WAVE2 is an essential regulator of the actin cytoskeleton through its ability to stimulate Arp2/3-dependent actin polymerization downstream of activated Rac1 in different cell types (Suetsugu et al., 2003
; Takenawa and Miki, 2001
). The embryonic lethality in WAVE2 knock-out mice is associated with deficient lamellipodia and surface ruffling in cells and with impaired cell migration of embryonic fibroblast and in endothelial cells lacking WAVE expression and therefore highlights the importance of WAVE-mediated actin polymerization (Yamazaki et al., 2003
). Moreover, in a previous report, WAVE2 was reported to be important in CSF-1-induced actin reorganization and cell migration in macrophages (Abou Kheir et al., 2005
). While WAVE2 has a well-documented function in cell migration, WAVE2 regulation has been proven to be very complicated since Rac1 does not bind directly to WAVE2, as in the case between Cdc42 and WASP/N-WASP proteins. Based mostly on in vitro studies, IRSp53 was reported as an important candidate in linking WAVE2 to Rac1 (Miki and Takenawa, 2002
; Miki et al., 2000
; Oda et al., 2005
; Suetsugu et al., 2006a
). However, whether IRSp53 stands as an essential intermediate between Rac1 and WAVE2 in vivo was not established.
In this study, IRSp53 was identified as an essential intermediate that links Rac1 to WAVE2 to mediate actin cytoskeleton reorganization and cell migration in macrophages. Consistent with WAVE2 being important in chemotaxis (Abou Kheir et al., 2005
), inhibition of the CSF-1-induced actin cytoskeleton rearrangement in cells with reduced IRSp53 expression also resulted in a reduced ability of cells to migrate towards CSF-1. Altogether, these data identified IRSp53 as an essential mediator of macrophage motility in response to CSF-1.
The small Rho GTPases Rac1 and Cdc42 have been shown to act as regulators of actin reorganization and to mediate lamellipodia and filopodia formation in different cell types respectively (Aspenstrom et al., 1996
). IRSp53 can bind to both Rac1 and Cdc42. The NH2-terminal RCB domain of IRSp53 binds to activated GTP-Rac1 and mediates lamellipodia formation in fibroblasts (Miki et al., 2000
), while the CRIB motif of IRSp53 binds to activated GTP-Cdc42 and mediates microspikes and filopodia formation in fibroblasts and neuronal cells (Govind et al., 2001
; Krugmann et al., 2001
). This study reports that in macrophages IRSp53 binds to GTP-Rac1 but not GTP-Cdc42 in vivo. Moreover, IRSp53 is involved in Rac1-mediated surface ruffling which was consistent with recent findings by Suetsugu et al in human carcinoma A431 cells (Suetsugu et al., 2006a
). Consistent with a role of IRSp53 in mediating Rac1 and WAVE2 interaction and with the lack of interaction with Cdc42 in macrophages, formation of Cdc42-mediated filopodia was not affected in cells with reduced amounts of IRSp53. Notably, while others reported that over-expression of IRSp53 induces filopodia formation; this was not the case in macrophages (data not shown). Our results do not invalidate the recent findings that IRSp53, along with Cdc42 and Eps8, were required for filopodia formation in Cos-7 and HeLa cells (Disanza et al., 2006
), instead they suggest that there may be some cell type specificity that regulates whether IRSp53 interacts with either Rac1 or Cdc42. Connelly et al. has demonstrated that Tiam1 can mediate the interaction between IRSp53 and Rac1 by enhancing IRSp53 binding to both active Rac1 and the WAVE2 scaffold. Moreover, they show that Tiam1 promotes IRSp53 localization to Rac1-induced lamellipodia rather than Cdc42-induced filopodia (Connolly et al., 2005
). Tiam1 is expressed in macrophages and was reported to play a role in Rac1-dependent events (Abell et al., 2004
; Mizrahi et al., 2005
). Therefore, these results may explain the differences observed in the interaction of IRSp53 with either Rac1 or Cdc42.
It is important to note that IRSp53 and WAVE2/Abi1 unit are required for certain actin-mediated processes like surface ruffling and cell migration () but not in others like FcγR-mediated phagocytosis, filopodia and podosome formation (Fig. S3
), suggesting a specific and complex regulation of actin-dependent events in macrophages. The fact that none of the aforementioned proteins was found to localize in podosomes and to play a role in their formation is consistent with a unique role of WASP in those structures (Jones et al., 2002
; Linder and Aepfelbacher, 2003
). Moreover, WAVE2, Abi1 and IRSp53 had no role in the actin-dependent phagocytosis of IgG-opsonized erythrocytes in macrophages (this study and (Abou Kheir et al., 2005
). However, the lack of a role for these proteins in phagocytosis is perplexing since it has been shown that Rac1 is required for FcγR-mediated phagocytosis (Caron and Hall, 1998
; Cox et al., 1997
; Yamauchi et al., 2004
). These results would suggest that Rac1-dependent actin polymerization downstream of the Fcγ receptor is independent of WAVE2 and thereby must be mediated through a different mechanism. This alternative pathway of Rac-dependent actin polymerization may explain the lack of complete inhibition that is observed following inhibition of WAVE2 activity by multiple mechanisms (Abou Kheir et al., 2005
). More work would need to be done to uncover the mechanism of this WAVE2-independent yet Rac1-dependent actin polymerization.
As mentioned before, WAVE proteins differ from WASP and N-WASP by lacking the GTPase-binding domain that mediates the direct interaction with Rac1 (Takenawa and Miki, 2001
). Although WASP regulation appears to be mediated by an auto inhibitory state that is relieved by Cdc42 binding and tyrosine phosphorylation in response to stimuli (Cory et al., 2002
; Prehoda et al., 2000
), WAVE regulation is still under debate and understanding how WAVE proteins can relay signals from activated Rac1 to actin cytoskeleton represents a current area of active investigation. Recently, many groups showed that WAVE1/2 exists in a multi-protein complex that contains Nap1, PIR121/Sra1, Hspc300 and Abi1 where Abi1 comprises the core of the complex (Eden et al., 2002
; Gautreau et al., 2004
; Innocenti et al., 2004
; Steffen et al., 2004
). Although the architecture of the WAVE complex was the same, contrasting models have been proposed regarding its regulation. Initial reports suggested that WAVE1 would be kept inactive in this multi-protein complex and that Rac1 binding to PIR121 may trigger a conformational change that induces the dissociation of the complex and the release of active WAVE1 (Eden et al., 2002
). Subsequently, later reports suggested that the WAVE2 multi-protein complex remained intact when bound to Rac1 and was relocalized to active sites of actin assembly within the cell (Innocenti et al., 2004
; Steffen et al., 2004
). However, the fact that reducing the amount of any protein in the Abi1 complex alters WAVE2 endogenous levels makes it difficult to study the role of Abi1 complex in mediating the interaction between Rac1 and WAVE2 in vivo. Shi et al showed that WAVE2 was necessary for efficient invasion of epithelial cells (MDCK) by Salmonella
. Although this entry promotes the formation of IRSp53/WAVE2 complex, it is the Abi1/WAVE2 complex and not the IRSp53 that was required for the Salmonella internalization (Shi et al., 2005
). However, more recently, Suetsugu et al suggested that IRSp53, along with Rac1 and PIP3
, is required for the optimization of WAVE2-dependent actin assembly (Suetsugu et al., 2006a
). In macrophages, WAVE2 and Abi1 exists in the same molecular complex in a constitutive manner where Abi1 is required for WAVE2 stability and function downstream of CSF-1 (Abou Kheir et al., 2005
). While we can not completely eliminate a role for the Abi1 complex in cooperating in the interaction between Rac1 and WAVE2, this report presents evidence that supports the role for IRSp53 as the major factor linking Rac to WAVE2. We demonstrate that IRSp53 binds to the stable WAVE2/Abi1 sub complex in a Rac1-dependent manner and cells with reduced amounts of IRSp53 were indistinguishable from cells with reduced amounts of WAVE2 or Abi1 in terms of the ability to form F-actin rich surface ruffles and to migrate in response to CSF-1. Furthermore, transient expression of dominant negative WAVE2 protein (WAVE2ΔV) in cells with reduced amounts of IRSp53 did not have an additive effect on the ability of cells to ruffle in response to CSF-1 (). This observation, along with the observation that the level of WAVE2 and Abi1 associated with Rac1Q61L in IRSp53 shRNA-treated cells was significantly decreased and there was no interaction of Rac1 with WAVE2 when the WAVE2 binding site of IRSp53 was deleted (), suggested that Rac1/IRSp53/WAVE2/Abi1 form a single complex and act along the same pathway to activate Arp2/3-mediated actin assembly. Presumably, this macromolecular complex contains PIR121 and Nap1 as shown by others (Innocenti et al, 2004
; Steffan et al 2004
), and as indicated by Commassie staining of immunoprecipitates from RAW/LR5 cell lysates co-expressing GFP-tagged IRSp53 and Myc-tagged Rac1Q61L using anti-GFP antibodies where two bands corresponding to the molecular weights of PIR121 and Nap1 were detected (Fig. S5C
). The fact that WAVE2ΔWHD, but not WAVE2Δproline, fully rescued the CSF-1 ruffling ability of WAVE2 shRNA-treated cells indicates that IRSp53, but not the Abi1 complex (along with PIR121 and Nap1) plays a major role in mediating the interaction between Rac1 and WAVE2 in macrophages in vivo.
Since WAVE2 has been shown to be constitutively active in a purified state, the regulation of WAVE2-dependent actin polymerization in vivo has proven to be very difficult. One of the proposed models suggests that WAVE2 activity might be regulated simply by membrane translocation upon binding of Rac1 to the stable WAVE2 complex (Innocenti et al., 2004
; Stradal et al., 2004
). Results from RAW/LR5 cells as well as from Cos-7 cells showed that membrane targeting of WAVE2 did not induce actin polymerization and protrusions (this report). This suggests that membrane recruitment by itself is insufficient and there are additional levels of WAVE2 regulation. One might envision a model where Rac1 binding to WAVE2 might induce a conformational change that would increase the affinity of Arp2/3 to the CA domain accompanied by membrane translocation that will result in site-restricted actin polymerization. The dynamics of Arp2/3 binding to the CA domain of WAVE2 in quiescent or stimulated cells still needs to be examined. Furthermore, potential unidentified positive or negative regulators like SH3 domain-containing or proline- rich proteins might play a role in regulating the activity of WAVE2 complex.
In addition to its function in mediating its interaction with WAVE2 it may be possible that IRSp53 has additional functions that are required for the extension of actin rich membrane protrusions. The Rac1 binding/IMD domain of IRSp53 shows homology to BAR domains but forms a “zeppelin-shaped” dimer. When this domain is present in liposomes or expressed in cells it induces outward membrane deformations in the direction opposite to that seen with BAR domains (Millard et al., 2005
; Suetsugu et al., 2006b
). These protrusions require activated Rac1 but can occur in the absence of actin polymerization. This outward membrane deformation may be a necessary component in the formation of an actin rich protrusion. Therefore, IRSp53 may be required to initiate membrane protrusion as well as inducing the activation of WAVE2 dependent actin polymerization. Interestingly, the SH3 domain of IRSp53 has been shown to mediate the interaction with Eps8 and to enhance Eps8/Abi1/Sos-1 signaling to Rac1 (Funato et al., 2004
). More recently, Yanagida-Asanuma et al showed that synaptopodin, an actin-associated protein, directly binds to IRSp53 and suppresses Cdc42:IRSp53:Mena-initiated filopodia formation by blocking the binding of Cdc42 and Mena to IRSp53 in kidney podocytes (Yanagida-Asanuma et al., 2007
). This suggests that the interactions of IRSp53 alone are complex and need further investigations. It is worth mentioning that IMD proteins like IRSp53 are not conserved in lower eukaryotic organisms such as Dictyostelium discoideum and therefore the regulation of association between WAVE2, Abi1 complex, Rac1 and Cdc42 is likely to be organism specific as well as cell-type dependent.
Post-translational modification like phosphorylation has also been suggested to play a role in WAVE2 regulation. Phosphorylation of WAVE2 by Abl kinase has been reported to play a role in WAVE2 regulation (Leng et al., 2005
). Yet, no difference was detected in the phosphorylation status of WAVE2 upon CSF-1 stimulation of RAW/LR5 cells (data not shown), suggesting that phosphorylation of WAVE2 does not play a role in its regulation in these cells. However, phosphorylation of WAVE2 might be involved in other cell types. This highlights how complex the regulation of WAVE proteins is and how it might vary between different experimental conditions and different cell types. Based on our in vivo results in macrophages, we propose that the stable WAVE2/Abi1 sub complex exists in an inactive state in the cytoplasm. After extracellular stimulation (like CSF-1) and Rac1 binding to WAVE2, mediated by IRSp53, the now active complex is recruited to the leading edge of the cell and induces a site-specific actin polymerization required for cell protrusion and motility ().
A model for WAVE2 interaction and regulation in macrophages
In conclusion, our results suggest that IRSp53 is the major mediator that links Rac1 to WAVE2 in vivo and its function is crucial for CSF-1-induced F-actin rich protrusions and cell migration in macrophages. Our data also suggest that the mechanism of WAVE2 activation by Rac1 through IRSp53 is more complex and that membrane recruitment alone is insufficient for WAVE2 dependent actin polymerization. The details of the regulation of WAVE function have not been fully delineated and this issue awaits further investigations.