Differential Phosphorylation of RhoGDI Mediates the Distinct Cycling of Cdc42 and Rac1 to Regulate Second-phase Insulin Secretion

Cdc42 cycling through GTP/GDP states is critical for its function in the second/granule mobilization phase of insulin granule exocytosis in pancreatic islet beta cells, although the identities of the Cdc42 cycling proteins involved remain incomplete. Using a tandem affinity purification-based mass spectrometry screen for Cdc42 cycling factors in beta cells, RhoGDI was identified. RNA interference-mediated depletion of RhoGDI from isolated islets selectively amplified the second phase of insulin release, consistent with the role of RhoGDI as a Cdc42 cycling factor. Replenishment of RhoGDI to RNA interference-depleted cells normalized secretion, confirming the action of RhoGDI to be that of a negative regulator of Cdc42 activation. Given that RhoGDI also regulates Rac1 activation in beta cells, and that Rac1 activation occurs in a Cdc42-dependent manner, the question as to how the beta cell utilized RhoGDI for differential Cdc42 and Rac1 cycling was explored. Co-immunoprecipitation was used to determine that RhoGDI-Cdc42 complexes dissociated upon stimulation of beta cells with glucose for 3 min, correlating with the timing of glucose-induced Cdc42 activation and the onset of RhoGDI tyrosine phosphorylation. Glucose-induced disruption of RhoGDI-Rac1 complexes occurred subsequent to this, coincident with Rac1 activation, which followed the onset of RhoGDI serine phosphorylation. RhoGDI-Cdc42 complex dissociation was blocked by mutation of RhoGDI residue Tyr-156, whereas RhoGDI-Rac1 dissociation was blocked by RhoGDI mutations Y156F and S101A/S174A. Finally, expression of a triple Y156F/S101A/S174A-RhoGDI mutant specifically inhibited only the second/granule mobilization phase of glucose-stimulated insulin secretion, overall supporting the integration of RhoGDI into the activation cycling mechanism of glucose-responsive small GTPases.     

YES,a Src Family Kinase,Is a Proximal Glucose-specific Activator of Cell Division Cycle Control Protein 42 (Cdc42) in Pancreatic Islet β Cells

Second-phase insulin secretion sustains insulin release in the face of hyperglycemia associated with insulin resistance, requiring the continued mobilization of insulin secretory granules to the plasma membrane. Cdc42, the small Rho family GTPase recognized as the proximal glucose-specific trigger to elicit second-phase insulin secretion, signals downstream to activate the p21-activated kinase (PAK1), which then signals to Raf-1/MEK/ERK to induce filamentous actin (F-actin) remodeling, to ultimately mobilize insulin granules to the plasma membrane. However, the steps required to initiate Cdc42 activation in a glucose-specific manner in β cells have remained elusive. Toward this, we identified the involvement of the Src family kinases (SFKs), based upon the ability of SFK inhibitors to block glucose-stimulated Cdc42 and PAK1 activation events as well as the amplifying pathway of glucose-stimulated insulin release, in MIN6 β cells. Indeed, subsequent studies performed in human islets revealed that SFK phosphorylation was induced only by glucose and within 1 min of stimulation before the activation of Cdc42 at 3 min. Furthermore, pervanadate treatment validated the phosphorylation event to be tyrosine-specific. Although RT-PCR showed β cells to express five different SFK proteins, only two of these, YES and Fyn kinases, were found localized to the plasma membrane, and of these two, only YES kinase underwent glucose-stimulated tyrosine phosphorylation. Immunodetection and RNAi analyses further established YES kinase as a proximal glucose-specific signal in the Cdc42-signaling cascade. Identification of YES kinase provides new insight into the mechanisms underlying the sustainment of insulin secretion via granule mobilization/replenishment and F-actin remodeling.     

Glucose regulates the cortical actin network through modulation of Cdc42 cycling to stimulate insulin secretion

Glucose-stimulated insulin granule exocytosis in pancreatic beta-cells involves cortical actin remodeling that results in the transient disruption of the interaction between polymerized actin with the plasma membrane t-SNARE (target membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex. To examine the mechanism underlying the initiation of cortical actin remodeling, we have used the actin nucleating/stabilizing agent jasplakinolide to show that remodeling is initiated at a step proximal to the ATP-sensitive K+ channels in the stimulus-secretion pathway. Confocal immunofluorescent microscopy revealed that cortical actin remodeling was required for glucose-stimulated insulin secretion. Furthermore, glucose was found to mediate the endogenous activation state of the Rho family GTPase Cdc42, a positive proximal effector of actin polymerization, resulting in a net decrease of Cdc42-GTP within 5 min of stimulation. Intriguingly, glucose stimulation resulted in the rapid and reversible glucosylation of Cdc42, suggesting that glucose inactivated Cdc42 by selective glucosylation to induce cortical actin rearrangement. Moreover, expression of the constitutively active form of Cdc42 (Q61L) inhibited glucose-stimulated insulin secretion, whereas the dominant negative form (T17N) was without effect, suggesting that glucose-stimulated insulin secretion requires Cdc42 cycling to the GDP-bound state. In contrast, KCl-stimulated insulin secretion was unaffected by the expression of dominant negative or constitutively active Cdc42 and ceased to modulate endogenous Cdc42 activation, consistent with glucose-dependent cortical actin remodeling. These findings reveal that glucose regulates the cortical actin network through modulation of Cdc42 cycling to induce insulin secretion in pancreatic beta-cells.     

Analysis of small GTPase signaling pathways using p21-activated kinase mutants that selectively couple to Cdc42

p21-activated kinase 1 (Pak1) is an effector for the small GTPases Cdc42 and Rac. Because Pak1 binds to and is activated by both these GTPases, it has been difficult to precisely delineate the signaling pathways that link extracellular stimuli to Pak1 activation. To separate activation of Pak1 by Cdc42 versus activation by Rac, we devised a genetic screen in yeast that enabled us to create and identify Pak1 mutants that selectively couple to Cdc42 but not Rac1. We recovered several such Pak1 mutants and found that the residues most often affected lie within the p21 binding domain, a region previously known to mediate Pak1 binding to GTPases, but that several mutations also map outside the borders of the p21 binding domain. Pak1 mutants that associate with Cdc42 but not Rac1 were also activated by Cdc42 but not Rac1. In rat 3Y1 cells expressing oncogenic Ha-Ras, the Pak1 mutants defective in Rac1 binding are not activated, suggesting that Ras signals through a GTPase other than Cdc42 to activate Pakl. Similar results were obtained when epidermal growth factor was used to activate Pak1. However, Pak1 mutants that are unable to bind Rac are nonetheless well activated by calf serum, implying that this stimulus may induce Pak activation independent of Rac.     

Cool-1/βPIX functions as a guanine nucleotide exchange factor in the cycling of Cdc42 to regulate insulin secretion

Second-phase insulin release requires the sustained mobilization of insulin granules from internal storage pools to the cell surface for fusion with the plasma membrane. However, the detailed mechanisms underlying this process remain largely unknown. GTP-loading of the small GTPase Cdc42 is the first glucose-specific activation step in the process, although how glucose triggers Cdc42 activation is entirely unknown. In a directed candidate screen for guanine nucleotide exchange factors (GEFs), which directly activate small GTPases, Cool-1/βPix was identified in pancreatic islet beta cells. In support of its role as the beta cell Cdc42 GEF, βPix coimmunoprecipitated with Cdc42 in human islets and MIN6 beta cells in a glucose-dependent manner, peaking just prior to Cdc42 activation. Furthermore, RNAi-mediated βPix reduction by 50% corresponded to full ablation of glucose-induced Cdc42 activation and significant attenuation of basal and glucose-stimulated insulin secretion. Of the two Cdc42 guanine nucleotide dissociation inhibitor (GDI) proteins identified in beta cells, βPix competed selectively with caveolin-1 (Cav-1) but not RhoGDI in coimmunoprecipitation and GST-Cdc42-GDP interaction assays. However, a phospho-deficient Cav-1-Y14F mutant failed to compete with βPix; Cav-1(Tyr14) is an established phosphorylation site for Src kinase. Taken together, these data support a new model, wherein glucose stimulates Cav-1 and induces its dissociation from Cdc42, possibly via Src kinase activation to phosphorylate Cav-1(Tyr14), to promote Cdc42-βPix binding and Cdc42 activation, and to trigger downstream signaling and ultimately sustain insulin release.     

Phosphorylation of RhoGDI by Pak1 mediates dissociation of Rac GTPase

Selective activation of Rac GTPase signaling pathways requires the specific release of Rac from RhoGDI complexes. We identified a RhoGDI kinase from bovine brain as p21-activated kinase (Pak). Pak1 binds and phosphorylates RhoGDI both in vitro and in vivo at Ser101 and Ser174. This resulted in dissociation of Rac1-RhoGDI, but not RhoA-RhoGDI, complexes, as determined by in vitro assays of complexation and in vivo by coimmunoprecipitation analysis. We observed that Cdc42-induced Rac1 activation is inhibited by expression of Pak1 autoinhibitory domain. The dissociation of Rac1 from RhoGDI and its subsequent activation stimulated by PDGF or EGF is also attenuated by Pak1 autoinhibitory domain, and this is dependent on the ability of RhoGDI to be phosphorylated at Ser101/174. These results support a role for Pak1-mediated RhoGDI phosphorylation as a mechanism for Cdc42-mediated Rac activation, and suggest the possibility of Rac-induced positive feed-forward regulation of Rac activity.     

Activation of LIM-kinase by Pak1 couples Rac/Cdc42 GTPase signalling to actin cytoskeletal dynamics

Extracellular signals regulate actin dynamics through small GTPases of the Rho/Rac/Cdc42 (p21) family. Here we show that p21-activated kinase (Pak1) phosphorylates LIM-kinase at threonine residue 508 within LIM-kinase's activation loop, and increases LIM-kinase-mediated phosphorylation of the actin-regulatory protein cofilin tenfold in vitro. In vivo, activated Rac or Cdc42 increases association of Pak1 with LIM-kinase; this association requires structural determinants in both the amino-terminal regulatory and the carboxy-terminal catalytic domains of Pak1. A catalytically inactive LIM-kinase interferes with Rac-, Cdc42- and Pak1-dependent cytoskeletal changes. A Pak1-specific inhibitor, corresponding to the Pak1 autoinhibitory domain, blocks LIM-kinase-induced cytoskeletal changes. Activated GTPases can thus regulate actin depolymerization through Pak1 and LIM-kinase.     

Autophosphorylation-dependent degradation of Pak1, triggered by the Rho-family GTPase, Chp

The Paks (p21-activated kinases) Pak1, Pak2 and Pak3 are among the most studied effectors of the Rho-family GTPases, Rac, Cdc42 (cell division cycle 42) and Chp (Cdc42 homologous protein). Pak kinases influence a variety of cellular functions, but the process of Pak down-regulation, following activation, is poorly understood. In the present study, we describe for the first time a negative-inhibitory loop generated by the small Rho-GTPases Cdc42 and Chp, resulting in Pak1 inhibition. Upon overexpression of Chp, we unexpectedly observed a T-cell migration phenotype consistent with Paks inhibition. In line with this observation, overexpression of either Chp or Cdc42 caused a marked reduction in the level of Pak1 protein in a number of different cell lines. Chp-induced degradation was accompanied by ubiquitination of Pak1, and was dependent on the proteasome. The susceptibility of Pak1 to Chp-induced degradation depended on its p21-binding domain, kinase activity and a number of Pak1 autophosphorylation sites, whereas the PIX- (Pak-interacting exchange factor) and Nck-binding sites were not required. Together, these results implicate Chp-induced kinase autophosphorylation in the degradation of Pak1. The N-terminal domain of Chp was found to be required for Chp-induced degradation, although not for Pak1 activation, suggesting that Chp provides a second function, distinct from kinase activation, to trigger Pak degradation. Collectively, our results demonstrate a novel mechanism of signal termination mediated by the Rho-family GTPases Chp and Cdc42, which results in ubiquitin-mediated degradation of one of their direct effectors, Pak1.     

Function of the Rho family GTPases in Ras-stimulated Raf activation.

Ras plays an essential role in activation of Raf kinase which is directly responsible for activation of the MEK-ERK kinase pathway. A direct protein-protein interaction between Ras and the N-terminal regulatory domain of Raf is critical for Raf activation. However, association with Ras is not sufficient to activate Raf in vitro, indicating that Ras must activate some other biochemical events leading to activation of Raf. We have observed that RasV12Y32F and RasV12T35S mutants fail to activate Raf, yet retain the ability to interact with Raf. In this report, we showed that RasV12Y32F and RasV12T35S can cooperate with members of the Rho family GTPases to activate Raf while alone the Rho family GTPase is not effective in Raf activation. A dominant negative mutant of Rac or RhoA can block Raf activation by Ras. The effect of Rac or Cdc42 can be substituted by the Pak kinase, which is a direct downstream target of Rac/Cdc42. Furthermore, expression of a kinase inactive mutant of Pak or the N-terminal inhibitory domain of Pak1 can block the effect of Rac or Cdc42. In contrast, Pak appears to play no direct role in relaying the signal from RhoA to Raf, indicating that RhoA utilizes a different mechanism than Rac/Cdc42. Membrane-associated but not cytoplasmic Raf can be activated by Rac or RhoA. Our data support a model by which the Rho family small GTPases play an important role to mediate the activation of Raf by Ras. Ras, at least, has two distinct functions in Raf activation, recruitment of Raf to the plasma membrane by direct binding and stimulation of Raf activating kinases via the Rho family GTPases.     

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.     

A Pak- and Pix-dependent branch of the SDF-1alpha signalling pathway mediates T cell chemotaxis across restrictive barriers

Pak (p21-activated kinase) serine/threonine kinases have been shown to mediate directional sensing of chemokine gradients. We hypothesized that Pak may also mediate chemokine-induced shape changes, to facilitate leucocyte chemotaxis through restrictive barriers, such as the extracellular matrix. A potent inhibitor, Pak(i), was characterized and used to probe the role of Pak-family kinases in SDF-1alpha (stromal-cell derived factor-1alpha/CXCL12)-induced chemotaxis in a T cell model. Pak(i) potently inhibited SDF-1alpha-induced Pak activation by a bivalent mechanism, as indicated by its complete inactivation upon point mutation of two binding sites, but partial inactivation upon mutation of either site alone. Importantly, Pak(i) was not toxic to cells over the time frame of our experiments, since it did not substantially affect cell surface expression of CXCR4 (CXC chemokine receptor 4) or integrins, cell cycle progression, or a number of ligand-induced responses. Pak(i) produced dose-dependent inhibition of SDF-1alpha-induced migration through rigid filters bearing small pores; but unexpectedly, did not substantially affect the magnitude or kinetics of chemotaxis through filters bearing larger pores. SDF-1alpha-induced Pak activation was partly dependent on PIX (Pak-interactive exchange factor); correspondingly, an allele of beta-PIX that cannot bind Pak inhibited SDF-1alpha-induced chemotaxis through small, but not large pores. By contrast, other key players in chemotaxis: G(i), PI3K (phosphoinositide 3-kinase), and the Rho-family G-proteins, Rac and Cdc42 (cell division cycle 42), were required for SDF-1alpha-induced migration regardless of the barrier pore-size. These studies have revealed a distinct branch of the SDF-1alpha signalling pathway, in which the Rac/Cdc42 effector, Pak, and its partner, PIX, specifically regulate the cellular events required for chemokine-induced migration through restrictive barriers.     

监测活细胞中诱导激活的Rac、Cdc42时空图像的FRET单分子探针的构建

以PCR方法从人脑cDNA基因文库扩增Rac1、Cdc42 cDNA全序列及其效应蛋白基因Pak1、N-WASP的GTP酶联结区域(GBD)序列,从dsRed1-N1质粒扩增红色荧光蛋白dsRed1cDNA全序列.将cDNA序列依次定向克隆至pECFP-N1质粒载体,获得基于FRET原理,包含dsRed1,Pak1或N-WASP的GBD,Rac1或Cdc42,ECFP编码序列的单分子探针.在dsRed1的C末端加入一段CAAM法尼基化基序,构建包含EGFP,Pak1的GBD,Rac1或Cdc42,dsRed1-CAAM的质膜特异表达的单分子探针.采用这两种探针,可用于监测活细胞中诱导激活的Rac1、Cdc42信号转导通路的3D时空图像,检测待测蛋白分子的GEF或GAP活性.     

Cdc42 and Rac stimulate exocytosis of secretory granules by activating the IP(3)/calcium pathway in RBL-2H3 mast cells

We have expressed dominant-active and dominant-negative forms of the Rho GTPases, Cdc42 and Rac, using vaccinia virus to evaluate the effects of these mutants on the signaling pathway leading to the degranulation of secretory granules in RBL-2H3 cells. Dominant-active Cdc42 and Rac enhance antigen-stimulated secretion by about twofold, whereas the dominant-negative mutants significantly inhibit secretion. Interestingly, treatment with the calcium ionophore, A23187, and the PKC activator, PMA, rescues the inhibited levels of secretion in cells expressing the dominant-negative mutants, implying that Cdc42 and Rac act upstream of the calcium influx pathway. Furthermore, cells expressing the dominant-active mutants exhibit elevated levels of antigen-stimulated IP(3) production, an amplified antigen-stimulated calcium response consisting of both calcium release from internal stores and influx from the extracellular medium, and an increase in aggregate formation of the IP(3) receptor. In contrast, cells expressing the dominant-negative mutants display the opposite phenotypes. Finally, we are able to detect an in vitro interaction between Cdc42 and PLCgamma1, the enzyme immediately upstream of IP(3) formation. Taken together, these findings implicate Cdc42 and Rac in regulating the exocytosis of secretory granules by stimulation of IP(3) formation and calcium mobilization upon antigen stimulation.     

Cdc42 and the guanine nucleotide exchange factors Ect2 and trio mediate Fn14-induced migration and invasion of glioblastoma cells

Malignant glioblastomas are characterized by their ability to infiltrate into normal brain. We previously reported that binding of the multifunctional cytokine TNF-like weak inducer of apoptosis (TWEAK) to its receptor fibroblast growth factor-inducible 14 (Fn14) induces glioblastoma cell invasion via Rac1 activation. Here, we show that Cdc42 plays an essential role in Fn14-mediated activation of Rac1. TWEAK-treated glioma cells display an increased activation of Cdc42, and depletion of Cdc42 using siRNA abolishes TWEAK-induced Rac1 activation and abrogates glioma cell migration and invasion. In contrast, Rac1 depletion does not affect Cdc42 activation by Fn14, showing that Cdc42 mediates TWEAK-stimulated Rac1 activation. Furthermore, we identified two guanine nucleotide exchange factors (GEF), Ect2 and Trio, involved in TWEAK-induced activation of Cdc42 and Rac1, respectively. Depletion of Ect2 abrogates both TWEAK-induced Cdc42 and Rac1 activation, as well as subsequent TWEAK-Fn14-directed glioma cell migration and invasion. In contrast, Trio depletion inhibits TWEAK-induced Rac1 activation but not TWEAK-induced Cdc42 activation. Finally, inappropriate expression of Fn14 or Ect2 in mouse astrocytes in vivo using an RCAS vector system for glial-specific gene transfer in G-tva transgenic mice induces astrocyte migration within the brain, corroborating the in vitro importance of the TWEAK-Fn14 signaling cascade in glioblastoma invasion. Our results suggest that the TWEAK-Fn14 signaling axis stimulates glioma cell migration and invasion through two GEF-GTPase signaling units, Ect2-Cdc42 and Trio-Rac1. Components of the Fn14-Rho GEF-Rho GTPase signaling pathway present innovative drug targets for glioma therapy.     

A direct interaction between Cdc42 and vesicle-associated membrane protein 2 regulates SNARE-dependent insulin exocytosis

In pancreatic beta cells, insulin granule exocytosis is regulated by SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein (SNAP) receptor) proteins, and this is coupled to cortical F-actin reorganization via the Rho family GTPase Cdc42 by an unknown mechanism. We investigated interactions among the target SNARE protein Syntaxin 1A and the vesicle-associated membrane SNARE protein (VAMP2) with Cdc42 and compared these structural interactions with their functional importance to glucose-stimulated insulin secretion in MIN6 beta cells. Subcellular fractionation analyses revealed a parallel redistribution of Cdc42 and VAMP2 from the granule fraction to the plasma membrane in response to glucose that temporally corresponded with the glucose-induced activation of Cdc42. Moreover, within these fractions Cdc42 and VAMP2 were found to co-immunoprecipitate under basal and glucose-stimulated conditions, suggesting that they moved as a complex. Furthermore, VAMP2 bound both GST-Cdc42-GTPgammaS and GST-Cdc42-GDP, indicating that the Cdc42-VAMP2 complex could form under both cytosolic GDP-bound Cdc42 and plasma membrane GTP-bound Cdc42 conformational conditions. In vitro binding analyses showed that VAMP2 bound directly to Cdc42 and that a heterotrimeric complex with Syntaxin 1A could also be formed. Deletion analyses of VAMP2 revealed that only the N-terminal 28 residues were required for Cdc42 binding. Expression of this 28-residue VAMP2 peptide in MIN6 beta cells resulted in the specific impairment of glucose-stimulated insulin secretion, indicating a functional importance for the Cdc42-VAMP2 interaction. Taken together, these data suggest a mechanism whereby glucose activates Cdc42 to induce the targeting of intracellular Cdc42-VAMP2-insulin granule complexes to Syntaxin 1A at the plasma membrane.     

alphaPix stimulates p21-activated kinase activity through exchange factor-dependent and -independent mechanisms

Activation of p21-activated kinases (Paks) is achieved through binding of the GTPases Rac or Cdc42 to a conserved domain in the N-terminal regulatory region of Pak. Additional signaling components are also likely to be important in regulating Pak activation. Recently, a family of Pak-interacting guanine nucleotide exchange factors (Pix) have been identified and which are good candidates for regulating Pak activity. Using an active, truncated form of alphaPix (amino acids 155-545), we observe stimulation of Pak1 kinase activity when alphaPix155-545 is co-expressed with Cdc42 and wild-type Pak1 in COS-1 cells. This activation does not occur when we co-express a Pak1 mutant unable to bind alphaPix. The activation of wild-type Pak1 by alphaPix155-545 also requires that alphaPix155-545 retain functional exchange factor activity. However, the Pak1(H83,86L) mutant that does not bind Rac or Cdc42 is activated in the absence of GTPase by alphaPix155-545 and by a mutant of alphaPix155-545 that no longer has exchange factor activity. Pak1 activity stimulated in vitro using GTPgammaS-loaded Cdc42 was also enhanced by recombinant alphaPix155-545 in a binding-dependent manner. These data suggest that Pak activity can be modulated by physical interaction with alphaPix and that this specific effect involves both exchange factor-dependent and -independent mechanisms.     

Targeting and activation of Rac1 are mediated by the exchange factor beta-Pix

Rho guanosine triphosphatases (GTPases) are critical regulators of cytoskeletal dynamics and control complex functions such as cell adhesion, spreading, migration, and cell division. It is generally accepted that localized GTPase activation is required for the proper initiation of downstream signaling events, although the molecular mechanisms that control targeting of Rho GTPases are unknown. In this study, we show that the Rho GTPase Rac1, via a proline stretch in its COOH terminus, binds directly to the SH3 domain of the Cdc42/Rac activator beta-Pix (p21-activated kinase [Pak]-interacting exchange factor). The interaction with beta-Pix is nucleotide independent and is necessary and sufficient for Rac1 recruitment to membrane ruffles and to focal adhesions. In addition, the Rac1-beta-Pix interaction is required for Rac1 activation by beta-Pix as well as for Rac1-mediated spreading. Finally, using cells deficient for the beta-Pix-binding kinase Pak1, we show that Pak1 regulates the Rac1-beta-Pix interaction and controls cell spreading and adhesion-induced Rac1 activation. These data provide a model for the intracellular targeting and localized activation of Rac1 through its exchange factor beta-Pix.