Epidermal growth factor induction of front-rear polarity and migration in keratinocytes is mediated by integrin-linked kinase and ELMO2

Epidermal growth factor (EGF) is a potent chemotactic and mitogenic factor for epidermal keratinocytes, and these properties are central for normal epidermal regeneration after injury. The involvement of mitogen-activated protein kinases as mediators of the proliferative effects of EGF is well established. However, the molecular mechanisms that mediate motogenic responses to this growth factor are not clearly understood. An obligatory step for forward cell migration is the development of front-rear polarity and formation of lamellipodia at the leading edge. We show that stimulation of epidermal keratinocytes with EGF, but not with other growth factors, induces development of front-rear polarity and directional migration through a pathway that requires integrin-linked kinase (ILK), Engulfment and Cell Motility-2 (ELMO2), integrin β1, and Rac1. Furthermore, EGF induction of front-rear polarity and chemotaxis require the tyrosine kinase activity of the EGF receptor and are mediated by complexes containing active RhoG, ELMO2, and ILK. Our findings reveal a novel link between EGF receptor stimulation, ILK-containing complexes, and activation of small Rho GTPases necessary for acquisition of front-rear polarity and forward movement.     

Emerging role of ILK and ELMO2 in the integration of adhesion and migration pathways

Integrins and their associated proteins are essential components of the cellular machinery that modulates adhesion and migration. In particular, integrin-linked kinase (ILK), which binds to the cytoplasmic tail of β1 integrins, is required for migration in a variety of cell types. We previously identified engulfment and motility 2 (ELMO2) as an ILK-binding protein in epidermal keratinocytes. Recently, we investigated the biological role of the ILK/ELMO2 complexes, and found that they exist in the cytoplasm. ILK/ELMO2 species are recruited by active RhoG to the plasma membrane, where they induce Rac1 activation and formation of lamellipodia at the leading edge of migrating cells. A large number of growth factors and cytokines induce keratinocyte migration. However, we found that formation of RhoG/ELMO2/ILK complexes occurs selectively upon stimulation by epidermal growth factor, but not by transforming growth factor-β1 or keratinocyte growth factor. Herein we discuss the relevance of these complexes to our understanding of the molecular mechanisms involved in cell migration, as well as their potential functions in morphogenesis and tissue regeneration following injury.     

Emerging role of ILK and ELMO2 in the integration of adhesion and migration pathways

Integrins and their associated proteins are essential components of the cellular machinery that modulates adhesion and migration. In particular, integrin-linked kinase (ILK), which binds to the cytoplasmic tail of β1 integrins, is required for migration in a variety of cell types. We previously identified engulfment and motility 2 (ELMO2) as an ILK-binding protein in epidermal keratinocytes. Recently, we investigated the biological role of the ILK/ELMO2 complexes, and found that they exist in the cytoplasm. ILK/ELMO2 species are recruited by active RhoG to the plasma membrane, where they induce Rac1 activation and formation of lamellipodia at the leading edge of migrating cells. A large number of growth factors and cytokines induce keratinocyte migration. However, we found that formation of RhoG/ELMO2/ILK complexes occurs selectively upon stimulation by epidermal growth factor, but not by transforming growth factor-β1 or keratinocyte growth factor. Herein we discuss the relevance of these complexes to our understanding of the molecular mechanisms involved in cell migration, as well as their potential functions in morphogenesis and tissue regeneration following injury.     

An ELMO2-RhoG-ILK network modulates microtubule dynamics

ELMO2 belongs to a family of scaffold proteins involved in phagocytosis and cell motility. ELMO2 can simultaneously bind integrin-linked kinase (ILK) and RhoG, forming tripartite ERI complexes. These complexes are involved in promoting β1 integrin–dependent directional migration in undifferentiated epidermal keratinocytes. ELMO2 and ILK have also separately been implicated in microtubule regulation at integrin-containing focal adhesions. During differentiation, epidermal keratinocytes cease to express integrins, but ERI complexes persist. Here we show an integrin-independent role of ERI complexes in modulation of microtubule dynamics in differentiated keratinocytes. Depletion of ERI complexes by inactivating the Ilk gene in these cells reduces microtubule growth and increases the frequency of catastrophe. Reciprocally, exogenous expression of ELMO2 or RhoG stabilizes microtubules, but only if ILK is also present. Mechanistically, activation of Rac1 downstream from ERI complexes mediates their effects on microtubule stability. In this pathway, Rac1 serves as a hub to modulate microtubule dynamics through two different routes: 1) phosphorylation and inactivation of the microtubule-destabilizing protein stathmin and 2) phosphorylation and inactivation of GSK-3β, which leads to the activation of CRMP2, promoting microtubule growth. At the cellular level, the absence of ERI species impairs Ca²⁺-mediated formation of adherens junctions, critical to maintaining mechanical integrity in the epidermis. Our findings support a key role for ERI species in integrin-independent stabilization of the microtubule network in differentiated keratinocytes.     

Dock4 is regulated by RhoG and promotes Rac-dependent cell migration

Cell migration is essential for normal development and many pathological processes including tumor metastasis. Rho family GTPases play important roles in this event. In particular, Rac is required for lamellipodia formation at the leading edge during migration. Dock4 is a member of the Dock180 family proteins, and Dock4 mutations are present in a subset of human cancer cell lines. However, the function and the regulatory mechanism of Dock4 remain unclear. Here we show that Dock4 is regulated by the small GTPase RhoG and its effector ELMO and promotes cell migration by activating Rac1. Dock4 formed a complex with ELMO, and expression of active RhoG induced translocation of the Dock4-ELMO complex from the cytoplasm to the plasma membrane and enhanced the Dock4- and ELMO-dependent Rac1 activation and cell migration. On the other hand, RNA interference-mediated knockdown of Dock4 in NIH3T3 cells reduced cell migration. Taken together, these results suggest that Dock4 plays an important role in the regulation of cell migration through activation of Rac1, and that RhoG is a key upstream regulator for Dock4.     

Phagocytosis of apoptotic cells is regulated by a UNC-73/TRIO-MIG-2/RhoG signaling module and armadillo repeats of CED-12/ELMO

BACKGROUND: Phagocytosis of cells undergoing apoptosis is essential during development, cellular turnover, and wound healing. Failure to promptly clear apoptotic cells has been linked to autoimmune disorders. C. elegans CED-12 and mammalian ELMO are evolutionarily conserved scaffolding proteins that play a critical role in engulfment from worm to human. ELMO functions together with Dock180 (a guanine nucleotide exchange factor for Rac) to mediate Rac-dependent cytoskeletal reorganization during engulfment and cell migration. However, the components upstream of ELMO and Dock180 during engulfment remain elusive. RESULTS: Here, we define a conserved signaling module involving the small GTPase RhoG and its exchange factor TRIO, which functions upstream of ELMO/Dock180/Rac during engulfment. Complementary studies in C. elegans show that MIG-2 (which we identify as the homolog of mammalian RhoG) and UNC-73 (the TRIO homolog) also regulate corpse clearance in vivo, upstream of CED-12. At the molecular level, we identify a novel set of evolutionarily conserved Armadillo (ARM) repeats within CED-12/ELMO that mediate an interaction with activated MIG-2/RhoG; this, in turn, promotes Dock180-mediated Rac activation and cytoskeletal reorganization. CONCLUSIONS: The combination of in vitro and in vivo studies presented here identify two evolutionarily conserved players in engulfment, TRIO/UNC73 and RhoG/MIG-2, and the TRIO --> RhoG signaling module is linked by ELMO/CED-12 to Dock180-dependent Rac activation during engulfment. This work also identifies ARM repeats within CED-12/ELMO and their role in linking RhoG and Rac, two GTPases that function in tandem during engulfment.     

The Par-Tiam1 complex controls persistent migration by stabilizing microtubule-dependent front-rear polarity

BACKGROUND: The establishment and maintenance of cell polarity is crucial for many biological functions and is regulated by conserved protein complexes. The Par polarity complex consisting of Par3, Par6, and PKCzeta, in conjunction with Tiam1-mediated Rac signaling, controls apical-basal cell polarity in contacting epithelial cells. Here we tested the hypothesis that the Par complex, in conjunction with Tiam1, controls "front-rear" polarity during the persistent migration of freely migrating keratinocytes. RESULTS: Wild-type (WT) epidermal keratinocytes lacking cell-cell contacts are stably front-rear polarized and migrate persistently. In contrast, Tiam1-deficient (Tiam1 KO) and (si)Par3-depleted keratinocytes are generally unpolarized and migrate randomly because front-rear polarity is short lived. Immunoprecipitation experiments show that in migrating keratinocytes, Tiam1 associates with Par3 and PKCzeta. Moreover, Par3, PKCzeta, and Tiam1 proteins are enriched at the leading edges of polarized keratinocytes. Tiam1 KO keratinocytes are impaired in chemotactic migration toward growth factors, whereaes haptotactic migration is similar to WT. Par3 depletion or the blocking of PKCzeta signaling in WT keratinocytes impairs chemotaxis but has no additional effect on Tiam1 KO cells. The migratory and morphological defects in keratinocytes with impaired Par-Tiam1 function closely resemble cells with pharmacologically destabilized microtubules (MTs). Indeed, MTs in Tiam1 KO keratinocytes and WT cells treated with a PKCzeta inhibitor are unstable, thereby negatively influencing directional but not random migration. CONCLUSIONS: We conclude that the Par-Tiam1 complex stabilizes front-rear polarization of noncontacting migratory cells, thereby stimulating persistent and chemotactic migration, whereas in contacting keratinocytes, the same complex controls the establishment of long-lasting apical-basal polarity. These findings underscore a remarkable flexibility of the Par polarity complex that, depending on the biological context, controls distinct forms of cellular polarity.     

Identification of two signaling submodules within the CrkII/ELMO/Dock180 pathway regulating engulfment of apoptotic cells

Removal of apoptotic cells is a dynamic process coordinated by ligands on apoptotic cells, and receptors and other signaling proteins on the phagocyte. One of the fundamental challenges is to understand how different phagocyte proteins form specific and functional complexes to orchestrate the recognition/removal of apoptotic cells. One evolutionarily conserved pathway involves the proteins cell death abnormal (CED)-2/chicken tumor virus no. 10 (CT10) regulator of kinase (Crk)II, CED-5/180 kDa protein downstream of chicken tumor virus no. 10 (Crk) (Dock180), CED-12/engulfment and migration (ELMO) and MIG-2/RhoG, leading to activation of the small GTPase CED-10/Rac and cytoskeletal remodeling to promote corpse uptake. Although the role of ELMO : Dock180 in regulating Rac activation has been well defined, the function of CED-2/CrkII in this complex is less well understood. Here, using functional studies in cell lines, we observe that a direct interaction between CrkII and Dock180 is not required for efficient removal of apoptotic cells. Similarly, mutants of CED-5 lacking the CED-2 interaction motifs could rescue engulfment and migration defects in CED-5 deficient worms. Mutants of CrkII and Dock180 that could not biochemically interact could colocalize in membrane ruffles. Finally, we identify MIG-2/RhoG (which functions upstream of Dock180 : ELMO) as a possible point of crosstalk between these two signaling modules. Taken together, these data suggest that Dock180/ELMO and CrkII act as two evolutionarily conserved signaling submodules that coordinately regulate engulfment.     

Dock180 and ELMO1 proteins cooperate to promote evolutionarily conserved Rac-dependent cell migration

Cell migration is essential throughout embryonic and adult life. In numerous cell systems, the small GTPase Rac is required for lamellipodia formation at the leading edge and movement ability. However, the molecular mechanisms leading to Rac activation during migration are still unclear. Recently, a mammalian superfamily of proteins related to the prototype member Dock180 has been identified with homologues in Drosophila and Caenorhabditis elegans. Here, we addressed the role of Dock180 and ELMO1 proteins, which function as a complex to mediate Rac activation, in mammalian cell migration. Using mutants of Dock180 and ELMO1 in a Transwell assay as well as transgenic rescue of a C. elegans mutant lacking CED-5 (Dock180 homologue), we identified specific regions of Dock180 and ELMO1 required for migration in vitro and in a whole animal model. In both systems, the Dock180.ELMO1 complex formation and the ability to activate Rac were required. We also found that ELMO1 regulated multiple Dock180 superfamily members to promote migration. Interestingly, deletion mutants of ELMO1 missing their first 531 or first 330 amino acids that can still bind and cooperate with Dock180 in Rac activation failed to promote migration, which correlated with the inability to localize to lamellipodia. This finding suggests that Rac activation by the ELMO.Dock180 complex at discrete intracellular locations mediated by the N-terminal 330 amino acids of ELMO1 rather than generalized Rac activation plays a role in cell migration.     

Characterization of a novel interaction between ELMO1 and ERM proteins

ERMs are closely related proteins involved in cell migration, cell adhesion, maintenance of cell shape, and formation of microvilli through their ability to cross-link the plasma membrane with the actin cytoskeleton. ELMO proteins are also known to regulate actin cytoskeleton reorganization through activation of the small GTPbinding protein Rac via the ELMO-Dock180 complex. Here we showed that ERM proteins associate directly with ELMO1 as purified recombinant proteins in vitro and at endogenous levels in intact cells. We mapped ERM binding on ELMO1 to the N-terminal 280 amino acids, which overlaps with the region required for binding to the GTPase RhoG, but is distinct from the C-terminal Dock180 binding region. Consistent with this, ELMO1 could simultaneously bind both radixin and Dock180, although radixin did not alter Rac activation via the Dock180-ELMO complex. Most interestingly, radixin binding did not affect ELMO binding to active RhoG and a trimeric complex of active RhoG-ELMO-radixin could be detected. Moreover, the three proteins colocalized at the plasma membrane. Finally, in contrast to most other ERM-binding proteins, ELMO1 binding occurred independently of the state of radixin C-terminal phosphorylation, suggesting an ELMO1 interaction with both the active and inactive forms of ERM proteins and implying a possible role of ELMO in localizing or retaining ERM proteins in certain cellular sites. Together these data suggest that ELMO1-mediated cytoskeletal changes may be coordinated with ERM protein crosslinking activity during dynamic cellular functions.     

Hippo kinases maintain polarity during directional cell migration in Caenorhabditis elegans

Precise positioning of cells is crucial for metazoan development. Despite immense progress in the elucidation of the attractive cues of cell migration, the repulsive mechanisms that prevent the formation of secondary leading edges remain less investigated. Here, we demonstrate that Caenorhabditis elegans Hippo kinases promote cell migration along the anterior–posterior body axis via the inhibition of dorsal–ventral (DV) migration. Ectopic DV polarization was also demonstrated in gain‐of‐function mutant animals for C. elegans RhoG MIG‐2. We identified serine 139 of MIG‐2 as a novel conserved Hippo kinase phosphorylation site and demonstrated that purified Hippo kinases directly phosphorylate MIG‐2^S139. Live imaging analysis of genome‐edited animals indicates that MIG‐2^S139 phosphorylation impedes actin assembly in migrating cells. Intriguingly, Hippo kinases are excluded from the leading edge in wild‐type cells, while MIG‐2 loss induces uniform distribution of Hippo kinases. We provide evidence that Hippo kinases inhibit RhoG activity locally and are in turn restricted to the cell body by RhoG‐mediated polarization. Therefore, we propose that the Hippo–RhoG feedback regulation maintains cell polarity during directional cell motility.     

P-cadherin promotes collective cell migration via a Cdc42-mediated increase in mechanical forces

Collective cell migration (CCM) is essential for organism development, wound healing, and metastatic transition, the primary cause of cancer-related death, and it involves cell–cell adhesion molecules of the cadherin family. Increased P-cadherin expression levels are correlated with tumor aggressiveness in carcinoma and aggressive sarcoma; however, how P-cadherin promotes tumor malignancy remains unknown. Here, using integrated cell biology and biophysical approaches, we determined that P-cadherin specifically induces polarization and CCM through an increase in the strength and anisotropy of mechanical forces. We show that this mechanical regulation is mediated by the P-cadherin/β-PIX/Cdc42 axis; P-cadherin specifically activates Cdc42 through β-PIX, which is specifically recruited at cell–cell contacts upon CCM. This mechanism of cell polarization and migration is absent in cells expressing E- or R-cadherin. Thus, we identify a specific role of P-cadherin through β-PIX–mediated Cdc42 activation in the regulation of cell polarity and force anisotropy that drives CCM.     

ILK modulates epithelial polarity and matrix formation in hair follicles

Hair follicle morphogenesis requires coordination of multiple signals and communication between its epithelial and mesenchymal constituents. Cell adhesion protein platforms, which include integrins and integrin-linked kinase (ILK), are critical for hair follicle formation. However, their precise contribution to this process is poorly understood. We show that in the absence of ILK, the hair follicle matrix lineage fails to develop, likely due to abnormalities in development of apical–basal cell polarity, as well as in laminin-511 and basement membrane assembly at the tip of the hair bud. These defects also result in impaired specification of hair matrix and absence of precortex and inner sheath root cell lineages. The molecular pathways affected in ILK-deficient follicles are similar to those in the absence of epidermal integrin β1 and include Wnt, but not sonic hedgehog, signaling. ILK-deficient hair buds also show abnormalities in the dermal papilla. Addition of exogenous laminin-511 restores morphological and molecular markers associated with hair matrix formation, indicating that ILK regulates hair bud cell polarity and functions upstream from laminin-511 assembly to regulate the developmental progression of hair follicles beyond the germ stage.     

The inositol 5-phosphatase SHIP2 is an effector of RhoA and is involved in cell polarity and migration

Cell migration is essential for various physiological and pathological processes. Polarization in motile cells requires the coordination of several key signaling molecules, including RhoA small GTPases and phosphoinositides. Although RhoA participates in a front-rear polarization in migrating cells, little is known about the functional interaction between RhoA and lipid turnover. We find here that src-homology 2-containing inositol-5-phosphatase 2 (SHIP2) interacts with RhoA in a GTP-dependent manner. The association between SHIP2 and RhoA is observed in spreading and migrating U251 glioma cells. The depletion of SHIP2 attenuates cell polarization and migration, which is rescued by wild-type SHIP2 but not by a mutant defective in RhoA binding. In addition, the depletion of SHIP2 impairs the proper localization of phosphatidylinositol 3,4,5-trisphosphate, which is not restored by a mutant defective in RhoA binding. These results suggest that RhoA associates with SHIP2 to regulate cell polarization and migration.     

Ephexin4 and EphA2 mediate cell migration through a RhoG-dependent mechanism

EphA2, a member of the Eph receptor family, is frequently overexpressed in a variety of human cancers, including breast cancers, and promotes cancer cell motility and invasion independently of its ligand ephrin stimulation. In this study, we identify Ephexin4 as a guanine nucleotide exchange factor (GEF) for RhoG that interacts with EphA2 in breast cancer cells, and knockdown and rescue experiments show that Ephexin4 acts downstream of EphA2 to promote ligand-independent breast cancer cell migration and invasion toward epidermal growth factor through activation of RhoG. The activation of RhoG recruits its effector ELMO2 and a Rac GEF Dock4 to form a complex with EphA2 at the tips of cortactin-rich protrusions in migrating breast cancer cells. In addition, the Dock4-mediated Rac activation is required for breast cancer cell migration. Our findings reveal a novel link between EphA2 and Rac activation that contributes to the cell motility and invasiveness of breast cancer cells.     

The tumor suppressor Lgl1 regulates front-rear polarity of migrating cells

Cell migration is a highly integrated, multistep process that plays an important role in physiological and pathological processes. The migrating cell is highly polarized, with complex regulatory pathways that integrate its component processes spatially and temporally.¹ The Drosophila tumor suppressor, Lethal (2) giant larvae (Lgl), regulates apical-basal polarity in epithelia and asymmetric cell division.² But little is known about the role of Lgl in establishing cell polarity in migrating cells. Recently, we showed that the mammalian Lgl1 interacts directly with non-muscle myosin IIA (NMIIA), inhibiting its ability to assemble into filaments in vitro.³ Lgl1 also regulates the cellular localization of NMIIA, the maturation of focal adhesions, and cell migration.³ We further showed that phosphorylation of Lgl1 by aPKCζ prevents its interaction with NMIIA and is important for Lgl1 and acto-NMII cytoskeleton cellular organization.⁴ Lgl is a critical downstream target of the Par6-aPKC cell polarity complex; we showed that Lgl1 forms two distinct complexes in vivo, Lgl1-NMIIA and Lgl1-Par6-aPKCζ in different cellular compartments.⁴ We further showed that aPKCζ and NMIIA compete to bind directly to Lgl1 through the same domain. These data provide new insights into the role of Lgl1, NMIIA, and Par6-aPKCζ in establishing front-rear polarity in migrating cells. In this commentary, I discuss the role of Lgl1 in the regulation of the acto-NMII cytoskeleton and its regulation by the Par6-aPKCζ polarity complex, and how Lgl1 activity may contribute to the establishment of front-rear polarity in migrating cells.     

Emerging role of paxillin-PKL in regulation of cell adhesion,polarity and migration

Cell adhesion and motility is of fundamental importance during development, normal physiology and pathologic conditions such as tumor metastasis. Focal adhesion proteins and their dynamic interactions play a critical role in the regulation of directed cell migration upon exposure to extracellular guidance cues. Using a combination of pharmacological inhibitors, knockout and knockdown cells and mutant protein expression, we recently reported that following adhesion and growth factor stimulation the dynamic interaction between paxillin and PKL(GIT2) is regulated by Src/FAK-dependent phosphorylation of PKL and that this interaction is necessary for the coordination of Rho family GTPase signaling controlling front-rear cell polarity and thus directional migration. Herein, we discuss the implications of these observations.Key words: FAK, Src, PTP-PEST, PIX, PAK, Arf6, Rac1, cell polarity, cell migration, tyrosine phosphorylation     

An evolutionarily conserved autoinhibitory molecular switch in ELMO proteins regulates Rac signaling

Dedicator of cytokinesis (DOCK) proteins are guanine nucleotide exchange factors (GEFs) controlling the activity of Rac1/Cdc42 during migration, phagocytosis, and myoblast fusion [1-4]. Engulfment and cell motility (ELMO) proteins bind a subset of DOCK members and are emerging as critical regulators of Rac signaling [5-10]. Although formation of a DOCK180/ELMO complex is not essential for Rac1 activation, ELMO mutants deficient in binding to DOCK180 are unable to promote cytoskeleton remodeling [11]. How ELMO regulates signaling through DOCK GEFs is poorly understood. Here, we identify an autoinhibitory switch in ELMO presenting homology to a regulatory unit described for Dia formins. One part of the switch, composed of a Ras-binding domain (RBD) and Armadillo repeats, is positioned N-terminally while the other is housed in the C?terminus. We demonstrate interaction between these fragments, suggesting autoinhibition of ELMO. Using a bioluminescence resonance energy transfer biosensor, we establish that ELMO undergoes conformational changes upon disruption of autoinhibition. We found that engagement of ELMO to RhoG, or with DOCK180, promotes the relief of autoinhibition in ELMO. Functionally, we found that ELMO mutants with impaired autoregulatory activity promote cell?elongation. These results demonstrate an unsuspected level of regulation for Rac1 signaling via autoinhibition of ELMO.     

Galectin-3– and phospho-caveolin-1–dependent outside-in integrin signaling mediates the EGF motogenic response in mammary cancer cells

In murine mammary epithelial cancer cells, galectin-3 binding to β1,6-acetylglucosaminyltransferase V (Mgat5)–modified N-glycans restricts epidermal growth factor (EGF) receptor mobility in the plasma membrane and acts synergistically with phospho-caveolin-1 to promote integrin-dependent matrix remodeling and cell migration. We show that EGF signaling to RhoA is galectin-3 and phospho-caveolin-1 dependent and promotes the formation of transient, actin-rich, circular dorsal ruffles (CDRs), cell migration, and fibronectin fibrillogenesis via Src- and integrin-linked kinase (ILK)–dependent signaling. ILK, Src, and galectin-3 also mediate EGF stimulation of caveolin-1 phosphorylation. Direct activation of integrin with Mn²⁺ induces galectin-3, ILK, and Src-dependent RhoA activation and caveolin-1 phosphorylation. This suggests that in response to EGF, galectin-3 enables outside-in integrin signaling stimulating phospho-caveolin-1–dependent RhoA activation, actin reorganization in CDRs, cell migration, and fibronectin remodeling. Similarly, caveolin-1/galectin-3–dependent EGF signaling induces motility, peripheral actin ruffling, and RhoA activation in MDA-MB-231 human breast carcinoma cells, but not HeLa cells. These studies define a galectin-3/phospho-caveolin-1/RhoA signaling module that mediates integrin signaling downstream of growth factor activation, leading to actin and matrix remodeling and tumor cell migration in metastatic cancer cells.