Rap1 translates chemokine signals to integrin activation,cell polarization,and motility across vascular endothelium under flow

Chemokines arrest circulating lymphocytes within the vasculature through the rapid up-regulation of leukocyte integrin adhesive activity, promoting subsequent lymphocyte transmigration. However, the key regulatory molecules regulating this process have remained elusive. Here, we demonstrate that Rap1 plays a pivotal role in chemokine-induced integrin activation and migration. Rap1 was activated by secondary lymphoid tissue chemokine (SLC; CCL21) and stromal-derived factor 1 (CXCL4) treatment in lymphocytes within seconds. Inhibition of Rap1 by Spa1, a Rap1-specific GTPase-activating protein, abrogated chemokine-stimulated lymphocyte rapid adhesion to endothelial cells under flow via intercellular adhesion molecule 1. Expression of a dominant active Rap1V12 in lymphocytes stimulated shear-resistant adhesion, robust cell migration on immobilized intercellular adhesion molecule 1 and vascular cell adhesion molecule 1, and transendothelial migration under flow. We also demonstrated that Rap1V12 expression in lymphocytes induced a polarized morphology, accompanied by the redistribution of CXCR4 and CD44 to the leading edge and uropod, respectively. Spa1 effectively suppressed this polarization after SLC treatment. This unique characteristic of Rap1 may control chemokine-induced lymphocyte extravasation.     

Role of Epac1, an exchange factor for Rap GTPases, in endothelial microtubule dynamics and barrier function

Rap1 GTPase activation by its cAMP responsive nucleotide exchange factor Epac present in endothelial cells increases endothelial cell barrier function with an associated increase in cortical actin. Here, Epac1 was shown to be responsible for these actin changes and to colocalize with microtubules in human umbilical vein endothelial cells. Importantly, Epac activation with a cAMP analogue, 8-pCPT-2'O-Me-cAMP resulted in a net increase in the length of microtubules. This did not require cell-cell interactions or Rap GTPase activation, and it was attributed to microtubule growth as assessed by time-lapse microscopy of human umbilical vein endothelial cell expressing fluorophore-linked microtubule plus-end marker end-binding protein 3. An intact microtubule network was required for Epac-mediated changes in cortical actin and barrier enhancement, but it was not required for Rap activation. Finally, Epac activation reversed microtubule-dependent increases in vascular permeability induced by tumor necrosis factor-alpha and transforming growth factor-beta. Thus, Epac can directly promote microtubule growth in endothelial cells. This, together with Rap activation leads to an increase in cortical actin, which has functional significance for vascular permeability.     

Rap1 controls cell adhesion and cell motility through the regulation of myosin II

We have investigated the role of Rap1 in controlling chemotaxis and cell adhesion in Dictyostelium discoideum. Rap1 is activated rapidly in response to chemoattractant stimulation, and activated Rap1 is preferentially found at the leading edge of chemotaxing cells. Cells expressing constitutively active Rap1 are highly adhesive and exhibit strong chemotaxis defects, which are partially caused by an inability to spatially and temporally regulate myosin assembly and disassembly. We demonstrate that the kinase Phg2, a putative Rap1 effector, colocalizes with Rap1-guanosine triphosphate at the leading edge and is required in an in vitro assay for myosin II phosphorylation, which disassembles myosin II and facilitates filamentous actin-mediated leading edge protrusion. We suggest that Rap1/Phg2 plays a role in controlling leading edge myosin II disassembly while passively allowing myosin II assembly along the lateral sides and posterior of the cell.     

Rudhira/BCAS3 is a cytoskeletal protein that controls Cdc42 activation and directional cell migration during angiogenesis

Cell migration is a common cellular process in angiogenesis and tumor metastasis. Rudhira/BCAS3 (Breast Cancer Amplified Sequence 3) is a conserved protein expressed in the embryonic vasculature and malignant tumors. Here, we show for the first time that Rudhira plays an active role in directional cell migration. Rudhira depletion in endothelial cells inhibits Matrigel-induced tube formation and retards healing of wounded cell monolayers. We demonstrate that during wound healing, Rudhira rapidly re-localizes and promotes Cdc42 activation and recruitment to the leading edge of migrating cells. Rudhira deficient cells show impaired downstream signaling of Cdc42 leading to dramatic changes in actin organization and classic cell polarity defects such as loss of microtubule organizing center (MTOC) and Golgi re-orientation. Biochemical assays and co-localization studies show that Rudhira interacts with microtubules as well as intermediate filaments. Thus, Rudhira could control directional cell migration and angiogenesis by facilitating crosstalk between cytoskeletal elements.     

TIMP-2 promotes cell spreading and adhesion via upregulation of Rap1 signaling

We previously demonstrated that TIMP-2 treatment of human microvascular endothelial cells (hMVECs) activates Rap1 via the pathway of paxillin-Crk-C3G. Here, we show that TIMP-2 overexpression in hMVECs by adenoviral infection enhances Rap1 expression, leading to further increase in Rap1-GTP. TIMP-2 expression, previously reported to inhibit cell migration, also leads to cell spreading accompanied with increased cell adhesion. HMVECs stably expressing Rap1 display a similar phenotype as hMVECs-TIMP-2, whereas the expression of inactive Rap1 mutant, Rap1(38N), leads to elongated appearance with greatly reduced cell adhesion. Furthermore, the phenotype of hMVECs-Rap1(38N) was not reversed by TIMP-2 overexpression. TIMP-2 greatly promotes the association of Rap1 with actin. Therefore, these findings suggest that TIMP-2 mediated alteration in cell morphology requires Rap1, TIMP-2 may recruit Rap1 to sites of actin cytoskeleton remodeling necessary for cell spreading, and enhanced cell adhesion by TIMP-2 expression may hinder cell migration.     

Rap1 GTPase inhibits leukocyte transmigration by promoting endothelial barrier function

The passage of leukocytes out of the blood circulation and into tissues is necessary for the normal inflammatory response, but it also occurs inappropriately in many pathological situations. This process is limited by the barrier presented by the junctions between adjacent endothelial cells that line blood vessels. Here we show that activation of the Rap1 GTPase in endothelial cells accelerated de novo assembly of endothelial cell-cell junctions and increased the barrier function of endothelial monolayers. In contrast, depressing Rap1 activity by expressing Rap1GAP led to disassembly of these junctions and increased their permeability. We also demonstrate that endogenous Rap1 was rapidly activated at early stages of junctional assembly, confirming the involvement of Rap1 during junctional assembly. Intriguingly, elevating Rap1 activity selectively within endothelial cells decreased leukocyte transendothelial migration, whereas inhibiting Rap1 activity by expression of Rap1GAP increased leukocyte transendothelial migration, providing physiological relevance to our hypothesis that Rap1 augments barrier function of inter-endothelial cell junctions. Furthermore, these results suggest that Rap1 may be a novel therapeutic target for clinical conditions in which an inappropriate inflammatory response leads to disease.     

Characterization of interactions of adapter protein RAPL/Nore1B with RAP GTPases and their role in T cell migration

Using a model of integrin-triggered random migration of T cells, we show that stimulation of LFA-1 integrins leads to the activation of Rap1 and Rap2 small GTPases. We further show that Rap1 and Rap2 have distinct roles in adhesion and random migration of these cells and that an adapter protein from the Ras association domain family (Rassf), RAPL, has a role downstream of Rap2 in addition to its link to Rap1. Further characterization of the RAPL protein and its interactions with small GTPases from the Ras family shows that RAPL forms more stable complexes with Rap2 and classical Ras proteins compared with Rap1. The different interaction pattern of RAPL with Rap1 and Rap2 is not affected by the disruption of the C-terminal SARAH domain that we identified as the alpha-helical region responsible for RAPL dimerization in vitro and in cells. Based on mutagenesis and three-dimensional modeling, we propose that interaction surfaces in RAPL-Rap1 and RAPL-Rap2 complexes are different and that a single residue in the switch I region of Rap proteins (residue 39) contributes considerably to the different kinetics of these protein-protein interactions. Furthermore, the distinct role of Rap2 in migration of T cells is lost when this critical residue is converted to the residue present in Rap1. Together, these observations suggest a wider role for Rassf adapter protein RAPL and Rap GTPases in cell motility and show that subtle differences between highly similar Rap proteins could be reflected in distinct interactions with common effectors and their cellular function.     

Rap1-mediated lymphocyte function-associated antigen-1 activation by the T cell antigen receptor is dependent on phospholipase C-gamma1

The small GTPase, Rap1, is a potent activator of leukocyte integrins and enhances the adhesive activity of lymphocyte function-associated antigen-1 (LFA-1) when stimulated by the T cell receptor (TCR) or chemokines. However, the mechanism by which Rap1 is activated remains unclear. Here, we demonstrate that phospholipase C (PLC)-gamma1 plays a critical role in the signaling pathway leading to Rap1 activation triggered by the TCR. In Jurkat T cells, TCR cross-linking triggered persistent Rap1 activation, and SDF-1 (CXCL12) activated Rap1 transiently. A phospholipase C inhibitor, U73122, abrogated Rap1 activation triggered by both the TCR and SDF-1 (CXCL12). PLC-gamma1-deficient Jurkat T cells showed a marked reduction of TCR-triggered Rap1 activation and adhesion to intercellular adhesion molecule-1 (ICAM-1) mediated by LFA-1. In contrast, SDF-1-triggered Rap1 activation and adhesion were not affected in these cells. Transfection of these cells with an expression plasmid encoding PLC-gamma1 restored Rap1 activation by the TCR and the ability to adhere to ICAM-1, accompanied by polarized LFA-1 surface clustering colocalized with regulator of adhesion and polarization enriched in lymphoid tissues (RAPL). Furthermore, when expressed in Jurkat cells, CalDAG-GEFI, a calcium and diacylglycerol-responsive Rap1 exchange factor, associated with Rap1, and resulted in enhanced Rap1 activation and adhesion triggered by the TCR. Our results demonstrate that TCR activation of Rap1 depends on PLC-gamma1. This activity is likely to be mediated by CalDAG-GEFI, which is required to activate LFA-1.     

Regulation by Afadin of Cyclical Activation and Inactivation of Rap1, Rac1, and RhoA Small G Proteins at Leading Edges of Moving NIH3T3 Cells

Cyclical activation and inactivation of Rho family small G proteins, such as Rho, Rac, and Cdc42, are needed for moving cells to form leading edge structures in response to chemoattractants. However, the mechanisms underlying the dynamic regulation of their activities are not fully understood. We recently showed that another small G protein, Rap1, plays a crucial role in the platelet-derived growth factor (PDGF)-induced formation of leading edge structures and activation of Rac1 in NIH3T3 cells. We showed here that knockdown of afadin, an actin-binding protein, in NIH3T3 cells resulted in a failure to develop leading edge structures in association with an impairment of the activation of Rap1 and Rac1 and inactivation of RhoA in response to PDGF. Overexpression of a constitutively active mutant of Rap1 (Rap1-CA) and knockdown of SPA-1, a Rap1 GTPase-activating protein that was negatively regulated by afadin by virtue of binding to it, in afadin-knockdown NIH3T3 cells restored the formation of leading edge structures and the reduction of the PDGF-induced activation of Rac1 and inactivation of RhoA, suggesting that the inactivation of Rap1 by SPA-1 is responsible for inhibition of the formation of leading edge structures. The effect of Rap1-CA on the restoration of the formation of leading edge structures and RhoA inactivation was diminished by additional knockdown of ARAP1, a Rap-activated Rho GAP, which localized at the leading edges of moving NIH3T3 cells. These results indicate that afadin regulates the cyclical activation and inactivation of Rap1, Rac1, and RhoA through SPA-1 and ARAP1.Cell migration is a spatiotemporally regulated process involving the formation and disassembly of protrusions, such as filopodia and lamellipodia, ruffles, focal complexes, and focal adhesions. At the leading edges of moving cells, the continuous formation and disassembly of these protrusive structures are tightly regulated by the actions of the Rho family small G proteins, including RhoA, Rac1, and Cdc42. RhoA regulates the formation of stress fibers and focal adhesions, whereas Rac1 and Cdc42 regulate the formation of lamellipodia and filopodia, respectively (1, 2). In addition, both Rac1 and Cdc42 regulate the formation of focal complexes (3, 4). In order to have cells keep moving, each member of the Rho family small G proteins should cyclically be active and inactive as these leading edge structures are dynamically formed and disassembled. Rac1 and Cdc42 must be activated and RhoA must be inactivated at focal complexes, and vice versa at focal adhesions. Thus, the cyclical activation and inactivation of the Rho family small G proteins are critical for turnover of the transformation of focal complexes into focal adhesions during cell movement. The activities of these small G proteins are tightly regulated by guanine nucleotide exchange factors and GTPase-activating proteins (GAPs).² It is likely that signals from receptors and integrins cooperatively regulate the dynamics of this spatial and temporal activation and inactivation of the Rho family small G proteins. However, the molecular mechanisms of their cyclical activation and inactivation through the regulation of guanine nucleotide exchange factors and GAPs at the leading edges remain largely unknown.We recently showed that platelet-derived growth factor (PDGF) receptor (PDGFR), integrin α_vβ₃, and Necl-5 associate with each other and form a complex and that this complex is clustered at the leading edges of directionally moving NIH3T3 cells in response to PDGF (5, 6). We also demonstrated that PDGF induces the activation of Rap1, which then induces the activation of Rac1 (7). Overexpression of Rap1GAP to inactivate Rap1 inhibits the PDGF-induced formation of leading edge structures, cell movement, and activation of Rac1, suggesting that, in addition to the activation of Rap1, the subsequent activation of Rac1 and presumably the inactivation of RhoA may be critical for the PDGF-induced migration of NIH3T3 cells.Afadin is a nectin- and F-actin-binding protein that is involved in the formation of adherens junctions in cooperation with nectin and cadherin (8). Afadin has multiple domains: two Ras association (RA) domains, a forkhead-associated domain, a dilute domain, a PSD-95-Dlg-1-ZO-1 domain, three proline-rich domains, and an F-actin-binding domain at the C terminus and localizes to adherens junctions in epithelial cells (9). Afadin-knock-out mice showed impaired formation of the cell-cell junction during embryogenesis (10, 11). Although Ras small G protein was initially identified as an interacting molecule with the RA domain of afadin (12), other studies demonstrate that afadin binds GTP-bound Rap1 with a higher affinity than GTP-bound Ras or GTP-bound Rap2 (13, 14). In addition to the functional role of afadin in the organization of cell-cell adhesion, we recently found that, in NIH3T3 cells that do not form cell-cell junctions, afadin did not associate with nectin, localized at the leading edges during cell movement, and was involved in their directional, but not random, movement. The interaction of afadin with Rap1 at the leading edge was necessary for the PDGF-induced directional movement of NIH3T3 cells. Thus, in addition to that in the formation of adherens junctions, afadin plays another role in directional cell movement in NIH3T3 cells.In a series of studies using afadin-knockdown NIH3T3 cells, we found that neither lamellipodia, ruffles, nor focal complexes are formed, suggesting that Rap1 may be inactivated and, conversely, RhoA may be activated in the reduced state of afadin. Here we first examined this possibility and found that Rap1 is indeed inactivated, whereas RhoA is activated in afadin-knockdown NIH3T3 cells. To understand the mechanisms of how the activities of Rap1 and RhoA are regulated in afadin-knockdown NIH3T3 cells, we searched for afadin-interacting proteins that could potentially regulate Rap1 activity and sought Rap1 targets that might regulate RhoA activity. We focused on SPA-1 and ARAP1 and found that these proteins coordinately regulate the activities of these small G proteins. SPA-1 is a GAP for Rap1 that interacts with afadin (15), whereas ARAP1 is a Rho GAP that binds Rap1 and could be activated by virtue of this binding (16). We describe here how afadin regulates the cyclical activation and inactivation of Rap1, Rac1, and RhoA through SPA-1 and ARAP1 at the leading edges of moving NIH3T3 cells. We conclude that afadin is critical for the coordinated regulation of the activation of Rap1 and Rac1 and subsequent inactivation of RhoA necessary for cell movement.     

Regulation of Rap1 activity by RapGAP1 controls cell adhesion at the front of chemotaxing cells

Spatial and temporal regulation of Rap1 is required for proper myosin assembly and cell adhesion during cell migration in Dictyostelium discoideum. Here, we identify a Rap1 guanosine triphosphatase–activating protein (GAP; RapGAP1) that helps mediate cell adhesion by negatively regulating Rap1 at the leading edge. Defects in spatial regulation of the cell attachment at the leading edge in rapGAP1⁻ (null) cells or cells overexpressing RapGAP1 (RapGAP1^OE) lead to defective chemotaxis. rapGAP1⁻ cells have extended chemoattractant-mediated Rap1 activation kinetics and decreased MyoII assembly, whereas RapGAP1^OE cells show reciprocal phenotypes. We see that RapGAP1 translocates to the cell cortex in response to chemoattractant stimulation and localizes to the leading edge of chemotaxing cells via an F-actin–dependent pathway. RapGAP1 localization is negatively regulated by Ctx, an F-actin bundling protein that functions during cytokinesis. Loss of Ctx leads to constitutive and uniform RapGAP1 cortical localization. We suggest that RapGAP1 functions in the spatial and temporal regulation of attachment sites through MyoII assembly via regulation of Rap1–guanosine triphosphate.     

Rap1-dependent pathways coordinate cytokinesis in Dictyostelium

Cytokinesis is the final step of mitosis when a mother cell is separated into two daughter cells. Major cytoskeletal changes are essential for cytokinesis; it is, however, not well understood how the microtubules and actomyosin cytoskeleton are exactly regulated in time and space. In this paper, we show that during the early stages of cytokinesis, in rounded-up Dictyostelium discoideum cells, the small G-protein Rap1 is activated uniformly at the cell cortex. When cells begin to elongate, active Rap1 becomes restricted from the furrow region, where the myosin contractile ring is subsequently formed. In the final stages of cytokinesis, active Rap1 is only present at the cell poles. Mutant cells with decreased Rap1 activation at the poles showed strongly decreased growth rates. Hyperactivation of Rap1 results in severe growth delays and defective spindle formation in adherent cells and cell death in suspension. Furthermore, Rap mutants show aberrant regulation of the actomyosin cytoskeleton, resulting in extended furrow ingression times and asymmetrical cell division. We propose that Rap1 drives cytokinesis progression by coordinating the three major cytoskeletal components: microtubules, actin, and myosin II. Importantly, mutated forms of Rap also affect cytokinesis in other organisms, suggesting a conserved role for Rap in cell division.     

MAP1A light chain 2 interacts with exchange protein activated by cyclic AMP 1 (EPAC1) to enhance Rap1 GTPase activity and cell adhesion

We have recently demonstrated that light chain 2 (LC2) of the microtubule-associated protein MAP1A interacts with the cyclic AMP (cAMP)-binding domain of exchange protein directly activated by cyclic AMP 1 (EPAC1). In the present study we used a simultaneous expression system and found that LC2 enhances both basal and 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3':5'-cyclic monophosphate (8-CPT-2Me-cAMP)-stimulated Rap1 activation by EPAC1. LC2 is known to stabilize microtubules; therefore we examined whether microtubules enhanced Rap1 activation by LC2. Nocodazole inhibited Rap1 activity in cells transfected with EPAC1 alone but had little effect on Rap1 activity in cells transfected with both EPAC1 and LC2. This indicates that part of the actions of LC2 in enhancing EPAC1 activity may be through stabilization of microtubules. We also found that in cells transfected with LC2, Rap1 was more sensitive to activation by 8-CPT-2Me-cAMP. Moreover, LC2 enhanced the ability of transfected and endogenous EPAC1 to interact with cyclic AMP-agarose, indicating that LC2 elicits conformational changes in the cAMP domain of EPAC1, enhancing its ability to be activated by cyclic AMP. We also found that disruption of the interaction of endogenous EPAC1 and LC2 with antibodies to the cAMP domain of EPAC1 abolished Rap1 activity in PC12 cell lysates, demonstrating the importance of LC2 for EPAC1 activation in these cells. Consistent with a role of EPAC1 in controlling integrin activity, we found that cell adhesion to laminin was enhanced in LC2- and EPAC1-transfected cells stimulated with 8-CPT-2Me-cAMP. LC2 is therefore a biological enhancer of EPAC1 activity toward Rap1 and associated downstream signaling mechanisms.     

MAGI-1 is required for Rap1 activation upon cell-cell contact and for enhancement of vascular endothelial cadherin-mediated cell adhesion

Rap1 is a small GTPase that regulates adherens junction maturation. It remains elusive how Rap1 is activated upon cell-cell contact. We demonstrate for the first time that Rap1 is activated upon homophilic engagement of vascular endothelial cadherin (VE-cadherin) at the cell-cell contacts in living cells and that MAGI-1 is required for VE-cadherin-dependent Rap1 activation. We found that MAGI-1 localized to cell-cell contacts presumably by associating with beta-catenin and that MAGI-1 bound to a guanine nucleotide exchange factor for Rap1, PDZ-GEF1. Depletion of MAGI-1 suppressed the cell-cell contact-induced Rap1 activation and the VE-cadherin-mediated cell-cell adhesion after Ca2+ switch. In addition, relocation of vinculin from cell-extracellular matrix contacts to cell-cell contacts after the Ca2+ switch was inhibited in MAGI-1-depleted cells. Furthermore, inactivation of Rap1 by overexpression of Rap1GAPII impaired the VE-cadherin-dependent cell adhesion. Collectively, MAGI-1 is important for VE-cadherin-dependent Rap1 activation upon cell-cell contact. In addition, once activated, Rap1 upon cell-cell contacts positively regulate the adherens junction formation by relocating vinculin that supports VE-cadherin-based cell adhesion.     

Guanine Nucleotide Exchange Factor-H1 Regulates Cell Migration via Localized Activation of RhoA at the Leading Edge

Cell migration involves the cooperative reorganization of the actin and microtubule cytoskeletons, as well as the turnover of cell–substrate adhesions, under the control of Rho family GTPases. RhoA is activated at the leading edge of motile cells by unknown mechanisms to control actin stress fiber assembly, contractility, and focal adhesion dynamics. The microtubule-associated guanine nucleotide exchange factor (GEF)-H1 activates RhoA when released from microtubules to initiate a RhoA/Rho kinase/myosin light chain signaling pathway that regulates cellular contractility. However, the contributions of activated GEF-H1 to coordination of cytoskeletal dynamics during cell migration are unknown. We show that small interfering RNA-induced GEF-H1 depletion leads to decreased HeLa cell directional migration due to the loss of the Rho exchange activity of GEF-H1. Analysis of RhoA activity by using a live cell biosensor revealed that GEF-H1 controls localized activation of RhoA at the leading edge. The loss of GEF-H1 is associated with altered leading edge actin dynamics, as well as increased focal adhesion lifetimes. Tyrosine phosphorylation of focal adhesion kinase and paxillin at residues critical for the regulation of focal adhesion dynamics was diminished in the absence of GEF-H1/RhoA signaling. This study establishes GEF-H1 as a critical organizer of key structural and signaling components of cell migration through the localized regulation of RhoA activity at the cell leading edge.     

Galpha12/13 is essential for directed cell migration and localized Rho-Dia1 function

Scratch-wound assays are frequently used to study directed cell migration, a process critical for embryogenesis, invasion, and tissue repair. The function and identity of trimeric G-proteins in cell behavior during wound healing is not known. Here we show that Galpha12/13, but not Galphaq/11 or Galphai, is indispensable for coordinated and directed cell migration. In mouse embryonic fibroblasts endogenous Rho activity is present at the rear of migrating cells but also at the leading edge, whereas it is undetectable at the cell front of Galpha12/13-deficient mouse embryonic fibroblasts. Spatial activation of Rho at the wound edge can be stimulated by lysophosphatidic acid. Active Rho colocalizes with the diaphanous-related formin Dia1 at the cell front. Galpha12/13-deficient cells lack Dia1 localization to the wound edge and are unable to form orientated, stable microtubules during wound healing. Knock down of Dia1 reveals its requirement for microtubule stabilization as well as polarized cell migration. Thus, we identified Galpha12/13-proteins as essential components linking extracellular signals to localized Rho-Dia1 function during directed cell movement.     

The Rap GTPases regulate B cell migration toward the chemokine stromal cell-derived factor-1 (CXCL12): potential role for Rap2 in promoting B cell migration

Stromal cell-derived factor-1 (SDF-1) is a potent chemoattractant for B cells and B cell progenitors. Although the binding of SDF-1 to its receptor, CXCR4, activates multiple signaling pathways, the mechanism by which SDF-1 regulates cell migration is not completely understood. In this report we show that activation of the Rap GTPases is important for B cells to migrate toward SDF-1. We found that treating B cells with SDF-1 resulted in the rapid activation of both Rap1 and Rap2. Moreover, blocking the activation of Rap1 and Rap2 via the expression of a Rap-specific GTPase-activating protein significantly reduced the ability of B cells to migrate toward SDF-1. Conversely, expressing a constitutively active form of Rap2 increased SDF-1-induced B cell migration. Thus, the Rap GTPases control cellular processes that are important for B cells to migrate toward SDF-1.     

RA-GEF-1, a guanine nucleotide exchange factor for Rap1, is activated by translocation induced by association with Rap1*GTP and enhances Rap1-dependent B-Raf activation

We previously identified RA-GEF-1, a novel guanine nucleotide exchange factor (GEF) for Rap1 with the ability to associate with Rap1.GTP at its Ras/Rap1-associating (RA) domain. Because it possesses a PSD-95/DlgA/ZO-1 (PDZ) domain, it was also named PDZ-GEF. In this report, we have examined the role of the RA domain of this protein in Rap1-mediated cellular responses. A mutant of RA-GEF-1 (RA-GEF-1DeltaRA) carrying a 21-residue deletion at its RA domain fully retains the in vitro GEF activity toward Rap1 but completely loses the Rap1 binding activity. In contrast, RA-GEF-1DeltaRA, expressed in COS-7 cells, exhibits a 3-fold reduction in its in vivo GEF activity toward Rap1 compared with wild-type RA-GEF-1 as examined by the Rap1 pull-down assay. Correspondingly, when coexpressed with wild-type Rap1, RA-GEF-1DeltaRA is unable to further activate B-Raf, whereas RA-GEF-1 stimulates B-Raf as efficiently as activated Rap1. Consistent with these observations, coexpression of activated Rap1 induces translocation of RA-GEF-1, which is otherwise located in the cytoplasm, to the perinuclear compartment, where Rap1 is also predominantly localized. This localization almost coincides with that of the Golgi apparatus, which was detected by anti-trans-Golgi-network 38 antibody. RA-GEF-1DeltaRA fails to show the translocation. These results indicate that RA-GEF-1 defines a novel category of GEF that is translocated to a particular subcellular compartment by association with the GTP-bound form of a small GTPase and catalyzes activation of the GDP-bound form present in the compartment, thereby causing an amplification of cellular responses induced by the small GTPase.     

Bmx is a downstream Rap1 effector in VEGF-induced endothelial cell activation

We had previously shown that Rap1 mediates certain of the signaling pathways involved in VEGF-induced endothelial cell migration, although the downstream Rap1 effectors are not known. Towards the goal of identifying those effectors, we utilized a commercially available antibody array filter to identify proteins that either directly interact with Rap1 or interact indirectly through a multi-protein complex. The protocol identified 10 possible Rap1-interacting proteins, including the Bmx non-receptor tyrosine kinase. The conclusion that VEGF treatment leads to a Rap1/Bmx complex was confirmed by an experiment in which cell lysates from VEGF and control cells were immunoprecipitated with Bmx antibodies and Western blotting was done using anti-Rap1 antibodies. VEGF treatment led to the recruitment of Bmx to the CAS scaffolding protein, and inhibition of the Bmx kinase blocked VEGF-induced cell migration. Formation of a Rap1/Bmx complex was not observed in cells transfected with an expression vector for a dominant-negative Rap1, indicating that Bmx is a downstream Rap1 effector in VEGF-induced endothelial cell activation.     

cAMP-induced Epac-Rap activation inhibits epithelial cell migration by modulating focal adhesion and leading edge dynamics

Epithelial cell migration is a complex process crucial for embryonic development, wound healing and tumor metastasis. It depends on alterations in cell–cell adhesion and integrin–extracellular matrix interactions and on actomyosin-driven, polarized leading edge protrusion. The small GTPase Rap is a known regulator of integrins and cadherins that has also been implicated in the regulation of actin and myosin, but a direct role in cell migration has not been investigated. Here, we report that activation of endogenous Rap by cAMP results in an inhibition of HGF- and TGFβ-induced epithelial cell migration in several model systems, irrespective of the presence of E-cadherin adhesion. We show that Rap activation slows the dynamics of focal adhesions and inhibits polarized membrane protrusion. Importantly, forced integrin activation by antibodies does not mimic these effects of Rap on cell motility, even though it does mimic Rap effects in short-term cell adhesion assays. From these results, we conclude that Rap inhibits epithelial cell migration, by modulating focal adhesion dynamics and leading edge activity. This extends beyond the effect of integrin affinity modulation and argues for an additional function of Rap in controlling the migration machinery of epithelial cells.