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Cell movement begins with a leading edge protrusion, which is stabilized by nascent adhesions and retracted by mature adhesions. The ERK-MAPK (extracellular signal regulated kinasemitogen-activated protein kinase) localizes to protrusions and adhesions, but how it regulates motility is not understood. We demonstrate ERK controls protrusion initiation and protrusion speed. Lamellipodial protrusions are generated via the WRC (WAVE2 Regulatory Complex), which activates the Arp2/3 actin nucleator for actin assembly. The WRC must be phosphorylated to be activated, but the sites and kinases that regulate its intermolecular changes and membrane recruitment are unknown. We show ERK co-localizes with the WRC at lamellipodial leading edges and directly phosphorylates two WRC components: WAVE2 and Abi1. The phosphorylations are required for functional WRC interaction with Arp2/3 and actin during cell protrusion. Thus, ERK coordinates adhesion disassembly with WRC activation and actin polymerization to promote productive leading edge advancement during cell migration.
Cells move via cycles of leading edge protrusion and adhesion to the ECM (extracellular matrix), cell body translocation and posterior retraction. The Arp2/3 complex drives leading edge protrusion by nucleating actin monomers to assemble a dense meshwork of short branched filaments called the lamellipodium (Insall and Machesky, 2009; Pollard and Cooper, 2009). As the lamellipodium protrudes, small dynamic nascent adhesions continuously form within the branched actin network (Choi et al., 2008). These adhesions are multi-protein complexes that include Paxillin and FAK, which regulate ECM coupling to actin (Vicente-Manzanares et al., 2009). A looser network of long unbranched actin filaments, the lamella, abuts the lamellipodium (Ponti et al., 2004). At the lamellipodium/lamella interface, older adhesions either disassemble or mature into larger, elongated adhesions that provide traction force (Choi et al., 2008). Myosin II, which can be activated by Myosin Light Chain Kinase (MLCK), couples to older adhesions and aids their maturation. Myosin II also functions in mature adhesions to regulate lamella retraction and in the cell cortex to regulate cell body translocation (Vicente-Manzanares et al., 2009). In order to achieve productive forward movement, protrusions must form, adhesions assemble and disassemble, and the cell body must move in a very synchronized manner. Along these lines, the rates of nascent adhesion turnover and lamellipodium advancement have been found to directly correlate (Choi et al., 2008).
How the cytoskeleton and adhesion machinery are regulated in order to achieve such coordination is just beginning to be understood. In the lamellipodium, Arp2/3 activity is triggered by its binding to the VCA (verprolin-homology, cofilin-homology and acidic) region of WAVE2 (WASp and verprolin homologous protein 2). WAVE2 functions in a stable WRC that contains HSPC300, Abi (Abl-interactor) 1 or 2, Nap1 (NCK-associated protein), and Sra-1 (specifically Rac1-associated protein) (Insall and Machesky, 2009; Takenawa and Suetsugu, 2007). These WRC components are required for each other's stability and leading edge localization (Takenawa and Suetsugu, 2007). Advances in purifying and assaying the WRC have led to the conclusion that it is inactive towards Arp2/3, until phospholipids bind WAVE2 and the activated form of Rac (Rac-GTP) binds Sra-1 (Derivery et al., 2009; Ismail et al., 2009; Lebensohn and Kirschner, 2009). These regulators recruit and cluster the WRC at the plasma membrane (Padrick and Rosen, 2010). A recent WAVE1-containing WRC crystal structure revealed Rac-GTP binding additionally allosterically activates the WRC by inducing a conformational change that releases intermolecular VCA sequestration (Chen et al., 2010). WRC phosphorylation is also required for activation (Chen et al., 2010; Lebensohn and Kirschner, 2009). Abl phosphorylation of WAVE2 Tyr150 participates in WRC activation by destabilizing the inhibited WRC conformation and releasing the VCA domain (Chen et al., 2010; Leng et al., 2005; Stuart et al., 2006). The involvement of other phosphosphorylations is likely, but the phosphorylation sites and their activating kinases are unknown (Lebensohn and Kirschner, 2009).
The ERK-MAPK pathway is one of the principal signaling cascades by which cells respond to extracellular and intracellular cues. Growth factors activate ERK-MAPK by signaling through their cognate receptors at the cell surface to the small GTPase Ras. Ras recruits the MAP kinase kinase kinase Raf, which phosphorylates and activates the MAP kinase kinase MEK, which then phosphorylates and activates ERK (McKay and Morrison, 2007; Pullikuth and Catling, 2007). Depending on the cell type and stimulus, Ras can also activate PI(3)K (phosphatidylinositol 3-kinase), which functions with its downstream effector kinase Akt to regulate migration speed and directionality (Kolsch et al., 2008). ERK activity can also be induced via ECM signaling during adhesion. In this pathway, the small GTPases Rac and Cdc42 activate PAK (p21-activated kinase), which phosphorylates MEK to make it a more efficient Raf substrate (Coles and Shaw, 2002; Eblen et al., 2002; Slack-Davis et al., 2003).
ERK signaling is required for cell motility in diverse systems, yet its chief function during motility is unknown. ERK regulates the disassembly of older adhesions via its phosphorylation and activation of FAK (focal adhesion kinase), Paxillin and MLCK (Ishibe et al., 2003; Klemke et al., 1997; Liu et al., 2002; Vomastek et al., 2007; Webb et al., 2004). ERK has also been found to be required for membrane ruffling and extension (Brahmbhatt and Klemke, 2003; Scott et al., 2006). Thus, we hypothesized ERK regulates the formation of lamellipodial protrusions and thereby links protrusion with adhesion turnover. We found ERK specifically regulates protrusion initiation, and protrusion and migration speed. ERK co-localizes with and directly phosphorylates the WRC on WAVE2 and Abi1. These phosphorylations promote functional WRC binding to Arp2/3 and actin and are required for lamellipodial protrusion. Thus, ERK regulates WRC activation to promote actin polymerization for rapid edge advancement during cell motility.
We determined if ERK activity was required for EGF (epidermal growth factor)-stimulated protrusion in normal HMECs (human mammary epithelial cells) using time-lapse microscopy. In HMECs, EGF is a strong activator of the ERK-MAPK pathway and a weak activator of PI(3)K, as assessed by phospho-ERK and phospho-Akt levels (Figure 1A). We manually quantified the EGF-induced change in HMEC cell surface area as a read-out of cell protrusion and retraction. Unmodified HMECs exhibited minimal protrusive activity (Figure 1B, V). Because the small GTPase Rac is required for lamellipodial protrusion (Heasman and Ridley, 2008), we stably infected and selected the HMECs with virus expressing PuromycinR and constitutively-active Rac mutant Q61L. RacQ61L expression did not affect the levels of active ERK (phospho-ERK), excluding Rac regulation of ERK via the PAK–MEK pathway (Figure 1A). EGF induced a protrusion and retraction cycle in HMEC/RacQ61L cells that closely mimicked the published dynamics of murine mammary adenocarcinoma cells (Figures 1A and 1B, (Segall et al., 1996)). Pre-treatment of HMEC/RacQ61L cells with a MEK inhibitor (U0126) completely abrogated their protrusion (Figures 1B and 1C). Thus, ERK activity is required for cell protrusion.
We further dissected ERK's role in protrusion by quantifying the effects of ERK inhibition on the exploratory dynamics of growing PtK1 cells (Machacek and Danuser, 2006). We used GFP as a cytoplasmic marker and edge tracking software to calculate the percentage and speed of protrusion and retraction. U0126 pre-treatment abrogated protrusion and caused the cell edges to exhibit overall retraction throughout a 10 min timelapse (Figure 1D). At any given time, 44% of control-treated PtK1 cell edges were protruding, while only 7% of the U0126-treated membranes protruded. Strikingly, the remaining protrusions in MEK-inhibited cells exhibited significantly reduced instantaneous protrusion velocity (7.8 +/− 0.8 nm/s for control cells versus 2.2 +/− 0.4 for U0126-reated cells, means and SEM for 5 cells, p=0.0003). In contrast, retraction velocity was not greatly affected (5.5 +/− 2.3 nm/s for control and 6.2 +/− 2.4 for U0126, means and SEM for 5 cells, p=0.7). Thus, though ERK inhibition results in overall retraction, this is due to down-regulation of protrusive activity rather than an increase in contractility.
We hypothesized ERK might signal through the Arp2/3 machinery to promote protrusion. We first determined if ERK promotes cell protrusion by augmenting ERK activity in HMECs. Cells expressing hyperactive B-RafV600E or over-expressing the EGFR (EGF receptor) exhibited enhanced EGF-stimulated cell protrusion, similar to constitutively active RacQ61L (Figures S1A and S1B). We used these cells to address the requirement for Rac and the WRC in ERK-driven protrusions. In both HMEC/B-RafV600E and HMEC/EGFR cells, dominant-negative RacT17N blocked EGF-stimulated protrusion without affecting phospho-ERK levels, although RacT17N did reduce EGFR expression (Figures 2A and 2B). When we reduced the levels of individual WRC components with pooled or individual siRNAs, the abundance of the other WRC complex members was also reduced (Figures 2C and S2A), likely via proteasomal degradation (Kunda et al., 2003). Loss of these WRC components blocked HMEC/EGFR protrusion similar to reduction of ERK (Figure 2D). We also repeated the experiment with Alexa555-labeled siRNA and analyzed only the Alexa555-labeled transfected subpopulation (Figures S2B and S2C). Again, loss of WRC components significantly inhibited protrusion. Thus, we conclude these WRC subunits are required for ERK-driven protrusion in the HMEC system.
We hypothesized that molecular cooperation between ERK and the WRC would require them to be in the same place at the same time during lamellipodial protrusion. We assessed their localization in HMEC/EGFR cells 2.5 min after EGF stimulation, when the protrusions are still growing. At this time point, HMECs retained large amounts of cortical actin and exhibited small areas of ruffled edges and cytoplasmic pERK. 76% (19/25) of randomly-selected cells exhibited peaks in the intensity of WAVE2 stain at the edge, suggesting these cells had responded to the EGF and translocated WAVE2 to the membrane. 21% (4/19) of these protruding cells exhibited pERK staining preferentially at the cell edge (Figures 3A, S3A, and S3B). While these results are not surprising given that ERK regulates many disparate biological processes in different subcellular locales (McKay and Morrison, 2007; Shaul and Seger, 2007), we aimed to confirm any WAVE2 and pERK co-localization during protrusion in a system with less heterogeneity. We thus analyzed pERK and WAVE2 localization in Cos-7 cells, which exhibit a more uniform protrusion response to EGF: large lamellipodia with WAVE2 localized to the edge and minimal cortical actin (Figure 3B, 25/25 randomly-selected cells). Measuring the intensity of each stain with respect to distance from the cell edge verified that both WAVE2 and phospho-ERK intensities peaked at the edge and decreased coincidently with the actin intensity peak (Figures 3B and S3B, 24/25 cells). pERK intensity then increased further inside the cell, consistent with active ERK localization to the cytoplasmic and nuclear compartments (Figures 3B, S3A, and S3B, (McKay and Morrison, 2007).
We assessed the co-localization of WAVE2 and phospho-ERK via an object-based co-localization method (Lachmanovich et al., 2003). We calculated the probability of finding WAVE2 stain punctae within 0.2 μm (the resolution of diffraction-limited microscopy) of any given pERK stain puncta. This probability was 25% (+/− 3% SEM) higher than a random distribution of equally dense points for HMECs, and 28% (+/− 4% SEM) higher for Cos-7 cells, indicating they are significantly closer together than would occur by chance. The relative probabilities of the distance between pERK and WAVE2 decreased to 1 (equal to that of chance) and then to less than 1 (less likely than chance) at further distances (Figure 3C). Thus, WAVE2 and pERK indeed co-localize.
In both systems, ERK inhibition caused a re-structuring of actin into stress fibers and large punctate structures reminiscent of adhesions (Figures 3A and 3B). However, ERK inhibition did not impact WAVE2 localization to the cell edge. 6/10 HMECs and 9/10 randomly-selected Cos-7 cells pre-treated with U0126 exhibited peaks in WAVE2 intensity at the cell edge (Figures 3A and 3B). We further confirmed ERK localizes to the edge of lamellipodial protrusions by co-staining with Paxillin, a component of lamellipodial nascent adhesions. Phospho-ERK stain overlapped with lamellipodial nascent adhesions and also large mature adhesions, consistent with previous studies (Fincham et al., 2000; Ishibe et al., 2003; Vomastek et al., 2007) (Figures 3D and S3C). Inhibition or loss of ERK or WAVE2 caused indistinguishable cellular phenotypes: the edges lacked any sign of protrusion and were instead surrounded by large adhesions (Figures 3D and S3C).
We hypothesized ERK cooperates with the WRC in cell protrusion by regulating WRC activation. ERK has been suggested to both phosphorylate (Danson et al., 2007) and not phosphorylate (Lebensohn and Kirschner, 2009) WAVE2. We tested whether ERK phosphorylates the WRC by in vivo labeling and in vitro kinase assay. When the WRC was immunoprecipitated from 32P-labeled EGF-stimulated HMEC/EGFR cells, ERK-dependent phosphorylation was detected on WAVE2 and Abi1, but not Nap1 or Sra-1 (Figure 4A). In vitro, GST-ERK2 phosphorylated WAVE2 in the WRC, but not un-complexed WAVE2 (Figure 4B). Further, ERK2 phosphorylation of WAVE2 and Abi1 increased with increasing amounts of Nap1/Sra-1 in the WRC substrate (Figure 4C).
In order to identify the sites that ERK phosphorylates on WAVE2 and Abi1, we used mass spectrometry to analyze endogenous WRC purified from EGF-stimulated HMEC/EGFR cells and in vitro phosphorylated WRC purified from 293F cells (Figures S4A and S4B). ERK interacts with its scaffolds and substrates via ERK's CD (common docking) and/or DBP (DEF motif binding pocket) binding to D domains and DEF motifs within the interacting protein (Dimitri et al., 2005; Lee et al., 2004). We used ERK2 mutants that cannot bind D (D319N) domains or DEF (Y261A) motifs as negative controls for the in vitro-phosphorylated WRC mass spectrometry analysis, as they exhibited minimal phosphorylation of the WRC (Figure 4D). We counted the number of times the spectra for each site are observed in the different ERK2 samples, as it is suggestive of their relative abundance.
S308, S343, T346 and S351 were previously inferred to be ERK phosphorylation sites, as their point mutation abrogates growth factor-induced WAVE2 phosphorylation and bandshift (Danson et al., 2007; Nakanishi et al., 2007). S482 and S484 were also originally reported to be ERK sites (Nakanishi et al., 2007), but have since been shown to be phosphorylated by Casein Kinase 2, not ERK2 (Pocha and Cory, 2009). S482 and S484 are followed by Asp and Glu residues, creating motifs selected against by ERK2 and preferred by Casein Kinase 2 (Hutti et al., 2004). Our mass spectrometry analysis identified pS296 in both the wildtype and D319N ERK2 samples. We found WAVE2 pS308 only in the wildtype ERK2 sample (Figure S5 and Table S1). This suggests WAVE2 S296 and S308 are phosphorylated in an ERK-dependent manner. S343, T346 and S351 have not been reported in any mass spectrometry analysis, likely because they reside in a poly-proline rich region refractory to tryptic and chymotryptic digestion. We made antibodies to phospho-S308, -S343, -T346 and -S351 and confirmed they specifically detected their respective sites using alanine mutants as controls (Figure 5A). We used these antibodies to test whether ERK regulated phosphorylation of these sites in vivo. In HMECs, each site exhibited U0126-sensitive phosphorylation with EGF stimulation (Figure 5B). Cos-7 cells exhibited ERK-dependent phosphorylation of S343, T346 and S351, but not S308. The S308, T346, and S351 sites exhibited relatively high basal phosphorylation, suggesting residual ERK activity or a different Pro-directed kinase maintains their phosphorylation in the starved state. In Cos-7 cells, mutation of the three common WAVE2 sites to Ala residues (3A) abrogated EGF-induced U0126-sensitive in vivo labeling, although total basal phosphorylation remained high (Figures S4C). Mutation of S482 and S484 to Ala did not affect EGF-inducible U0126-sensitive phosphorylation. Thus, pS343, pT346, and pS351 account for the majority of ERK-induced phosphate transfer to WAVE2.
Our mass spectrometry also identified Abi1 S183 and S225 on Abi1 (numbering corresponds to Abi1 isoform 1) as sites phosphorylated on endogenous protein and in the wildtype ERK-dependent in vitro phosphorylated sample (Figure S6 and Table S1). pS216 was also a suspected, although not definitive, site (data not shown). These sites were not found in the ERK2 CD or DBP mutant samples. Interestingly, 5 additional Abi1 residues are predicted ERK-phosphosites (T265, S267, S392, T394 and S410) (Obenauer et al., 2003). These additional sites also lie in regions refractory to tryptic and chymotryptic digests. S392 or T394, and S410 were recently identified using Asp-N digestions (Ross Tomaino, personal communication) (Lebensohn and Kirschner, 2009). We mutated each of these sites individually and assayed their contribution to the in vitro ERK phosphorylation of GST-Abi1. Although no one site contributed significantly to the overall ERK phosphorylation of Abi1, mutation of some sites did induce a mobility shift in the Abi1 protein that is consistent with reduced phosphorylation (Figure 5C, S216A, T265A and T394A). We combined mutation of the 3 sites identified by our mass spectrometry with mutation of T265 and S410, a site identified in previous mass spectrometry experiments of the WRC (Lebensohn and Kirschner, 2009). ERK phosphorylation of this 5A mutant was reduced, but not abolished (Figure 5C). When the remaining 3 Scansite-predicted sites were additionally mutated, the Abi1 8A mutant exhibited only residual (0.2% of wild-type) phosphorylation by ERK2 (Figure 5C). To further validate that this sheer number of sites were truly phosphorylated by ERK, we expressed and immunoprecipitated the 5A and 8A mutants from 32P-labeled Cos-7 cells (Figure 5D). While the 5A retained EGF-inducible U0126-sensitive phosphate transfer, basal and EGF-induced phosphorylation of the 8A mutant were abolished. We made antibodies to phospho-S225 and confirmed its recognition of wildtype but not alanine-mutated Abi1 (Figure 5E). The antibody also detected U0126-sensitive phosphorylation of endogenous Abi1 in both HMECs and Cos-7 cells (Figure 5F).
We sought to understand how ERK phosphorylation of WAVE2 and Abi1 might regulate Arp2/3 activity and cell protrusion. Our immunofluorescence data indicate ERK does not regulate WRC activation by controlling its localization to the membrane (Figure 3). We therefore hypothesized ERK promotes WRC activation by regulating its allosteric activation. All the ERK phosphorylation sites on WAVE2 and Abi1 reside within their respective Pro-rich domains (PRD), unstructured domains that were genetically deleted in the WRC crystal structure (Chen et al., 2010). Phosphorylation of the PRDs would disrupt their interaction with SH3 and PLP binding domains, potentially altering WRC activation.
We asked whether ERK phosphorylation of WAVE2 and Abi1 could regulate WRC interaction with Arp2/3 or actin. We immunoprecipitated the WRC from 293T cells and found EGF induced an ERK-dependent increase in Arp2/3 and actin co-immunoprecipitation (Figure 6A). Consistent with these data, mutating all 4 WAVE2 and 8 Abi1 sites to phospho-mimetic Asp/Glu residues (WRC-D/E) increased Arp3 and actin co-immunoprecipitation in the starved state above that seen with wildtype WRC under EGF-stimulation (Figure 6B). The increased WRC-D/E interaction with Arp2/3 and actin was not further induced with EGF stimulation. These data suggest EGF-induced ERK phosphorylation of the WRC promotes WRC interaction with Arp2/3 and actin. Somewhat surprisingly though, mutation of the sites to Ala's (WRC-A) also increased basal WRC co-immunoprecipitation with Arp3 and actin, though not to extent of the phospho-mimetic mutations. We then dissected which phosphorylation sites contribute to the basal and regulated WRC interaction with Arp2/3 and actin by assaying the individual WAVE2 and Abi1 phospho-site mutants. Mutation of the WAVE2 sites alone induced un-regulated WRC interaction with Arp2/3 and actin similar to that of WRC-A (Figure 6C). In contrast, mutation of the Abi1 sites reduced both basal and EGF-stimulated WRC interaction with Arp3 and actin.
These data indicate ERK phosphorylation of Abi1 is required for basal and EGF-induced WRC interaction with the Arp2/3 complex. In addition, the basal phosphorylation of WAVE2 found in the starved U0126-treated states might be required for a conformation that permits a regulated release of Arp3 and actin. We hypothesized the de-phosphorylated WRC-A might function as a dominant negative by forming a more stable and un-regulatable interaction with Arp2/3, thereby preventing WRC and Arp2/3 cycling on and off of the expanding lamellipodial actin meshwork. In contrast, hyper-phosphorylated mimic WRC-D might cycle through binding and releasing Arp2/3 and actin faster than hypo-phosphorylated WRC, producing more productive Arp2/3 activation and actin polymerization.
To test this, we purified wildtype and mutant WRC from log-phase mammalian 293F cells, permitting the complexes to aquire activating phosphorylations found in steady-state cells (Lebensohn and Kirschner, 2009). The purified wildtype WAVE2 and Abi1 consisted of mixed populations of phospho-proteins (Figure 6D). In contrast, the WRC-A (4 WAVE2 and 8 Abi1 sites mutated to Ala's) and WRC-D/E (4 WAVE2 and 8 Abi1 sites mutated to phospho-mimetic Asp/Glu residues) complexes formed sharp bands of unphosphorylated or phospho-mimetic protein, respectively. WAVE2 concentration is 30–50 nM in wildtype cells (Suetsugu et al., 2006) and we microinjected a volume of 200 nM WRC that roughly equaled 30% of HMEC/EGFR cytoplasmic volume, for a final concentration of ~67 nM purified WRC. Cells microinjected with buffer or wildtype WRC exhibited EGF-stimulated lamellipodium protrusion that did not retract within the 15 min timecourse (Figure 6E). Strikingly, the cells injected with WRC-A did not protrude. This suggests that while the WRC-A protein can bind Arp2/3 and actin, it functions as a dominant negative by inhibiting proper Arp2/3 activity and actin polymerization cycles. In contrast, the cells injected with WRC-D/E exhibited more extensive protrusion (Figure 7B), suggesting the increased Arp3 and actin binding observed with the phospho-mimetic mutations hyper-activated WRC activation of Arp2/3 and actin polymerization.
We further assayed the function of ERK phosphorylation of the WRC in Cos-7 cell migration, which involves cell polarization, edge protrusion and adhesion and cell body translocation. Depending on the cell type, an increase in instantaneous protrusion may lead to an increase in migration speed. However, excessive protrusion will slow down motility, as the protrusive forces overcome the cells' ability to contract and move the cell body (Ji et al., 2008). Under growing conditions, Cos-7 cells move with an average speed of 0.26 +/− 0.02 μm/min (means and SD for 30 cells over 10 hours). Inhibition of ERK with U0126 reduces migration speed (0.17 +/− 0.01 μm/min, p=0.0004). We simultaneously reduced endogenous WAVE2 and Abi1 levels with Alexa555-labeled siRNA and rescued their expression with the wildtype and point mutant combinations by co-transfection (Figure S7A). Wildtype (WT) WAVE2 and Abi1 rescued the reduction in cell speed caused by wave2 and abi1 siRNA (Figure S7B). As expected, co-transfection of WAVE2 3A and Abi1 8A failed to rescue, while WAVE2 3D and Abi 8D increased migration speed above that of wildtype. Interestingly, WAVE2 3A and 3D also rescue cell motility, indicating that in the context of wildtype Abi1 and residual wildtype WAVE2, WAVE2 phosphosite mutants do not impact cell polarization, protrusion, adhesion, and cell body translocation. In contrast, expression of Abi1 8A does not rescue migration while Abi1 8D increases migration speed (Figure S7B). Thus, ERK phosphorylation of Abi1 is essential for functional WRC during cell motility.
We have found that a key function of ERK during cell motility is to regulate protrusion initiation and speed. Active ERK localizes to the leading edge of protruding lamellipodia and phosphorylates the WRC on multiple sites within the PRDs of WAVE2 and Abi1. We propose that cumulatively, these phosphorylations contribute to the Rac-induced WRC conformational change that exposes the VCA domain, leading to WRC binding and activation of Arp2/3 during lamellipodial protrusion (Chen et al., 2010; Ismail et al., 2009; Lebensohn and Kirschner, 2009).
The detection of ERK phosphorylation of WAVE2 and Abi1 by in vivo labeling suggested the presence of either a very efficient ERK site or multiple weaker sites. Yet, the sheer number of ERK sites identified on WAVE2 and Abi1 was surprising. ERK CD-WRC D domain and ERK DBP-WRC DEF motif interactions appear to assist ERK in interacting with the complex to phosphorylate so many sites. Interestingly, WAVE2 contains putative D and DEF domains, but Abi1 lacks any potential ERK interaction domains (Scansite (Obenauer et al., 2003)). Thus, ERK is likely recruited to Abi1 via interaction domains elsewhere in the complex, potentially via Nap1 and Sra-1, which harbor several putative ERK interaction domains (Scansite (Obenauer et al., 2003)). Consistent with this model, Nap1/Sra-1 augmented ERK phosphorylation of WAVE2 and Abi1.
It is somewhat confounding that a recent study concluded ERK was not the kinase responsible for phosphorylating several of the phospho-sites we identified on WAVE2 and Abi1 (Lebensohn and Kirschner, 2009). This conclusion was based upon the finding that WRCs purified from starved or EGF-stimulated A431 cells exhibited equivalent in vitro actin polymerization activity. While it is possible that not all of our identified Abi1 sites are phosphorylated at the same time in vivo, we suggest that in cell systems with extremely high EGFR levels, such as A431 cells, high basal ERK activity will maintain WRC phosphorylation during starvation. Along these lines, the mass spectrometry in the A431 study identified WAVE2 S308 and Abi1 S183, S216, S225, S392 and S410 phosphorylated equally in the starved and stimulated situations. In contrast, we found many of these sites to be phosphorylated in an ERK-dependent manner in vitro and in vivo. In another study, Danson et al. concluded that S343 is a substrate for JNK-MAPK, not ERK, and that T346 is a target of both kinases. This conclusion was based on an in vitro kinase assay in which ERK was unable to phosphorylate FLAG-WAVE2. In our hands, WAVE2 was not a great substrate for ERK2 unless it was incorporated in the WRC. Because we observe clear U0126-sensitivity at these sites in vivo, we conclude ERK phosphorylates both S343 and T346 and that JNK may also phosphorylate these sites under conditions in which JNK is active, such as cellular stress or starvation. Other Proline-directed kinases, such as Cdk5 which phosphorylates WAVE1 (Kim et al., 2006) or Gsk-3, may also participate in the basal-state WAVE2 and Abi1 phosphorylation.
Mutation of the ERK phosphorylation sites on Abi1 significantly inhibited WRC interaction with Arp2/3 and actin and cell protrusion and migration. Thus, phosphorylation of Abi1 is required for functional WRC. Mutation of the ERK phosphorylation sites on WAVE2 induced an unregulated WAVE2 interaction with Arp2/3 and actin, inhibited cell protrusion. The Arp2/3 co-immunoprecipitation data suggest a completely unphosphorylated WAVE2 functions differently than a hypo-phosphorylated WAVE2, such as the WAVE2 in starved and U0126-treated cells. As WAVE2 dimerizes to activate Arp2/3 (Padrick et al., 2008), phosphorylation of one of the 8 sites in the two Pro-rich regions is likely needed for proper cycling of Arp2/3 binding, activation, and release during rapid protrusion. We conclude the unregulated interaction of un-phosphorylated WAVE2 with Arp2/3 functions in a dominant-negative manner, as microinjection of the WRC-A blocks EGF-induced protrusion.
In a similar study, introduction of WAVE2 3A into NIH 3T3 cells had no effect PDGF-induced membrane ruffling (Danson et al., 2007). However, knockdown of WAVE2 levels with siRNA also did not affect membrane ruffling in these cells, suggesting NIH 3T3 system does not model lamellipodial protrusion. In a would healing assay, these authors found WAVE2 3A increased NIH 3T3 migration speed and decreased persistence by reducing Golgi apparatus polarization, a function independent of Rac activity. While a wound healing assay involves directionality and the loss of cell-cell adhesions, it is possible that this other function of WAVE2 could be at play in our migration assay, shadowing any defect in speed due to lamellipodia protrusion. Additionally, the WAVE2 3A mutant may retain enough activity in the presence of wildtype Abi1 and residual endogenous WAVE2 to complement the loss of migration speed induced by WAVE2 knockdown.
ERK regulation of WRC activation likely controls lamellipodial protrusion via two non-mutually exclusive mechanisms. First, WRC activation leads to Arp2/3 activation and generation of the actin meshwork that provides the pushing force for protrusion initiation and speed. Second, ERK may also control lamellipodium protrusion by regulating the actin polymerization necessary for nascent adhesion formation. Rac activation and actin filament assembly are required for nascent adhesion formation in the lamellipodium (Choi et al., 2008; Nayal et al., 2006). These adhesions generate adhesion forces necessary for stable and persistent lamellipodium advancement without ruffling (Giannone et al., 2007; Ji et al., 2008). ERK and Rac may act together at the leading edge to activate the WRC and Arp2/3 to initiate and reinforce nascent adhesions. Indeed, in spreading macrophages, ERK functions to stabilize the protruding lamellipodia and its inhibition causes the production of transient, unstable protrusions (Smith et al., 2008).
Inhibition of ERK and the WRC eliminates protrusion and nascent adhesion formation. The cell peripheries are encircled by enlarged adhesion and the cells contract. As ERK also regulates adhesion disassembly and maturation, via phosphorylation and activation of FAK and MLCK (Webb et al., 2004), ERK's role in promoting leading edge formation mechanistically links protrusion formation with mature adhesion disassembly. We propose ERK signaling balances lamellipodia protrusion/retraction and adhesion assembly/disassembly. Thus, extracellular cues for increased ERK activity coordinately increase actin polymerization to generate protrusion force and adhesion turnover for protrusion advancement.
HMECs were obtained from Cambrex, immortalized and cultured as previously described (Abe et al., 2009). HMECs were starved for 24 hours in DMEM/F-12, 35 mM Hepes, 0.5 μg/ml Hydrocortisone. PtK1 cells were cultured in F-12 medium, 10% FBS (fetal bovine serum). Cos-7 and 293T cells were cultured in DMEM, 10% FBS. Cos-7 cells were starved with 0.2% FBS and 293T cells with 0% FBS. 293F cells were cultured in Freestyle medium (Invirogen). Where indicated, cells were pre-treated with 20 nM U0126 for 2 h and stimulated with 20 ng/ml EGF for 2.5 or 5 min.
Antibodies obtained from commercial sources include those used in immunoblotting: anti-EGFR (Santa Cruz), B-Raf (Santa Cruz), p-ERK (Sigma), GAPDH (Ambion), Sra-1 (Upstate), Nap1 (Sigma), Abi1 (MBL), WAVE2 (Cell Signaling), immunoprecipitations: anti-Abi1 (Bethyl), and immunofluorescence: p-ERK (Sigma), ERK (Upstate) and WAVE2 (Cell Signaling). The ERK antibodies used in immunoblots were previously described (Murphy et al., 2002). The phospho-WAVE2 and phospho-Abi1 antibodies were synthesized and purified by Millipore: p-WAVE2 S308 (07-1511), -S343 (07-1512), -T346 (07-1513), -S351 (07-1514) and p-Abi1 S225 (07-2129). 20 nM mixes of validated duplexes from Qiagen were used for siRNA transfections.
HMECs and PtK1 cells were cultured on acid-washed glass coverslips and transferred to heated chambers on a Nikon Ti inverted fluorescence microscope. Images were collected every 10 sec using Metamorph software and a Hammamatsu ORCA-R2 camera. This sampling rate fully captures the protrusion/retraction behavior of morphologically active PtK1 cells (Machacek and Danuser, 2006).
HMECs were imaged in starve media using D.I.C. filters for 16 min. 20 ng/ml EGF (final concentration) was added after 1 min. 10 HMECs per experiment were manually traced and the surface area calculated using Image J software. Experimental and control groups contained 3–4 samples of 10 cells for 2-tailed, unpaired T-test analysis. WRC was diluted to 100 nM in PBS and co-injected with 0.25 mg/ml FITC-dextran. Cells were recovered for 2 h and then a single epi-fluorescence image was taken with a FITC-filter cube to document which cells were microinjected and then imaged with D.I.C. filters for 16 min. Microinjected experimental and control groups contained 3–4 samples of 15 cells for 2-tailed, unpaired T-tests.
PtK1 cells were imaged by epi-fluorescence in L-15 medium containing 20 mM Hepes and 0.03 U/ml Oxyrase with a 60× 1.4 N.A. objective and 1.5× magnification. PtK1 cell boundaries were extracted from the fluorescence microscopy data using adaptive thresholding combined with Canny edge-detection. These cell edges were then tracked as described previously (Machacek and Danuser, 2006). Edge velocities were calculated for each point along the cell edge. The edge was then sub-divided into 30 segments of equal length and these velocities were averaged for each segment, excluding three segments on each end to eliminate effects of the image boundary. These protrusion velocities were then combined into activity maps. Areas of these maps with positive or negative velocity correspond to protruding and retracting areas on the cell edge respectively. 2-tailed, unpaired T-tests were used to calculate significance between U0126 and control groups, with each group containing 5 cell edges segmented into 24 segments.
Cells were cultured on glass-bottomed Matek dishes. Wells were covered with mineral oil before transferring the plate to a 37° C incubation chamber on an inverted fluorescence microscope. Images were collected every 10 min with Metamorph software for 10 hours. Migration of the nucleus was calculated using the Track Points function in Metamorph. Every Alexa555-labeled cell that did not divide or move out of the field of view was included in the analysis (11–20 cells).
For confocal immunofluorescence, cells were fixed in 3.7% formaldehyde in PBS for 15 min, rinsed with PBS, and blocked and permeabilized in 3% bovine serum albumin, 0.2% Triton X-100 for 30 min. Primary antibodies were incubated in WIS (1% BSA, 0.2% Tween-20 in PBS) overnight at 4 °C. Cells were washed with WIS before incubation with fluorophore-conjugated secondary antibodies, DAPI and phalloidin for 1 hr. TIRF immunofluorescence was carried out as previously described (Vomastek et al., 2007). Images within each experiment were taken with the same exposure times and were scaled equivalently.
To examine stain with respect to distance from the cell edge, the edge was found via automatic intensity-histogram based thresholding of the phalloidin stain. The distance from this edge at each point in the cell interior was calculated via the distance transform. The average intensity in each channel was calculated for integer intervals of the distance transform, yielding the average intensity at each distance from the cell edge. As a validation of the cell-edge location, the average intensities were also calculated 20 pixels outside of the identified cell boundary. This confirmed that the fluorescence quickly decayed to background levels outside of the identified cell-edge location (Figure S2A). The co-localization of phospho-ERK and WAVE2 was analyzed via a nearest neighbor, object-based co-localization method (Lachmanovich et al., 2003). Punctae of WAVE2 and p-ERK staining were detected via laplacian-of-gaussian filtering followed by local maxima detection and local background estimation via low-pass filtering. Punctae 2 standard deviations above background intensity were analyzed. For each detected puncta in the p-ERK channel, the nearest puncta in the WAVE2 channel was found, allowing construction of a nearest-neighbor distance histogram. For each cell, a random distribution of punctae of identical density was also created. The relative nearest-neighbor distance probabilities were calculated by dividing the observed distance histogram by the random distribution histogram.
For metabolic labeling, cells were starved 20 hours in normal starve media and 2 h in phosphate-free media. HMECs were then labeled with 1 mCi/ml 32P orthophosphate for 2 h, stimulated, and lysed with 20 mM Tris-Cl, 125 mM NaCl, 1% Triton X-100, 1 mM EDTA containing protease and phosphatase inhibitors. The WRC was immunoprecipitated using Protein A/G-conjugated anti-Abi1 or anti-FLAG and was washed with lysis buffer before separation by SDS-PAGE and transfer to Nitrocellulose membrane. For in vitro kinase assays, 2.5 ng active GST-ERK2 (Upstate) or immunoprecipitated HA-ERK2 from 293T cells (stimulated with 100 ng/ml phorbol 12-myristate 13-acetate for 15 min) (Dimitri et al., 2005) were incubated with 125 μM ATP, 5 μCi γ-labeled ATP and either 2 μg GST-Abi1 or 0.5 μg FLAG-purified WRC at 30 °C for 15 or 30 min, respectively. Reactions were within the linear range for both time and substrate.
Coomasie-stained bands of WAVE2 and Abi1 were diced into 1mm cubes. Gel pieces were reduced, alkylated with iodoacetamide and subjected to in-gel digestion as described previously (Zappaterra et al., 2007), except a double digest using both trypsin and chymotrypsin was performed. Dried peptides were subjected to liquid chromatography tandem mass spectrometry in a linear ion trap (LTQ)-orbitrap hybrid mass spectrometer (Thermo Electron) set up and run as described previously (Ballif et al., 2008). Data were searched against databases for WAVE2 and Abi1 using Sequest; requiring no enzyme specificity; allowing precursor m/z tolerances of 30 ppm; allowing serine, threonine and tyrosine residues to be phosphorylated (+79.96633 Da); allowing methionine residues to be oxidized (+15.99492 Da); and requiring cysteine residues to be carbamiodomethylated (+57.02146 Da). Each identified phosphopeptide was manually examined for accuracy of the Sequest assignment, and had a measured monoisotopic mass different by less the 5 ppm m/z of the calculated monoisotopic mass.
To ensure we purified the in-tact WRC, we performed a 2-step purification in which first 6xHis-Sra-1 was affinity purified using Ni-NTA agarose and then WAVE2 was immunoprecipitated using anti-Protein C (for micro-injections) or FLAG (for kinase assay substrate) affinity matrix. 293F cells were transfected with equal concentrations of pcDNA3 plasmids encoding T7-Abi1, V5-Nap1, 6xHis-Sra-1, myc-HSPC300 and either Protein C-WAVE2 or FLAG-WAVE2. After 72 hours, cells were lysed in WRC buffer (25 mM Tris, pH 8, 20 mM Imidazole, 150 mM NaCl, 20% glycerol) containing 1% NP-40, 5 mM β-mercaptoethanol, and protease and phosphatase inhibitors. Lysates were cleared by ultra-centrifugation and subjected to Ni2+ affinity purification for 1 hour using Ni-NTA agarose (Qiagen). The beads were washed with 8 CVs of WRC buffer containing 20 mM Imidazole and 8 CVs of WRC buffer containing 40 mM Imidazole. The WRC was eluted from the agarose once with 2.5 CVs of WRC buffer containing 100 mM Imidazole and once with 2.5 CVs of WRC buffer containing 200 mM Imidazole. 1 mM CaCl2 was added to the combined eluate and anti-PC or anti-FLAG purification was carried out for 2 hours. PC immunoprecipitations were eluted with 5 mM EGTA and FLAG were eluted with 0.2 μg/ml 3× FLAG peptide (Sigma). PC purifications were buffer-exchanged into 10 mM Imidazole, pH 7, 100 mM KCl, 1 mM EGTA, 1 mM MgCl2, 1 mM DTT, and 20% glycerol using PD-10 desalting columns (Roche). Following concentration on Amicon Ultra-15 columns, WRC was snap frozen and stored at −80 °C. We quantified the purified WRCs using Coomasie-stained Nap1 and Sra-1 bands, which they did not exhibit phosphorylation-induced band-shifts.
293T cells were transfected with plasmids encoding the WRC (pCDNA3/Flag-WAVE2, pCDNA3/T7-Abi1, pCDNA3/V5-Nap1, pCDNA3/S-Sra1) using Calcium phosphate and starved of serum after 24 hours, for 24 hours. The cell were then washed with PBS containing 1mM CaCl2 and 1mM MgCl2 and lysed with 0.5% NP-40, 20mM Tris pH7.4, 125 mM NaCl, 2 mM MgCl2, 1mM EDTA and protease and phosphatase inhibitors. 2 mg of lysate was immunoprecipitated with anti-FLAG-conjugated beads at 4 °C for 40 min. Immunoprecipitates were washed three times with lysis buffer containing 0.1% NP-40.
We would like to thank Rana Anjum and Steven Gygi for their initial help with WRC Mass Spectrometry and Anne Marie Pendergast and Alexis Gautreau for the Abi1 and WAVE2 plasmids. The authors thank the Nikon Imaging Center at Harvard Medical School for help with light microscopy. This work was funded by NCI R37CA46595 (J.B.), a Susan G. Komen PDF (M.C.M.), the Vermont Genetics Network through NIH grant P20 RR16462 from the INBRE Program of the NCRR (B.A.B.), and the NIH Cell Migration Consortium U54GM64346 (G.D).
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