Signaling at the leading edge of migrating cells must be tightly regulated so as to coordinate this process. For proteins recruited to the leading edge phosphorylation, dephosphorylation, and interaction with downstream signaling partners control the motility response. Ultimately, these signaling pathways converge upon the Rho family of GTPases, as these proteins are important regulators of cell migration. In particular, Rac1 activation leads to the formation of new focal complexes at the leading edge and lamellipodia production. In response to migratory cues multiple signaling pathways can influence Rac activity. For example, integrin activation can activate Rac through the formation of a p130Cas–Crk–DOCK180–ELMO Rac GEF (Brugnera et al., 2002
) or a paxillin–Cool-PIX–p21PAK complex (Brown et al., 2002
; Feng et al., 2002
). Here, we show that primary MEFs lacking the LIM protein Ajuba are defective in cell migration, due to an inability to activate Rac1 as a result of defective localization, and thus, activation of the p130Cas–Crk–DOCK180-signaling complex.
Ajuba and p130Cas colocalize in cells, and coimmunoprecipitate. Immunofluorescent and biochemical evidence show that Ajuba null MEFs have reduced p130Cas (total protein and tyrosine phosphorylated p130Cas) at focal complexes of migrating cells, which is normalized following reexpression of Ajuba. Overexpression of p130Cas rescued the migration defect of Ajuba null cells. Mapping studies demonstrate that the PreLIM region of Ajuba mediates Ajuba's interaction with p130Cas, via the COOH-terminal 180–amino acid, major focal adhesion targeting domain, of p130Cas. When overexpressed in wt MEFs Ajuba's PreLIM region acted in a “dominant inhibitory” manner to block p130Cas localization to nascent focal complexes and inhibit cell migration. Furthermore, the PreLIM region of Ajuba also blocked the ability of p130Cas to rescue the migration defect in Ajuba null and p130Cas null cells. Together, these data suggest that Ajuba acts upstream of p130Cas in regulating Rac activation and thus cell motility.
Alternatively, overexpression of p130Cas in Ajuba null cells rescues migration through an Ajuba-independent pathway because p130Cas has more than one focal adhesion targeting domain (Harte et al., 2000
), however, only when overexpressed. But because the PreLIM region of Ajuba blocked endogenous p130Cas localization and function (migration regulation) in wt cells, and blocked the ability of p130Cas to rescue the motility defect of p130Cas null cells, we would argue that the Ajuba–p130Cas interaction is physiologically relevant. However, we cannot exclude that there are other pathways regulating p130Cas to adhesive sites in migrating cells: Ajuba-dependent (primary) and a secondary Ajuba-independent pathway, possibly through a direct interaction with FAK or other Ajuba-related LIM proteins.
In epithelial cells, Ajuba's recruitment from the cytosol to cell–cell junctions is regulated by the formation of new cell–cell adhesion (Marie et al., 2003
). Video imaging of live fibroblasts revealed that Ajuba also shuttles from cytosol to focal adhesions (unpublished data). This, and the fact that Ajuba interacts with the focal adhesion targeting domain in p130Cas, suggests that Ajuba might regulate recruitment p130Cas to the leading edge of migrating cells. Because the PreLIM region of Ajuba does not localize to adhesive sites, rather it is diffusely cytosolic (Marie et al., 2003
), yet blocks p130Cas localization suggests that Ajuba and p130Cas may associate in the cytosol before recruitment to adhesive complexes.
The Ajuba-related LIM proteins, Zyxin and TRIP6, were recently shown to also interact with HEF1–p130Cas proteins (Yi et al., 2002
). The functional consequences of these interactions have not been determined, however. Interestingly, the domains of HEF1 and Zyxin required for their interaction may be distinct from those of p130Cas and Ajuba. Why this difference in how these related proteins interact is not clear, but, overexpression of TRIP6 in fibroblasts inhibits cell migration, similar to knocking out Ajuba (Yi et al., 2002
), suggesting that Zyxin and TRIP6 may exert opposing effects to that of Ajuba upon migration, possibly through effects on p130Cas family members.
The p130Cas–Crk–DOCK180–ELMO-signaling complex is highly conserved across species. In both C. elegans
, mutations in the DOCK180 homologues result in phenotypes with defects in cell migration and phagocytosis (Nolan et al., 1998
; Reddien and Horvitz, 2000
). In mice, there are four other proteins related to Ajuba, with broad overlap in their cellular expression pattern. Probable functional redundancy between these proteins may explain the viability of Ajuba null mice. Database searches indicate that C. elegans
have one and two LIM proteins similar to Ajuba or Zyxin, respectively. Whether these LIM protein genes regulate Rac activity and cell migration in these organisms through the conserved Crk–DOCK180 pathway is a provocative possibility that remains to be determined.
FAK levels are also reduced in the lamellipodia of Ajuba null MEFs. Because p130Cas is recruited to FAK present at integrin complexes (Bouton et al., 2001
), the decreased FAK levels in lamellipodia of migrating Ajuba null MEFs could also account for the decrease in p130Cas. However, overexpression of FAK or a constitutively activated isoform of FAK (SuperFAK) did not rescue the migration defect of Ajuba null cells, or restore proper localization of p130Cas (unpublished data). The inability of SuperFAK to rescue could be explained by the signaling pathways selectively activated by this mutant. SuperFAK expression increased phosphorylation of paxillin and tensin, but not p130Cas (Gabarra-Niecko et al., 2002
). Because p130Cas, but not paxillin, rescue the migration defect in Ajuba null cells, it is perhaps not surprising that SuperFAK did not rescue the migration defect in Ajuba null cells. Ajuba did not interact with FAK in cells, so why FAK levels are reduced in the lamellipodia of Ajuba null cells is not clear.
That Ajuba was found to interact with the mitotic kinase Aurora A, and regulate its kinase activity (Hirota et al., 2003
), suggests that Ajuba may influence the activation (i.e., tyrosine phosphorylation) of FAK and p130Cas by cellular kinases (e.g., Src). Inherent to this argument is that the activation status of each protein is important for its appropriate, stable localization, however.
Focal complexes that form at the leading edge have a short half-life (Zaidel-Bar et al., 2003
). The reduction in pY397-FAK and p130Cas at focal complexes at the leading edge of Ajuba null cells suggests a potential role for Ajuba in focal complex assembly, maintenance, or turnover. Ajuba may anchor p130Cas to focal complexes, thereby strengthening the focal complex. In the absence of Ajuba the strength of adhesion to the ECM may be impaired resulting in decreased size or amount of lamellipodia produced. Alternatively, it is simply the reduction in activation of Rac1 in migrating Ajuba null cells that impairs actin polymerization and, or focal complex formation. Recent work by Bear et al. (2002)
, showed that the relation between net cell translocation and lamellipodia production are based on the persistence of lamellipodia protrusion.
Finally, paxillin, another LIM domain containing focal adhesion protein (distinct from Zyxin/Ajuba), also regulates migration (Turner, 1991
). Through an association with Cool-PIX and p21PAK, paxillin has been shown to activate Rac during migration (Brown et al., 2002
; Feng et al., 2002
). Ajuba null cells did not show any reduction in either the amount or activity of paxillin at the leading edge. This suggests that, although both p130Cas and paxillin activation at the leading edge of migrating cells leads to Rac activation they are not redundant pathways. The persistence of the paxillin–Rac signaling pathway in Ajuba null cells may also explain why the inhibition of cell migration in Ajuba null cells, ex vivo, did not result in a more dramatic whole animal phenotype.