 8% CO₂. Cells were transfected on a 1.2-liter scale with 1.2 mg of DNA and 1.6 ml of 293Fectin at a cell density of 1.1 × 10⁶/ml. Forty-eight hours later, cells were pelleted by centrifugation at 1,000 × g for 15 min and frozen on liquid nitrogen. Later, the cells were lysed on ice for 30 min with lysis buffer (20 mM Tris, pH 8.0/1 mM EDTA/1 mM EGTA/10% glycerol/150 mM NaCl/1% Nonident P-40/0.125% deoxycholate/1 mM PMSF/1 mM sodium orthovanadate/1 mM NaF/20 mM β-glycerophosphate). Lysates were centrifuged at 163,000 × g for 13 min at 4°C, and TAP-N-WASP was purified from the supernatant as described in ref. 51. A silver staining kit (Amersham Biosciences) was used according to the manufacturer's protocol for assessment of protein purity.

In Vitro Phosphorylation of Cortactin.

Recombinant human Src expressed and purified from Sf9 cells was purchased from Upstate Biotechnology (Lake Placid, NY) and used for in vitro phosphorylation of cortactin. Cortactin (2 μM) was incubated at room temperature for 1 h with 75 units of Src in 200 μl of reaction buffer. The reaction buffer included 100 mM Tris·HCl pH 7.2, 125 mM MgCl₂, 2 mM EGTA, 2.5 mM ATP, 0.25 mM sodium orthovanadate, 1 mM NaF, 2 mM DTT, and protease inhibitors (1 μg/ml pepstatin A/0.1 mM PMSF/10 μM leupeptin). For experiments that included nonphosphorylated cortactin, mock reactions were set up as above but without the addition of recombinant Src. To assess the stoichiometry of cortactin phosphorylation by Src, the above kinase reaction was carried out with the addition of 10 μCi (1 Ci = 37 GBq) of adenosine 5′-triphosphate [γ-³²P] (Perkin–Elmer, Downers Grove, IL) followed by gel electrophoresis. The radioactively labeled cortactin band was excised, and the amount of radioactivity was assessed by a scintillation counter.

Pyrene-Labeled Actin Assembly Assay.

Pyrene-actin polymerization assays were performed as described in ref. 19. All actin polymerization assays included 2.5 μM actin and 100 nM Arp2/3, and various assays included 0.1 nM N-WASp, 50 nM WIP, 200 nM Nck1, 100 nM cortactin, 10 μM PIP₂ in PC/PS vesicles, and 500 nM Cdc42 (unless otherwise stated). GST-viral cap antigen (250 nM) was included as a positive control for maximal Arp2/3 activation. Actin filament barbed end calculations were performed as described (39). Standard errors of the mean (SEM) were calculated from standard deviations and sample numbers (3 or more) in each condition. The quantity of recombinant N-WASp available was a limiting factor in assays with N-WASp. The average barbed end concentration generated in assays using the positive control for Arp2/3 complex (GST-viral cap antigen) was 28.7 ± 4.8 nM (n = 8; standard error of the mean).

SPR Analysis.

Solutions of cortactin and phosphocortactin (30 μg/ml) in immobilization buffer (10 mM sodium acetate pH 5.0) were used to couple ≈2,000 response units to CM5 SPR chips (BIAcore, Uppsala, Sweden) via amine coupling. The amine coupling kit (BIAcore) was used according to the manufacturer's instructions. The polymerization buffer used in actin assembly assays served as the buffer for carrying analyte and washing. The chips were regenerated after each experiment with 10 mM glycine·HCl, pH 2.2 at 20 μl/min for 1 min.

Abbreviations

c-Src

cellular Src

SPR

surface plasmon resonance.