Rac1 accumulates in the nucleus during the G2 phase of the cell cycle and promotes cell division

Rac1 regulates a wide variety of cellular processes. The polybasic region of the Rac1 C terminus functions both as a plasma membrane-targeting motif and a nuclear localization sequence (NLS). We show that a triproline N-terminal to the polybasic region contributes to the NLS, which is cryptic in the sense that it is strongly inhibited by geranylgeranylation of the adjacent cysteine. Subcellular fractionation demonstrated endogenous Rac1 in the nucleus and Triton X-114 partition revealed that this pool is prenylated. Cell cycle-blocking agents, synchronization of cells stably expressing low levels of GFP-Rac1, and time-lapse microscopy of asynchronous cells revealed Rac1 accumulation in the nucleus in late G2 and exclusion in early G1. Although constitutively active Rac1 restricted to the cytoplasm inhibited cell division, activated Rac1 expressed constitutively in the nucleus increased the mitotic rate. These results show that Rac1 cycles in and out of the nucleus during the cell cycle and thereby plays a role in promoting cell division.     

Yersinia pseudotuberculosis spatially controls activation and misregulation of host cell Rac1

Yersinia pseudotuberculosis binds host cells and modulates the mammalian Rac1 guanosine triphosphatase (GTPase) at two levels. Activation of Rac1 results from integrin receptor engagement, while misregulation is promoted by translocation of YopE and YopT proteins into target cells. Little is known regarding how these various factors interplay to control Rac1 dynamics. To investigate these competing processes, the localization of Rac1 activation was imaged microscopically using fluorescence resonance energy transfer. In the absence of translocated effectors, bacteria induced activation of the GTPase at the site of bacterial binding. In contrast, the entire cellular pool of Rac1 was inactivated shortly after translocation of YopE RhoGAP. Inactivation required membrane localization of Rac1. The translocated protease YopT had very different effects on Rac1. This protein, which removes the membrane localization site of Rac1, did not inactivate Rac1, but promoted entry of cleaved activated Rac1 molecules into the host cell nucleus, allowing Rac1 to localize with nuclear guanosine nucleotide exchange factors. As was true for YopE, membrane-associated Rac1 was the target for YopT, indicating that the two translocated effectors may compete for the same pool of target protein. Consistent with the observation that YopE inactivation requires membrane localization of Rac1, the presence of YopT in the cell interfered with the action of the YopE RhoGAP. As a result, interaction of target cells with a strain that produces both YopT and YopE resulted in two spatially distinct pools of Rac1: an inactive cytoplasmic pool and an activated nuclear pool. These studies demonstrate that competition between bacterial virulence factors for access to host substrates is controlled by the spatial arrangement of a target protein. In turn, the combined effects of translocated bacterial proteins are to generate pools of a single signaling molecule with distinct localization and activation states in a single cell.     

CYK4 inhibits Rac1-dependent PAK1 and ARHGEF7 effector pathways during cytokinesis

In mitosis, animal cells lose their adhesion to the surrounding surfaces and become rounded. During mitotic exit, they reestablish these adhesions and at the same time physically contract and divide. How these competing processes are spatially segregated at the cell cortex remains mysterious. To address this question, we define the specific effector pathways used by RhoA and Rac1 in mitotic cells. We demonstrate that the MKlp1-CYK4 centralspindlin complex is a guanosine triphosphatase-activating protein (GAP) for Rac1 and not RhoA and that CYK4 negatively regulated Rac1 activity at the cell equator in anaphase. Cells expressing a CYK4 GAP mutant had defects in cytokinesis and showed elevated staining for the cell adhesion marker vinculin. These defects could be rescued by depletion of ARHGEF7 and p21-activated kinase, Rac1-specific effector proteins required for cell adhesion. Based on these findings, we propose that CYK4 GAP activity is required during anaphase to inhibit Rac1-dependent effector pathways associated with control of cell spreading and adhesion.     

Regulation of actin cytoskeleton by Rap1 binding to RacGEF1

Rap1 is rapidly and transiently activated in response to chemoattractant stimulation and helps establish cell polarity by locally modulating cytoskeletons. Here, we investigated the mechanisms by which Rap1 controls actin cytoskeletal reorganization in Dictyostelium and found that Rap1 interacts with RacGEF1 in vitro and stimulates F-actin polymerization at the sites where Rap1 is activated upon chemoattractant stimulation. Live cell imaging using GFP-coronin, a reporter for F-actin, demonstrates that cells expressing constitutively active Rap1 (Rap1CA) exhibit a high level of F-actin uniformly distributed at the cortex including the posterior and lateral sides of the chemotaxing cell. Examination of the localization of a PH-domain containing PIP3 reporter, PhdA-GFP, and the activation of Akt/Pkb and other Ras proteins in Rap1CA cells reveals that activated Rap1 has no effect on the production of PIP3 or the activation of Akt/Pkb and Ras proteins in response to chemoattractant stimulation. Rac family proteins are crucial regulators in actin cytoskeletal reorganization. In vitro binding assay using truncated RacGEF1 proteins shows that Rap1 interacts with the DH domain of RacGEF1. Taken together, these results suggest that Rap1-mediated F-actin polymerization probably occurs through the Rac signaling pathway by directly binding to RacGEF1.     

Rac1-induced cell migration requires membrane recruitment of the nuclear oncogene SET

The Rho GTPase Rac1 controls cell adhesion and motility. The effector loop of Rac1 mediates interactions with downstream effectors, whereas its C-terminus binds the exchange factor beta-Pix, which mediates Rac1 targeting and activation. Here, we report that Rac1, through its C-terminus, also binds the nuclear oncogene SET/I2PP2A, an inhibitor of the serine/threonine phosphatase PP2A. We found that SET translocates to the plasma membrane in cells that express active Rac1 as well as in migrating cells. Membrane targeting of SET stimulates cell migration in a Rac1-dependent manner. Conversely, reduction of SET expression inhibits Rac1-induced migration, indicating that efficient Rac1 signalling requires membrane recruitment of SET. The recruitment of the SET oncogene to the plasma membrane represents a new feature of Rac1 signalling. Our results suggest a model in which Rac1-stimulated cell motility requires both effector loop-based downstream signalling and recruitment of a signalling amplifier, that is, SET, through the hypervariable C-terminus.     

The Nuclear Import of the Small GTPase Rac1 is Mediated by the Direct Interaction with Karyopherin α2

The small GTPase Rac1 is involved in multiple cytosolic functions but recent data point out that Rac1 also translocates to the nucleus to regulate signalling pathways that control gene expression and progression through the cell cycle. Here, we identify the nuclear import receptor karyopherin α2 (KPNA2) as a direct interaction partner of Rac1. The C‐terminal polybasic region of Rac1 contains a nuclear localization signal (NLS), whereas Rac2 and Rac3 lack a functional NLS and do not bind to KPNA2. The presence of the NLS in Rac1 determines the specificity of the interaction and is a prerequisite for the nuclear import. Although this interaction is independent of the Rac1 GDP/GTP loading, the induction of the translocation requires Rac1 activation. The activation of Rac1 via the cytotoxic necrotizing factor 1 and the concurrent inhibition of its proteasomal degradation are crucial for the nuclear accumulation of Rac1. Conversely, the reduction of KPNA2 expression inhibits the nuclear import of Rac1. For the first time, our results show a direct interaction between Rac1 and KPNA2 and argue for a KPNA2‐dependent nuclear import of Rac1. Liquid chromatography tandem mass spectrometry (LC‐MS/MS) analysis revealed that nuclear Rac1 coimmunoprecipitates with numerous proteins. In the nucleus, Rac1 may participate in a variety of so far uncharacterized processes.     

ARF1 controls Rac1 signaling to regulate migration of MDA-MB-231 invasive breast cancer cells

ADP-ribosylation factors (ARFs) are monomeric G proteins that regulate many cellular processes such as reorganization of the actin cytoskeleton. We have previously shown that ARF1 is overexpressed in highly invasive breast cancer cells and contribute to their enhanced migration. In this study, we propose to define the molecular mechanism by which ARF1 regulates this complex cellular response by investigating the role of this ARF GTPase on the activation process of Rac1, a Rho GTPase, associated with lamellipodia formation during cell migration. Here, we first show that inhibition of ARF1 or Rac1 expression markedly impacts the ability of MDA-MB-231 cells to migrate upon EGF stimulation. However, the effect of ARF1 depletion can be reversed by overexpression of the Rac1 active mutant, Rac1 Q⁶¹L. Depletion of ARF1 also impairs the ability of EGF stimulation to promote GTP-loading of Rac1. To further investigate the possible cross-talk between ARF1 and Rac1, we next examined whether they could form a complex. We observed that the two GTPases could directly interact independently of the nature of the nucleotide bound to them. EGF treatment however resulted in the association of Rac1 with its effector IRSp53, which was completely abrogated in ARF1 depleted cells. We present evidences that this ARF isoform is responsible for the plasma membrane targeting of both Rac1 and IRSp53, a step essential for lamellipodia formation. In conclusion, this study provides a new mechanism by which ARF1 regulates cell migration and identifies this GTPase as a promising pharmacological target to reduce metastasis formation in breast cancer patients.     

Regulation of Rac1 activation by the low density lipoprotein receptor-related protein

The low density lipoprotein receptor-related protein (LRP-1) binds and mediates the endocytosis of multiple ligands, transports the urokinase-type plasminogen activator receptor (uPAR) and other membrane proteins into endosomes, and binds intracellular adaptor proteins involved in cell signaling. In this paper, we show that in murine embryonic fibroblasts (MEFs) and L929 cells, LRP-1 functions as a major regulator of Rac1 activation, and that this activity depends on uPAR. LRP-1-deficient MEFs demonstrated increased Rac1 activation compared with LRP-1-expressing MEFs, and this property was reversed by expressing the VLDL receptor, a member of the same gene family as LRP-1, with overlapping ligand-binding specificity. Neutralizing the activity of LRP-1 with receptor-associated protein (RAP) increased Rac1 activation and cell migration in MEFs and L929 cells. The same parameters were unaffected by RAP in uPAR-/- MEFs, prepared from uPAR gene knockout embryos, and in uPAR-deficient LM-TK- cells. Untreated uPAR+/+ MEFs demonstrated substantially increased Rac1 activation compared with uPAR-/- MEFs. In addition to Rac1, LRP-1 suppressed activation of extracellular signal-regulated kinase (ERK) in MEFs; however, it was Rac1 (and not ERK) that was responsible for the effects of LRP-1 on MEF migration. Thus, LRP-1 regulates two signaling proteins in the same cell (Rac1 and ERK), both of which may impact on cell migration. In uPAR-negative cells, LRP-1 neutralization does not affect Rac1 activation, and other mechanisms by which LRP-1 may regulate cell migration are not unmasked.     

IQGAP1 translocates to the nucleus in early S-phase and contributes to cell cycle progression after DNA replication arrest

IQGAP1 is a plasma membrane-associated protein and an important regulator of the actin cytoskeleton, contributing to cell migration, polarity and adhesion. In this study, we demonstrate the nuclear translocation of IQGAP1 using confocal microscopy and cell fractionation. Moreover, we identify a specific pool of IQGAP1 that accumulates in the nucleus during late G1-early S phase of the cell cycle. The nuclear targeting of IQGAP1 was facilitated by N- and C-terminal sequences, and its ability to slowly shuttle between nucleus and cytoplasm/membrane was partly regulated by the CRM1 export receptor. The inhibition of GSK-3β also stimulated nuclear localization of IQGAP1. The dramatic nuclear accumulation of IQGAP1 observed when cells were arrested in G1/S phase suggested a possible role in cell cycle regulation. In support of this, we used immunoprecipitation assays to show that the nuclear pool of IQGAP1 in G1/S-arrested cells associates with DNA replication complex factors RPA32 and PCNA. More important, the siRNA-mediated silencing of IQGAP1 significantly delayed cell cycle progression through S phase and G2/M in NIH 3T3 cells released from thymidine block. Our findings reveal an unexpected regulatory pathway for IQGAP1, and show that a pool of this cytoskeletal regulator translocates into the nucleus in late G1/early S phase to stimulate DNA replication and progression of the cell cycle.     

Regulation of p21-activated kinase-independent Rac1 signal transduction by nischarin

Nischarin regulates Rac1-dependent cell motility by interaction with and inhibition of the p21-activated kinase (PAK1). In addition to regulating the activation of PAK1, Rac1 controls multiple downstream pathways to regulate cell growth and differentiation, as well as cell motility. Signaling by a constitutively activated Rac1 mutant deficient in PAK binding (Rac1Q61L-40C) was examined to determine whether Nischarin impinges on these other Rac1 effector pathways. Nischarin formed immunoprecipitatable complexes with Rac1Q61L and Rac1Q61L-40C when the proteins were co-expressed. In NIH3T3 cells, Rac1Q61L and Rac1Q61L-40C stimulation of a minimal NF-kappaB response element or the cyclin D1 promoter, a downstream target of NF-kappaB, was inhibited by co-expression of Nischarin. Additionally, suppression of endogenous Nischarin protein with small interfering RNA in PC12 cells enhanced Rac1Q61L and Rac1Q61L-40C activation of NF-kappaB. In further support of Nischarin suppressing PAK independent Rac signaling, foci formation in monolayers of NIH3T3 cells by Rac1Q61L-40C in cooperation with c-Raf/CAAX was inhibited by the presence of Nischarin. Nischarin alters the cellular localization of Rac1Q61L and Rac1Q61L-40C to vesicles and this positively correlates with the repression of the Rac1 signal. Thus, Nischarin, in addition to regulating the PAK strand of Rac1 signaling, can also regulate other links in the web of Rac1 signaling pathways.     

Isolation of Rho GTPase effector pathways during axon development

The Rho GTPases Rac1 and Cdc42 have been implicated in the regulation of axon outgrowth and guidance. However, the downstream effector pathways through which these GTPases exert their effects on axon development are not well characterized. Here, we report that axon outgrowth defects within specific subsets of motoneurons expressing constitutively active Drosophila Rac1 largely persist even with the addition of an effector-loop mutation to Rac1 that disrupts its ability to bind to p21-activated kinase (Pak) and other Cdc42/Rac1 interactive-binding (CRIB)-motif effector proteins. While hyperactivation of Pak itself does not lead to axon outgrowth defects as when Rac1 is constitutively activated, live analysis reveals that it can alter filopodial activity within specific subsets of neurons similar to constitutive activation of Cdc42. Moreover, we show that the axon guidance defects induced by constitutive activation of Cdc42 persist even in the absence of Pak activity. Our results suggest that (1) Rac1 controls axon outgrowth through downstream effector pathways distinct from Pak, (2) Cdc42 controls axon guidance through both Pak and other CRIB effectors, and (3) Pak's primary contribution to in vivo axon development is to regulate filopodial dynamics that influence growth cone guidance.     

EGF-induced ERK-activation downstream of FAK requires rac1-NADPH oxidase

Reactive oxygen species (ROS) function as signaling molecules mainly by reversible oxidation of redox-sensitive target proteins. ROS can be produced in response to integrin ligation and growth factor stimulation through Rac1 and its effector protein NADPH oxidase. One of the central roles of Rac1-NADPH oxidase is actin cytoskeletal rearrangement, which is essential for cell spreading and migration. Another important regulator of cell spread is focal adhesion kinase (FAK), a coordinator of integrin and growth factor signaling. Here, we propose a novel role for NADPH oxidase as a modulator of the FAK autophosphorylation site. We found that Rac1-NADPH oxidase enhanced the phosphorylation of FAK at Y397. This site regulates FAK's ability to act as a scaffold for EGF-mediated signaling, including activation of ERK. Accordingly, we found that EGF-induced activation of FAK at Y925, the following activation of ERK, and phosphorylation of FAK at the ERK-regulated S910-site depended upon NADPH oxidase. Furthermore, the inhibition of NADPH oxidase caused excessive focal adhesions, which is in accordance with ERK and FAK being modulators of focal adhesion dissociation. Our data suggest that Rac1 through NADPH oxidase is part of the signaling pathway constituted by FAK, Rac1, and ERK that regulates focal adhesion disassembly during cell spreading.     

PAK1 negatively regulates the activity of the Rho exchange factor NET1

Rho family small G-protein activity is controlled by guanine nucleotide exchange factors that stimulate the release of GDP, thus allowing GTP binding. Once activated, Rho proteins control cell signaling through interactions with downstream effector proteins, leading to changes in cytoskeletal organization and gene expression. The ability of Rho family members to modulate the activity of other Rho proteins is also intrinsic to these processes. In this work we show that the Rac/Cdc42hs-regulated protein kinase PAK1 down-regulates the activity of the RhoA-specific guanine nucleotide exchange factor NET1. Specifically, PAK1 phosphorylates NET1 on three sites in vitro: serines 152, 153, and 538. Replacement of serines 152 and 153 with glutamate residues down-regulates the activity of NET1 as an exchange factor in vitro and its ability to stimulate actin stress fiber formation in cells. Using a phospho-specific antibody that recognizes NET1 phosphorylated on serine 152, we show that PAK1 phosphorylates NET1 on this site in cells and that Rac1 stimulates serine 152 phosphorylation in a PAK1-dependent manner. Furthermore, coexpression of constitutively active PAK1 inhibits the ability of NET1 to stimulate actin polymerization only when serines 152 and 153 are present. These data provide a novel mechanism for the control of RhoA activity by Rac1 through the PAK-dependent phosphorylation of NET1 to reduce its activity as a guanine nucleotide exchange factor.     

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.     

RLIP76 (RalBP1) is an R-Ras effector that mediates adhesion-dependent Rac activation and cell migration

The Ras family of small GTPases regulates cell proliferation, spreading, migration and apoptosis, and malignant transformation by binding to several protein effectors. One such GTPase, R-Ras, plays distinct roles in each of these processes, but to date, identified R-Ras effectors were shared with other Ras family members (e.g., H-Ras). We utilized a new database of Ras-interacting proteins to identify RLIP76 (RalBP1) as a novel R-Ras effector. RLIP76 binds directly to R-Ras in a GTP-dependent manner, but does not physically associate with the closely related paralogues H-Ras and Rap1A. RLIP76 is required for adhesion-induced Rac activation and the resulting cell spreading and migration, as well as for the ability of R-Ras to enhance these functions. RLIP76 regulates Rac activity through the adhesion-induced activation of Arf6 GTPase and activation of Arf6 bypasses the requirement for RLIP76 in Rac activation and cell spreading. Thus, we identify a novel R-Ras effector, RLIP76, which links R-Ras to adhesion-induced Rac activation through a GTPase cascade that mediates cell spreading and migration.     

MEK1 restores migration of polyamine-depleted cells by retention and activation of Rac1 in the cytoplasm

We previously showed that polyamines are required for proliferation and migration both in vivo and in a cultured intestinal epithelial cell (IEC-6) model. Wounding of the IEC-6 monolayer induced transient ERK activation, which was further enhanced by EGF. EGF stimulated migration in control and polyamine-depleted cells, but the degree of stimulation was significantly less in polyamine-depleted cells. Inhibition of MEK1 inhibited basal as well as EGF-induced ERK activation and migration. Expression of constitutively active (CA)-MEK and dominant-negative (DN)-MEK had significant effects on F-actin structure. CA-MEK increased stress fiber and lamellipodia formation, while DN-MEK showed loss of stress fibers and abnormal actin cytoskeletal structure. Unlike EGF, CA-MEK significantly increased migration of both control and polyamine-depleted cells. The most important and significant finding in this study was that polyamine depletion caused localization of Rac1 and RhoA to the nuclear as well as perinuclear regions. Interestingly, CA-MEK completely reversed the subcellular distribution of Rac1 and RhoA proteins in polyamine-depleted cells. Polyamine depletion increased Rac1 in the nuclear fraction and decreased it in the cytoplasmic and membrane fractions of vector-transfected cells. CA-MEK prevented accumulation of Rac1 in the nucleus. Polyamine depletion significantly decreased Rac1 activity during 6-h migration in vector-transfected cells. Cells transfected with CA-MEK had almost identical levels of activated Rac1 in all three groups. These results suggest that polyamine depletion prevents activation of Rac1 and RhoA by sequestering them to the nucleus and that expression of constitutively active MEK reverses this effect, creating the cellular localization required for activation. epidermal growth factor; extracellular signal-regulated kinase; IEC-6 cells     

Phospholipase D activity regulates integrin-mediated cell spreading and migration by inducing GTP-Rac translocation to the plasma membrane

Small GTPase Rac is a crucial regulator of actin cytoskeletal rearrangement, and it plays an important role in cell spreading, migration, mitogenesis, phagocytosis, superoxide generation, and axonal growth. It is generally accepted that Rac activity is regulated by the guanosine triphosphate (GTP)/guanosine diphosphate (GDP) cycle. But, it is suggested that in addition to Rac-GTP loading, membrane localization is required for the initiation of downstream effector signaling. However, the molecular mechanisms that control the targeting of GTP-Rac to the plasma membrane remain largely unknown. Here, we have uncovered a signaling pathway linking phospholipase D (PLD) to the localized functions of Rac1. We show that PLD product phosphatidic acid (PA) acts as a membrane anchor of Rac1. The C-terminal polybasic motif of Rac1 is responsible for direct interaction with PA, and Rac1 mutated in this region is incapable of translocating to the plasma membrane and of activating downstream target p21-activated kinase upon integrin activation. Finally, we show that PA induces dissociation of Rho-guanine nucleotide dissociation inhibitor from Rac1 and that PA-mediated Rac1 localization is important for integrin-mediated lamellipodia formation, cell spreading, and migration. These results provide a novel molecular mechanism for the GTP-Rac1 localization through the elevating PLD activity, and they suggest a general mechanism for diverse cellular functions that is required localized Rac activation.