Activation of type I phosphatidylinositol 4-phosphate 5-kinase isoforms by the Rho GTPases, RhoA, Rac1, and Cdc42

Type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) catalyzes the formation of the phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP(2)), which is implicated in many cellular processes. The Rho GTPases, RhoA and Rac1, have been shown previously to activate PIP5K and to bind PIP5K. Three type I PIP5K isoforms (Ialpha,Ibeta, and Igamma) have been identified; however, it is unclear whether these isoforms are differentially or even sequentially regulated by Rho GTPases. Here we show that RhoA and Rac1, as well as Cdc42, but not the Ras-like GTPases, RalA and Rap1A, markedly stimulate PIP(2) synthesis by all three PIP5K isoforms expressed in human embryonic kidney 293 cells, both in vitro and in vivo. RhoA-stimulated PIP(2) synthesis by the PIP5K isoforms was mediated by the RhoA effector, Rho-kinase. Stimulation of PIP5K isoforms by Rac1 and Cdc42 was apparently independent of and additive with RhoA- and Rho-kinase, as shown by studies with C3 transferase and Rho-kinase mutants. RhoA, and to a lesser extent Rac1, but not Cdc42, interacted in a nucleotide-independent form with all three PIP5K isoforms. Binding of PIP5K isoforms to GTP-bound, but not GDP-bound, RhoA could be displaced by Rho-kinase, suggesting a direct and constitutive PIP5K-Rho GTPase binding, which, however, does not trigger PIP5K activation. In summary, our findings indicate that synthesis of PIP(2) by the three PIP5K isoforms is controlled by RhoA, acting via Rho-kinase, as well as Rac1 and Cdc42, implicating that regulation of PIP(2) synthesis has a central position in signaling by these three Rho GTPases.     

G-protein-coupled receptor activation induces the membrane translocation and activation of phosphatidylinositol-4-phosphate 5-kinase I alpha by a Rac- and Rho-dependent pathway.

Phosphatidylinositol 4,5-bisphosphate (PI4,5P(2)) mediates cell motility and changes in cell shape in response to extracellular stimuli. In platelets, it is synthesized from PI4P by PIP5K in response to stimulation of a G-protein-coupled receptor by an agonist, such as the thrombin. In the present study, we have addressed the pathway that induces PIP5K I alpha activation following the addition of thrombin. Under resting condition expressed PIP5K I alpha was predominantly localized in a perinuclear distribution. After stimulation of the thrombin receptor, PAR1, or overexpression of a constitutively active variant of G alpha(q), PIP5K I alpha translocated to the plasma membrane. Movement of PIP5K I alpha to the cell membrane was dependent on both GTP-bound Rac and Rho, but not Arf, because: 1) inactive GDP-bound variants of either Rac or Rho blocked the translocation induced by constitutively active G alpha(q), 2) constitutively GTP-bound active variants of Rac or Rho induced PIP5K I alpha translocation in the absence of other stimuli, and 3) constitutively active variants of Arf1 or Arf6 failed to induce membrane translocation of PIP5K I alpha. In addition, a dominant negative variant of Rho blocked the PIP5K I alpha membrane translocation induced by constitutively active Rac, whereas dominant negative variants of either Rac or Arf6 failed to block PIP5K I alpha membrane translocation induced by constitutively active Rho. This implies that the effect on PIP5K I alpha by Rac is indirect, and requires the activation of Rho. In contrast to the findings with PIP5K I alpha, the related lipid kinase PIP4K failed to undergo translocation after stimulation by small GTP-binding proteins Rac or Rho. We also tested whether membrane localization of PIP5K I alpha correlated with an increase in its lipid kinase activity and found that co-expressing of PIP5K I alpha with either constitutively active G alpha(q), Rac, or Rho led to a 5- to 7-fold increase in PIP5K I alpha activity. Thus, these findings suggest that stimulation of a G-protein-coupled receptor (PAR1) leads to the sequential activation of G alpha(q), Rac, Rho, and PIP5K I alpha. Once activated and translocated to the cell membrane, PIP5K I alpha becomes available to phosphorylate PI4P to generate PI4,5P(2) on the plasma membrane.     

Isoform-specific roles of the GTPase activating protein Nadrin in cytoskeletal reorganization of platelets

Cytoskeletal reorganization of activated platelets plays a crucial role in hemostasis and thrombosis and implies activation of Rho GTPases. Rho GTPases are important regulators of cytoskeletal dynamics and function as molecular switches that cycle between an inactive and an active state. They are regulated by GTPase activating proteins (GAPs) that stimulate GTP hydrolysis to terminate Rho signaling. The regulation of Rho GTPases in platelets is not explored. A detailed characterization of Rho regulation is necessary to understand activation and inactivation of Rho GTPases critical for platelet activation and aggregation. Nadrin is a RhoGAP regulating cytoplasmic protein explored in the central nervous system. Five Nadrin isoforms are known that share a unique GAP domain, a serine/threonine/proline-rich domain, a SH3-binding motif and an N-terminal BAR domain but differ in their C-terminus. Here we identified Nadrin in platelets where it co-localizes to actin-rich regions and Rho GTPases. Different Nadrin isoforms selectively regulate Rho GTPases (RhoA, Cdc42 and Rac1) and cytoskeletal reorganization suggesting that – beside the GAP domain – the C-terminus of Nadrin determines Rho specificity and influences cell physiology. Furthermore, Nadrin controls RhoA-mediated stress fibre and focal adhesion formation. Spreading experiments on fibrinogen revealed strongly reduced cell adhesion upon Nadrin overexpression. Unexpectedly, the Nadrin BAR domain controls Nadrin-GAP activity and acts as a guidance domain to direct this GAP to its substrate at the plasma membrane. Our results suggest a critical role for Nadrin in the regulation of RhoA, Cdc42 and Rac1 in platelets and thus for platelet adhesion and aggregation.     

ExoS Rho GTPase-activating protein activity stimulates reorganization of the actin cytoskeleton through Rho GTPase guanine nucleotide disassociation inhibitor

ExoS is a bifunctional Type III cytotoxin of Pseudomonas aeruginosa with N-terminal Rho GTPase-activating protein (RhoGAP) and C-terminal ADP-ribosyltransferase domains. Although the ExoS RhoGAP inactivates Cdc42, Rac, and RhoA in vivo, the relationship between ExoS RhoGAP and the eukaryotic regulators of Rho GTPases is not clear. The present study investigated the roles of Rho GTPase guanine nucleotide disassociation inhibitor (RhoGDI) in the reorganization of actin cytoskeleton mediated by ExoS RhoGAP. A green fluorescent protein-RhoGDI fusion protein was engineered and found to elicit actin reorganization through the inactivation of Rho GTPases. Green fluorescent protein-RhoGDI and ExoS RhoGAP cooperatively stimulated actin reorganization and translocation of Cdc42 from membrane to cytosol, and a RhoGDI mutant, RhoGDI(I177D), that is defective in extracting Rho GTPases off the membrane inhibited the actions of RhoGDI and ExoS RhoGAP on the translocation of Cdc42 from membrane to cytosol. A human RhoGDI small interfering RNA was transfected into HeLa cells to knock down 90% of the endogenous RhoGDI expression. HeLa cells with knockdown RhoGDI were resistant to the reorganization of the actin cytoskeleton elicited by type III-delivered ExoS RhoGAP. This indicates that ExoS RhoGAP and RhoGDI function in series to inactivate Rho GTPases, in which RhoGDI extracting GDP-bound Rho GTPases off the membrane and sequestering them in cytosol is the rate-limiting step in Rho GTPase inactivation. A eukaryotic GTPase-activating protein, p50RhoGAP, showed a similar cooperativity with RhoGDI on actin reorganization, suggesting that ExoS RhoGAP functions as a molecular mimic of eukaryotic RhoGAPs to inactivate Rho GTPases through RhoGDI.     

Shear stress-induced endothelial cell polarization is mediated by Rho and Rac but not Cdc42 or PI 3-kinases

Shear stress induces endothelial polarization and migration in the direction of flow accompanied by extensive remodeling of the actin cytoskeleton. The GTPases RhoA, Rac1, and Cdc42 are known to regulate cell shape changes through effects on the cytoskeleton and cell adhesion. We show here that all three GTPases become rapidly activated by shear stress, and that each is important for different aspects of the endothelial response. RhoA was activated within 5 min after stimulation with shear stress and led to cell rounding via Rho-kinase. Subsequently, the cells respread and elongated within the direction of shear stress as RhoA activity returned to baseline and Rac1 and Cdc42 reached peak activation. Cell elongation required Rac1 and Cdc42 but not phosphatidylinositide 3-kinases. Cdc42 and PI3Ks were not required to establish shear stress-induced polarity although they contributed to optimal migration speed. Instead, Rho and Rac1 regulated directionality of cell movement. Inhibition of Rho or Rho-kinase did not affect the cell speed but significantly increased cell displacement. Our results show that endothelial cells reorient in response to shear stress by a two-step process involving Rho-induced depolarization, followed by Rho/Rac-mediated polarization and migration in the direction of flow.     

Nadrin GAP activity is isoform- and target-specific regulated by tyrosine phosphorylation

Cytoskeletal reorganization is crucial for platelet adhesion and thrombus formation to avoid excessive bleeding. Major regulators of cytoskeletal dynamics are small GTPases of the Rho family. Rho GTPases become activated by G-protein coupled receptor activation, downstream of immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors and by outside-in signaling of integrins. They act as molecular switches and cycle between active and inactive states. GTPase activating proteins (GAPs) stimulate the hydrolysis of GTP to GDP to terminate Rho signaling. Nadrin is a RhoGAP that was recently identified in platelets. Five Nadrin isoforms are known consisting of a unique GAP and an N-terminal BAR domain responsible for the selective regulation of RhoA, Cdc42 and Rac1. Besides BAR domain mediated regulation of Nadrin GAP activity nothing is known about the regulation of Nadrin and the impact on cytoskeletal reorganization. Here we show that Nadrin becomes tyrosine phosphorylated upon platelet activation. We found Src family proteins (Src, Lyn, Fyn) to be responsible to control Nadrin GAP activity by phosphorylation. Interestingly, phosphorylation of Nadrin leads to tightly regulated Rho activation that was found to be Nadrin isoform- and (Rho) target-specific. Src-phosphorylation of Nadrin5 mediated inactivation of Cdc42 while RhoA and Rac1 became activated upon Src-mediated phosphorylation of Nadrin2. Our results suggest a critical role for spatial and temporal regulation of Nadrin and thus for the control of Rho GTPases in platelets.     

Rho-kinase induces association of adducin with the cytoskeleton in platelet activation

We examined whether adducin function is regulated through Rho-kinase after agonist stimulation in platelets. A variety of stimuli such as thrombin, STA(2) (a stable analog of TXA(2)), Ca(2+) ionophore, phorbol diester, and shear stress induced phosphorylation of alpha-adducin at Thr445. Preincubation with the Rho-kinase inhibitor Y-27632 in platelets inhibited agonist-induced phosphorylation of alpha-adducin. STA(2) stimulation led to a redistribution of adducin from Triton-insoluble (high speed) fraction (membrane skeleton) to Triton-insoluble (low speed) fraction (cytoskeleton) and detergent-soluble fraction. Phosphoadducin at Thr445 was selectively isolated in the cytoskeletal fraction, whereas phosphoadducin at Ser726 was mainly present in the Triton-soluble fraction. Y-27632 inhibition of STA(2)-induced alpha-adducin phosphorylation at Thr445 inhibited incorporation of alpha-adducin and spectrin into the platelet cytoskeleton, although Y-27632 did not affect phosphorylation of alpha-adducin at Ser726. These results suggest that Rho-kinase regulates the association of alpha-adducin and spectrin with the actin cytoskeleton in platelet activation.     

Targeting Rac1 by the Yersinia effector protein YopE inhibits caspase-1-mediated maturation and release of interleukin-1beta

Yersinia bacteria can take control of the host cell by injecting so-called Yop effector proteins into the cytosol of the cells to which they adhere. Using Yersinia enterocolitica strains that are deficient for one or more Yops, we could show that YopE and, to a lesser extent, YopT interfere with the caspase-1-mediated maturation of prointerleukin-1beta in macrophages. In addition, overexpression of YopE and YopT was shown to prevent the autoproteolytic activation of caspase-1 in a way that is dependent on their inhibitory effect on Rho GTPases. Expression of constitutive-active or dominant-negative Rho GTPase mutants or treatment with Rho GTPase inhibitors confirmed the role of Rho GTPases and, in particular, Rac1 in the autoactivation of caspase-1. Rac1-induced caspase-1 activation was mediated by its effect on LIM kinase-1, which is targeting the actin cytoskeleton. Rac-1 and LIM kinase-1 dominant-negative mutants were shown to inhibit caspase-1 activation induced by overexpression of Asc, which is a caspase-1-activating adaptor protein. Moreover, Rac1 as well as YopE and YopT significantly modulated caspase-1 oligomerization. These results highlight a previously unknown function of Rho GTPases in the activation of caspase-1 and give new insight on the role of YopE in immune-escape mechanisms of Yersinia.     

Activation of ERM proteins in vivo by Rho involves phosphatidyl-inositol 4-phosphate 5-kinase and not ROCK kinases.

When activated, ERM (ezrin, radixin, moesin) proteins are recruited to the plasma membrane, with concomitant carboxy-terminal threonine phosphorylation, where they crosslink actin filaments to the plasma membrane to form microvilli (reviewed in [1] [2] [3] [4] [5]). Here, we report that, when NIH3T3 or HeLa cells were transfected with a constitutively active mutant of the small GTPase RhoA (V14RhoA), microvilli were induced and the level of carboxy-terminal threonine-phosphorylated ERM proteins (CPERM) [6] [7] increased approximately 30-fold. This increase was not observed following transfection of constitutively active forms of two other Rho-family GTPases, Rac1 and Cdc42, or of a direct effector of Rho, Rho-kinase (also known as ROKalpha or ROCK-II) [8] [9] [10]. The V14RhoA-induced phosphorylation of ERM proteins was not suppressed by Y-27632, a specific inhibitor of ROCK kinases including Rho-kinase [11]. Overexpression of another direct effector of Rho, phosphatidylinositol 4-phosphate 5-kinase (PI4P5K) type Ialpha [12] [13] [14], but not a kinase-inactive mutant [15], increased approximately sixfold the level of CPERM, and induced microvilli. Together with the previous finding that the PI4P5K product phosphatidylinositol 4,5-bisphosphate (PIP(2)) activates ERM proteins in vitro [16], our data suggest that PIP(2), and not ROCK kinases, is involved in the RhoA-dependent activation of ERM proteins in vivo. The active state of ERM proteins is maintained through threonine phosphorylation by as yet undetermined kinases, leading to microvillus formation.     

p190-RhoGAP as an integral component of the Tiam1/Rac1-induced downregulation of Rho

The Rho family of small GTPases plays a central role in intracellular signal transduction, particularly in reorganization of the actin cytoskeleton. Rho activity induces cell contractility, whereas Rac promotes cellular protrusion, which counteracts Rho signaling. In this regard, the reciprocal balance between these GTPases determines cell morphology and migratory behavior. Here we demonstrate that Tiam1/Rac1 signaling is able to antagonize Rho activity directly at the GTPase level in COS-7 cells. p190-RhoGAP plays a central regulatory role in this signaling pathway. Interfering with its activation by Src-kinase-dependent tyrosine phosphorylation or its recruitment to the membrane through interaction with the SH2 domains of p120-RasGAP blocks the Tiam1-mediated rapid downregulation of Rho. This process is mediated by Rac1, but not by Rac2 or Rac3 isoforms. Our data provide evidence for a biochemical pathway of the reciprocal regulation of two related small GTPases, which are key elements in cell migration.     

Type Ialpha phosphatidylinositol-4-phosphate 5-kinase mediates Rac-dependent actin assembly

Action polymerization is essential for a variety of cellular processes including movement, cell division and shape change. The induction of actin polymerization requires the generation of free actin filament barbed ends, which results from the severing or uncapping of pre-existing actin filaments [1] [2], or de novo nucleation, initiated by the Arp2/3 complex [3] [4] [5] [6] [7]. Although little is known about the signaling pathways that regulate actin assembly, small GTPases of the Rho family appear to be necessary [8] [9] [10] [11]. In thrombin-stimulated platelets, the Rho family GTPase Rac1 induces actin polymerization by stimulating the uncapping of actin filament barbed ends [2]. The mechanism by which Rac regulates uncapping is unclear, however. We previously demonstrated that Rac interacts with a type I phosphatidylinositol-4-phosphate 5-kinase (PIP 5-kinase) in a GTP-independent manner [12] [13]. Because PIP 5-kinases synthesize phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)), a lipid that dissociates capping proteins from the barbed ends of actin filaments [14] [15] [16], they are good candidates for mediating the effects of Rac on actin assembly. Here, we have identified the Rac-associated PIP 5-kinase as the PIP 5-kinase isoforms alpha and beta. When added to permeabilized platelets, PIP 5-kinase alpha induced actin filament uncapping and assembly. In contrast, a kinase-inactive PIP 5-kinase alpha mutant failed to induce actin assembly and blocked assembly stimulated by thrombin or Rac. Furthermore, thrombin- or Rac-induced actin polymerization was inhibited by a point mutation in the carboxyl terminus of Rac that disrupts PIP 5-kinase binding. These results demonstrate that PIP 5-kinase alpha is a critical mediator of thrombin- and Rac-dependent actin assembly.     

Differential regulation of WASP and N-WASP by Cdc42, Rac1, Nck, and PI(4,5)P2

The Wiskott-Aldrich syndrome protein (WASP) and neural WASP (N-WASP) are key players in regulating actin cytoskeleton via the Arp2/3 complex. It has been widely reported that the WASP proteins are activated by Rho family small GTPase Cdc42 and that Rac1 acts through SCAR/WAVE proteins. However, a systematic study of the specificity of different GTPases for different Arp2/3 activators has not been conducted. In this study, we have expressed, purified, and characterized completely soluble, highly active, and autoinhibited full-length human WASP and N-WASP from mammalian cells. We show a novel N-WASP activation by Rho family small GTPase Rac1. This GTPase exclusively stimulates N-WASP and has no effects on WASP. Rac1 is a significantly more potent N-WASP activator than Cdc42. In contrast, Cdc42 is a more effective activator of WASP than N-WASP. Lipid vesicles containing PIP2 significantly improve actin nucleation by the Arp2/3 complex and N-WASP in the presence of Rac1 or Cdc42. PIP2 vesicles have no effect on WASP activity alone. Moreover, the inhibition of WASP-stimulated actin nucleation in the presence of Cdc42 and PIP2 vesicles has been observed. We found that adaptor proteins Nck1 or Nck2 are the most potent WASP and N-WASP activators with distinct effects on the WASP family members. Our in vitro data demonstrates differential regulation of full-length WASP and N-WASP by cellular activators that highlights fundamental differences of response at the protein-protein level.     

Regulation of platelet Rac1 and Cdc42 activation through interaction with calmodulin

Rac1 and Cdc42 are members of the Rho family of small GTPases and have been shown to induce lamellipodia and filopodia formation, respectively. This leads to changes in cytoskeleton organization and as a consequence affects cell migration. In the present work we demonstrate that endogenous Rac1 and Cdc42 interact with calmodulin (CaM) in a Ca(2+)-dependent fashion. The interaction of Rac1 and Cdc42 with CaM was shown to be direct. This novel interaction was further confirmed in platelets using co-immunoprecipitation studies. Using CaM database analysis and in vitro peptide competition assays we have identified a 14 amino acid region in Rac1 that is essential for CaM binding. The scrambled form of the peptide did not bind CaM demonstrating specificity of the predicted CaM binding region in Rac1. A similar region capable of binding CaM exists in Cdc42. Furthermore, using the optimal activation time-point for each GTPase, the role of CaM in the function of Rac1 and Cdc42 was examined. Results demonstrate that in human platelets, thrombin caused maximal activation of Rac1 and Cdc42 at ~60 s and ~25 s respectively. The potent CaM antagonist W7 abolished thrombin-mediated activation of Rac1. However, addition of W7 resulted in the activation of Cdc42 over basal and W7 did not inhibit thrombin-mediated activation of Cdc42. The less potent CaM inhibitor, W5, did not have any effect on Rac1 and Cdc42 activation. The results demonstrate that in platelets, binding of CaM to Rac1 increases its activation while its binding to Cdc42 reduces the activation of this GTPase. This suggests an important role for CaM in coordinating Rac1 and Cdc42 activation and in the regulation of cytoskeleton remodeling.     

Regulation of rho GTPases by crosstalk and neuronal activity in vivo

Proper development of neurons depends on synaptic activity, but the mechanisms of activity-dependent neuronal growth are not well understood. The small GTPases, RhoA, Rac, and Cdc42, regulate neuronal morphogenesis by controlling the assembly and stability of the actin cytoskeleton. We report an in situ method to determine endogenous Rho GTPase activity in intact Xenopus brain. We use this method to provide evidence for crosstalk between Rho GTPases in optic tectal cells. Moreover, crosstalk between the Rho GTPases appears to affect dendritic arbor development in vivo. Finally, we demonstrate that optic nerve stimulation regulates Rho GTPase activity in a glutamate receptor-dependent manner. These data suggest a link between glutamate receptor function, Rho GTPase activity, and dendritic arbor growth in the intact animal.     

Cell migration and signaling specificity is determined by the phosphatidylserine recognition motif of Rac1

The Rho guanosine triphosphatases (GTPases) control cell shape and motility and are frequently overexpressed during malignant growth. These proteins act as molecular switches cycling between active GTP- and inactive GDP-bound forms. Despite being membrane anchored via their isoprenylated C termini, Rho GTPases rapidly translocate between membrane and cytosolic compartments. Here, we show that the Rho GTPase Rac1 preferentially interacts with phosphatidylserine (PS)-containing bilayers through its polybasic motif (PBM). Rac1 isoprenylation contributes to membrane avidity but is not critical for PS recognition. The similar protein Cdc42 (cell division cycle 42), however, only associates with PS when prenylated. Conversely, other Rho GTPases such as Rac2, Rac3, and RhoA do not bind to PS even when they are prenylated. Cell stimulation with PS induces translocation of Rac1 toward the plasma membrane and stimulates GTP loading, membrane ruffling, and filopodia formation. This stimulation also promotes Cdc42 activation and phosphorylation of mitogen-activated protein kinase through Rac1/PS signaling. Consequently, the PBM specifically directs Rac1 to effect cytoskeletal rearrangement and cell migration by selective membrane phospholipid targeting.     

The novel Rho-family GTPase rif regulates coordinated actin-based membrane rearrangements

Small GTPases of the Rho family have a critical role in controlling cell morphology, motility and adhesion through dynamic regulation of the actin cytoskeleton [1,2]. Individual Rho GTPases have been shown to regulate distinct components of the cytoskeletal architecture; RhoA stimulates the bundling of actin filaments into stress fibres [3], Rac reorganises actin to produce membrane sheets or lamellipodia [4] and Cdc42 causes the formation of thin, actin-rich surface projections called filopodia [5]. We have isolated a new Rho-family GTPase, Rif (Rho in filopodia), and shown that it represents an alternative signalling route to the generation of filopodial structures. Coordinated regulation of Rho-family GTPases can be used to generate more complicated actin rearrangements, such as those underlying cell migration [6]. In addition to inducing filopodia, Rif functions cooperatively with Cdc42 and Rac to generate additional structures, increasing the diversity of actin-based morphology.     

Identification of P-Rex1 as a novel Rac1-guanine nucleotide exchange factor (GEF) that promotes actin remodeling and GLUT4 protein trafficking in adipocytes

Phosphoinositide 3-kinase (PI3K) signaling promotes the translocation of the glucose transporter, GLUT4, to the plasma membrane in insulin-sensitive tissues to facilitate glucose uptake. In adipocytes, insulin-stimulated reorganization of the actin cytoskeleton has been proposed to play a role in promoting GLUT4 translocation and glucose uptake, in a PI3K-dependent manner. However, the PI3K effectors that promote GLUT4 translocation via regulation of the actin cytoskeleton in adipocytes remain to be fully elucidated. Here we demonstrate that the PI3K-dependent Rac exchange factor, P-Rex1, enhances membrane ruffling in 3T3-L1 adipocytes and promotes GLUT4 trafficking to the plasma membrane at submaximal insulin concentrations. P-Rex1-facilitated GLUT4 trafficking requires a functional actin network and membrane ruffle formation and occurs in a PI3K- and Rac1-dependent manner. In contrast, expression of other Rho GTPases, such as Cdc42 or Rho, did not affect insulin-stimulated P-Rex1-mediated GLUT4 trafficking. P-Rex1 siRNA knockdown or expression of a P-Rex1 dominant negative mutant reduced but did not completely inhibit glucose uptake in response to insulin. Collectively, these studies identify a novel RacGEF in adipocytes as P-Rex1 that, at physiological insulin concentrations, functions as an insulin-dependent regulator of the actin cytoskeleton that contributes to GLUT4 trafficking to the plasma membrane.