In this study, we identified EGF as a physiological signal that induces recruitment of RhoG, ELMO2, and ILK to the plasma membrane to induce formation of lamellipodia and keratinocyte polarization, which also results in forward migration. In these cells, there appears to be a degree of selectivity in the mechanisms that couple EGF signaling with ILK/ELMO2 species. This is evidenced by the inability of exogenous ILK/ELMO2 to enhance cell polarization in response to stimulation by other growth factors, such as TGF-β1 and KGF. These observations are in keeping with the reported activation of multiple guanine-nucleotide exchange factors that stimulate RhoG in response to EGF but not to other growth factors (Samson et al., 2010
). Of note, EGF receptor stimulation has also been shown to induce activation of the EphA2 receptor tyrosine kinase, which in turn recruits RhoG/ELMO2 to promote migration of mammary carcinoma cells (Hiramoto-Yamaki et al., 2010
). Whether ILK is a component of the EphA2/RhoG/ELMO2 complexes in breast carcinoma cells remains to be investigated but could potentially place ILK as a key therapeutic target. Similarly, the possible involvement of EphA2 in EGF induction of keratinocyte polarization remains to be investigated.
Although ILK has been implicated in various growth factor signaling pathways (Hehlgans et al., 2007
), the direct involvement of ILK in EGF induction of cell polarization and forward movement is a novel function for this scaffold protein. ELMO2 has recently been reported to be a key player in EGF-induced membrane ruffling and polarization (Hiramoto-Yamaki et al., 2010
), and our findings provide evidence for a link between ILK and ELMO2 in EGF-induced cell polarization. It is significant that the formation of ILK/ELMO2 complexes can occur in the absence of EGF receptor stimulation (Ho et al., 2009
). Whether EGF receptor tyrosine kinase activity is sufficient for recruitment of this complex to the plasma membrane and/or subsequent downstream events remains to be established.
EGF receptor stimulation can rapidly activate RhoG, allowing it to interact with ELMO2 at the plasma membrane (Katoh and Negishi, 2003
; Hiramoto-Yamaki et al., 2010
). It is significant that, as shown in the present study, expression of RhoG F37A, which does not interact with ELMO2 and inhibits cell polarization and migration, does not interfere with ILK/ELMO2 association in cytoplasmic fractions. This suggests that RhoG activation downstream from EGF may serve to recruit ILK/ELMO2 to the plasma membrane rather than to catalyze the association between those two proteins.
ILK and ELMO2 have been independently implicated in regulation of Rac1 activity: the former, in complexes that contain α- or β-Pix, and the latter as a direct guanine-exchange factor when bound to Dock proteins (Katoh and Negishi, 2003
; Ho et al., 2009
; Hiramoto-Yamaki et al., 2010
). In our studies, Rac1
gene inactivation inhibits EGF-induced polarization. We observed similar effects upon expression of an ELMO2 deletion mutant incapable of interacting with Dock proteins (data not shown), suggesting that Rac1 may be activated directly downstream of ILK/ELMO2, potentially by ILK/ELMO2/Dock complexes. ELMO2/ILK may also serve as a hub to recruit other Rac1-activating proteins to induce formation of cell extensions and forward movement. How exactly Rac1 is activated downstream from ILK/ELMO2 remains to be determined.
Targeted inactivation of the Ilk
gene in epidermal keratinocytes severely affects their ability to spread, acquire front–rear polarity, and migrate (Lorenz et al., 2007
; Nakrieko et al., 2008
), emphasizing the importance of ILK for cell motion. Similarly, keratinocytes treated with ELMO2 shRNAs showed impaired ability to polarize in response to EGF, although they did not exhibit detectable defects in cell adhesion or spreading. These observations are consistent with the proposal that ELMO2 plays a minor role, if any, in keratinocyte attachment and spreading onto laminin 332 but is a key component of cell polarization in response to EGF. Alternatively, in the absence of ELMO2, ELMO1 may mediate some of these functions. Phenotypic redundancy between these two ELMO proteins has been suggested to occur in ELMO1-null mice (Elliott et al., 2010
The existence of cross-talk mechanisms between integrins and growth factor receptors is well established. Loss of β1 integrin impairs keratinocyte adhesion, spreading, and forward movement (Raghavan et al., 2003
). We now show that β1 integrin works in conjunction with EGF receptors to induce keratinocyte polarization. β1 integrins are essential for EGF signaling in various cell types, as they modulate endosomal trafficking of EGF receptors, which is necessary for optimal activation (Morello et al., 2011
). β1 integrins are also involved in formation and activation of complexes containing N-WASP and Cdc42, which are required for chemotaxis induced by platelet-derived growth factor (King et al., 2011
). The precise role of β1 integrins in the EGF/RhoG/ELMO2/ILK pathway may occur at various levels and is an important area for future research.
In conclusion, our data are consistent with a model in which EGF receptor stimulation promotes activation and recruitment of RhoG at the plasma membrane (). Active RhoG then associates with ELMO2/ILK complexes, resulting in activation of Rac1, formation of lamellipodia, and cell polarization, followed by forward migration. Aspects of this model that remain important areas for future research are how β1 integrins modulate this process and the events that link RhoG/ELMO2/ILK to Rac1 activation. ELMO2 forms complexes with several Dock proteins, including Dock4, which directly activate Rac1 (Ueda et al., 2008
). Whether this mechanism also functions in the context of the ILK/ELMO2 species remains to be defined.
FIGURE 8: Proposed model of EGF induction of keratinocyte polarization. EGF receptor stimulation mediates activation of RhoG and its localization to the plasma membrane. Active RhoG recruits ILK/ELMO2 complexes already present in the cytosol. The activation of (more ...)