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.