Oncogenic Dbl, Cdc42, and p21-activated kinase form a ternary signaling intermediate through the minimum interactive domains

Activation of many Rho family GTPase pathways involves the signaling module consisting of the Dbl-like guanine nucleotide exchange factors (GEFs), the Rho GTPases, and the Rho GTPase specific effectors. The current biochemical model postulates that the GEF-stimulated GDP/GTP exchange of Rho GTPases leads to the active Rho-GTP species, and subsequently the active Rho GTPases interact with and activate the effectors. Here we report an unexpected finding that the Dbl oncoprotein, Cdc42 GTPase, and PAK1 can form a complex through their minimum functional motifs, i.e., the Dbl-homolgy (DH) and Pleckstrin-homology domains of Dbl, Cdc42, and the PBD domain of PAK1. The Dbl-Cdc42-PAK1 complex is sensitive to the nucleotide-binding state of Cdc42 since either dominant negative or constitutively active Cdc42 readily disrupts the ternary binding interaction. The complex formation depends on the interactions between the DH domain of Dbl and Cdc42 and between Cdc42 and the PBD domain of PAK1 and can be reconstituted in vitro by using the purified components. Furthermore, the Dbl-Cdc42-PAK1 ternary complex is active in generating signaling output through the activated PAK1 kinase in the complex. The GEF-Rho-effector ternary intermediate is also found in other Dbl-like GEF, Rho GTPase, and effector interactions. Finally, PAK1, through the PDB domain, is able to accelerate the GEF-induced GTP loading onto Cdc42. These results suggest that signal transduction through Cdc42 and possibly other Rho family GTPases could involve tightly coupled guanine nucleotide exchange and effector activation mechanisms and that Rho GTPase effector may have a feedback regulatory role in the Rho GTPase activation.     

Maintenance of CDC42 GDP-bound state by Rho-GDI inhibits MAP kinase activation by the exchange factor Ras-GRF. evidence for Ras-GRF function being inhibited by Cdc42-GDP but unaffected by CDC42-GTP

The function of the Ras guanine nucleotide exchange factor Ras-GRF/cdc25(Mn) is subject to tight regulatory processes. We have recently shown that the activation of the Ras/MAPK pathway by Ras-GRF is controlled by the Rho family GTPase Cdc42 through still unknown mechanisms. Here, we report that retaining Cdc42 in its GDP-bound state by overexpressing Rho-GDI inhibits Ras-GRF-mediated MAPK activation. Conversely, Ras-GRF basal and LPA- or ionomycin-stimulated activities were unaffected by a constitutively active GTP-bound Cdc42. Moreover, the Cdc42 downstream effectors MLK3, ACK1, PAK1, and WASP had no detectable influence on Ras-GRF-mediated MAPK activation. In contrast, promoting GDP release from Cdc42 with the Rho family GEF Dbl or with ionomycin suppressed the restraint exerted by Cdc42 on Ras-GRF activity. We conclude that Cdc42-GDP inhibits Ras-GRF-induced MAPK activation, but neither Cdc42-GTP nor the Cdc42 downstream effectors affect Ras-GRF performance. Interestingly, the loss of the GDP-bound state by Cdc42 abolishes its inhibitory effects on Ras-GRF function. These results suggest that the Cdc42 mechanism of action may not be solely restricted to activation of downstream signaling cascades when GTP-loaded. Furthermore, the GDP-bound form may be acting as an inhibitory molecule down-modulating parallel signaling routes such as the Ras/MAPK pathway.     

A Cdc42 mutant specifically activated by intersectin

The Rho family GTPase Cdc42 functions as a molecular switch and controls many fundamental cellular processes such as cytoskeletal regulation, cell polarity, and vesicular trafficking. Guanine nucleotide exchange factors of the Dbl family activate Cdc42 and other Rho GTPases by catalyzing the removal of bound GDP, allowing for GTP loading, and subsequent effector recognition ultimately leading to downstream signaling events. Analysis of existing structural data reveals that the Dbl exchange factor intersectin engages a strictly conserved GTPase residue of Cdc42 (tyrosine 32) in a unique mode with respect to all other visualized exchange factor-Rho GTPase interfaces. To investigate this differential binding architecture, we analyzed the role of tyrosine 32 of Cdc42 in binding, and stimulation by Dbl family exchange factors. Deletion of the hydroxyl side chain of tyrosine 32 substantially increases the affinity of Cdc42 for intersectin, yet severely cripples interaction with Dbs, a normally potent exchange factor of Cdc42. Moreover, Cdc42(Y32F) is exclusively activated by intersectin, while virtually unresponsive to other Cdc42-activating exchange factors in vitro and in vivo. Further, the structural determinants unique to intersectin, which permit selective recognition and concomitant stimulation of Cdc42(Y32F), have been defined. Cdc42 and other individual Rho GTPases receive input stimulatory signals from a multitude of Dbl exchange factors, and therefore, Cdc42(Y32F) could act as a valuable reagent for understanding the specific influence of ITSN on Cdc42-mediated signaling phenomena.     

Unique structural and nucleotide exchange features of the Rho1 GTPase of Entamoeba histolytica

The single-celled human parasite Entamoeba histolytica possesses a dynamic actin cytoskeleton vital for its intestinal and systemic pathogenicity. The E. histolytica genome encodes several Rho family GTPases known to regulate cytoskeletal dynamics. EhRho1, the first family member identified, was reported to be insensitive to the Rho GTPase-specific Clostridium botulinum C3 exoenzyme, raising the possibility that it may be a misclassified Ras family member. Here, we report the crystal structures of EhRho1 in both active and inactive states. EhRho1 is activated by a conserved switch mechanism, but diverges from mammalian Rho GTPases in lacking a signature Rho insert helix. EhRho1 engages a homolog of mDia, EhFormin1, suggesting a role in mediating serum-stimulated actin reorganization and microtubule formation during mitosis. EhRho1, but not a constitutively active mutant, interacts with a newly identified EhRhoGDI in a prenylation-dependent manner. Furthermore, constitutively active EhRho1 induces actin stress fiber formation in mammalian fibroblasts, thereby identifying it as a functional Rho family GTPase. EhRho1 exhibits a fast rate of nucleotide exchange relative to mammalian Rho GTPases due to a distinctive switch one isoleucine residue reminiscent of the constitutively active F28L mutation in human Cdc42, which for the latter protein, is sufficient for cellular transformation. Nonconserved, nucleotide-interacting residues within EhRho1, revealed by the crystal structure models, were observed to contribute a moderating influence on fast spontaneous nucleotide exchange. Collectively, these observations indicate that EhRho1 is a bona fide member of the Rho GTPase family, albeit with unique structural and functional aspects compared with mammalian Rho GTPases.     

Activated PAK4 regulates cell adhesion and anchorage-independent growth

The serine/threonine kinase PAK4 is an effector molecule for the Rho GTPase Cdc42. PAK4 differs from other members of the PAK family in both sequence and function. Previously we have shown that an important function of this kinase is to mediate the induction of filopodia in response to activated Cdc42. Since previous characterization of PAK4 was carried out only with the wild-type kinase, we have generated a constitutively active mutant of the kinase to determine whether it has other functions. Expression of activated PAK4 in fibroblasts led to a transient induction of filopodia, which is consistent with its role as an effector for Cdc42. In addition, use of the activated mutant revealed a number of other important functions of this kinase that were not revealed by studying the wild-type kinase. For example, activated PAK4 led to the dissolution of stress fibers and loss of focal adhesions. Consequently, cells expressing activated PAK4 had a defect in cell spreading onto fibronectin-coated surfaces. Most importantly, fibroblasts expressing activated PAK4 had a morphology that was characteristic of oncogenic transformation. These cells were anchorage independent and formed colonies in soft agar, similar to what has been observed previously in cells expressing activated Cdc42. Consistent with this, dominant-negative PAK4 mutants inhibited focus formation by oncogenic Dbl, an exchange factor for Rho family GTPases. These results provide the first demonstration that a PAK family member can transform cells and indicate that PAK4 may play an essential role in oncogenic transformation by the GTPases. We propose that the morphological changes and changes in cell adhesion induced by PAK4 may play a direct role in oncogenic transformation by Rho family GTPases and their exchange factors.     

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.     

Cdc42 promotes G1 progression through p70 S6 kinase-mediated induction of cyclin E expression

The Rho family GTPase Cdc42 is recognized for its role in cellular proliferation and transformation. However, the mechanism by which it promotes cell cycle progression has remained undefined. Using an inducible expression system, we show that constitutively active Cdc42 (Cdc42V12) is sufficient by itself to induce anchorage-independent but not mitogen-independent growth in NIH3T3 cells. However, Cdc42V12 markedly accelerates activation of cyclin E-Cdk2 in response to mitogen. These effects were highly specific, as the kinetics of cyclin D-Cdk4 activation was unaltered. Cdc42V12 promotes Cdk2 activation by selectively inducing cyclin E expression without affecting other regulatory proteins such as the p27 Cdk inhibitor or Cdc25A. Furthermore, Cdc42V12 was able to activate a reporter gene driven by the cyclin E promoter in the absence of exogenous mitogen or adhesion. Cyclin E induction was sensitive to rapamycin but not inhibitors of mitogen-activated protein kinases, implicating p70 S6 kinase (p70S6k) as the relevant mediator. Consistent with this notion, wild type and constitutively active alleles of p70S6k were sufficient to activate the cyclin E promoter. In sum, these studies provide novel insights into the mechanism by which Cdc42 promotes G1 progression.     

Phosphatidylinositol 3-kinase, Cdc42, and Rac1 act downstream of Ras in integrin-dependent neurite outgrowth in N1E-115 neuroblastoma cells

Ras and Rho family GTPases have been ascribed important roles in signalling pathways determining cellular morphology and growth. Here we investigated the roles of the GTPases Ras, Cdc42, Rac1, and Rho and that of phosphatidylinositol 3-kinase (PI 3-kinase) in the pathway leading from serum starvation to neurite outgrowth in N1E-115 neuroblastoma cells. Serum-starved cells grown on a laminin matrix exhibited integrin-dependent neurite outgrowth. Expression of dominant negative mutants of Ras, PI 3-kinase, Cdc42, or Rac1 all blocked this neurite outgrowth, while constitutively activated mutants of Ras, PI 3-kinase, or Cdc42 were each sufficient to promote outgrowth even in the presence of serum. A Ras(H40C;G12V) double mutant which binds preferentially to PI 3-kinase also promoted neurite formation. Activated Ras(G12V)-induced outgrowth required PI 3-kinase activity, but activated PI 3-kinase-induced outgrowth did not require Ras activity. Although activated Rac1 by itself did not induce neurites, neurite outgrowth induced by activated Cdc42(G12V) was Rac1 dependent. Cdc42(G12V)-induced neurites appeared to lose their normal polarization, almost doubling the average number of neurites produced by a single cell. Outgrowth induced by activated Ras or PI 3-kinase required both Cdc42 and Rac1 activity, but Cdc42(G12V)-induced outgrowth did not need Ras or PI 3-kinase activity. Active Rho(G14V) reduced outgrowth promoted by Ras(G12V). Finally, expression of dominant negative Jun N-terminal kinase or extracellular signal-regulated kinase did not inhibit outgrowth, suggesting these pathways are not essential for this process. Our results suggest a hierarchy of signalling where Ras signals through PI 3-kinase to Cdc42 and Rac1 activation (and Rho inactivation), culminating in neurite outgrowth. Thus, in the absence of serum factors, Ras may initiate cell cycle arrest and terminal differentiation in N1E-115 neuroblastoma cells.     

Compartmentalized Ras proteins transform NIH 3T3 cells with different efficiencies

Ras GTPases were long thought to function exclusively from the plasma membrane (PM). However, a current model suggests that Ras proteins can compartmentalize to regulate different functions, and an oncogenic H-Ras mutant that is restricted to the endomembrane can still transform cells. In this study, we demonstrated that cells transformed by endomembrane-restricted oncogenic H-Ras formed tumors in nude mice. To define downstream targets of endomembrane Ras pathways, we analyzed Cdc42, which concentrates in the endomembrane and has been shown to act downstream of Ras in Schizosaccharomyces pombe. Our data show that cell transformation induced by endomembrane-restricted oncogenic H-Ras was blocked when Cdc42 activity was inhibited. Moreover, H-Ras formed a complex with Cdc42 on the endomembrane, and this interaction was enhanced when H-Ras was GTP bound or when cells were stimulated by growth factors. H-Ras binding evidently induced Cdc42 activation by recruiting and/or activating Cdc42 exchange factors. In contrast, when constitutively active H-Ras was restricted to the PM by fusing to a PM localization signal from the Rit GTPase, the resulting protein did not detectably activate Cdc42 although it activated Raf-1 and efficiently induced hallmarks of Ras-induced senescence in human BJ foreskin fibroblasts. Surprisingly, PM-restricted oncogenic Ras when expressed alone could only weakly transform NIH 3T3 cells; however, when constitutively active Cdc42 was coexpressed, together they transformed cells much more efficiently than either one alone. These data suggest that efficient cell transformation requires Ras proteins to interact with Cdc42 on the endomembrane and that in order for a given Ras protein to fully transform cells, multiple compartment-specific Ras pathways need to work cooperatively.     

C-terminal di-arginine motif of Cdc42 protein is essential for binding to phosphatidylinositol 4,5-bisphosphate-containing membranes and inducing cellular transformation

Rho GTPases regulate a diverse range of processes that are dependent on their proper cellular localization. The membrane localization of these GTPases is due in large part to their carboxyl-terminal geranylgeranyl moiety. In addition, most of the Rho family members contain a cluster of positively charged residues (i.e. a "polybasic domain"), directly preceding their geranylgeranyl moiety, and it has been suggested that this domain serves to fine-tune their localization among different cellular membrane sites. Here, we have taken a closer look at the role of the polybasic domain of Cdc42 in its ability to bind to membranes and induce the transformation of fibroblasts. A FRET assay for the binding of Cdc42 to liposomes of defined composition showed that Cdc42 associates more strongly with liposomes containing phosphatidylinositol 4,5-bisphosphate (PIP(2)) when compared either with uncharged control membranes or with liposomes containing a charge-equivalent amount of phosphatidylserine. The carboxyl-terminal di-arginine motif (Arg-186 and Arg-187) was shown to play an essential role in the binding of Cdc42 to PIP(2)-containing membranes. We further showed that substitutions for the di-arginine motif, when introduced within a constitutively active ("fast cycling") Cdc42(F28L) background, had little effect on the ability of the activated Cdc42 mutant to induce microspikes/filopodia in NIH 3T3 cells, whereas they eliminated its ability to transform fibroblasts. Taken together, these findings suggest that the di-arginine motif within the carboxyl terminus of Cdc42 is necessary for this GTPase to bind at membrane sites containing PIP(2), where it can initiate signaling activities that are essential for the oncogenic transformation of cells.     

The serine/threonine kinase PAK4 prevents caspase activation and protects cells from apoptosis

The serine/threonine kinase PAK4 was identified first as an effector molecule for the Rho GTPase Cdc42. PAK4 differs from other members of the PAK family both in sequence and function. Previously we have shown that an important function of this kinase is to mediate the induction of filopodia in response to activated Cdc42. Studies with a constitutively active PAK4 mutant have shown that it also has a role in promoting anchorage-independent growth, an important hallmark of oncogenic transformation. Here we show that another function of PAK4 is to protect cells against apoptotic cell death. Expression of wild-type or constitutively active PAK4 delays the onset of apoptosis in response to tumor necrosis factor alpha stimulation, UV irradiation, and serum starvation. Consistent with an antiapoptotic function, expression of PAK4 leads to an increase in phosphorylation of the proapoptotic protein Bad and an inhibition of caspase activation.     

Influencing cellular transformation by modulating the rates of GTP hydrolysis by Cdc42

The small GTPase Cdc42 has been implicated in a number of cellular responses ranging from the regulation of the actin cytoskeletal architecture to intracellular trafficking and cell cycle progression. Cdc42 mutants that constitutively exchange GDP for GTP but still hydrolyze GTP (called 'fast-cycling' mutants) promote cellular transformation, whereas Cdc42 mutants that are unable to hydrolyze GTP and are irreversibly trapped in the GTP-bound state often inhibit cell growth. In this work, we have set out to further establish that Cdc42 needs to cycle between its 'on' and 'off' states to stimulate cell growth, by examining the consequences of manipulating its GTP-binding/GTP hydrolytic cycle in two different ways. One approach was to examine whether substitutions that act in a manner opposite to the 'fast cyclers', and extend the lifetime of the activated GTP-bound state by slowing the GTP hydrolytic reaction (i.e., 'slow-cycling' mutations), positively influence cell growth. Indeed we show that one such slow-cycling mutant, Cdc42[Y32A], which is insensitive to Cdc42GAP but still exhibits a measurable intrinsic GTP hydrolytic activity, gives rise to increased levels of activated Cdc42 in NIH 3T3 cells. We go on to show that the Y32A mutant stimulates the actin cytoskeletal changes that lead to filopodia formation, confer growth advantages to fibroblasts under low serum conditions, and enable cells to grow to high densities when exposed to normal levels of serum. The second approach was to determine whether the transforming activity of the fast-cycling Cdc42[F28L] mutant can be reversed by compensating for its accelerated nucleotide exchange reaction through the expression of the GTPase-activating protein (Cdc42GAP) and the ensuing stimulation of GTP hydrolytic activity. We showed that expression of the limit functional domain of Cdc42GAP inhibited Cdc42[F28L]-induced transformation, as well as selectively reversed the transformed phenotypes caused by the hyperactivation of wild-type Cdc42 in cells expressing the oncogenic version of Dbl (for Diffuse B cell lymphoma), a guanine nucleotide exchange factor for Cdc42 and the related Rac and Rho GTPases. Overall, the results reported here establish the requirement for Cdc42 to cycle between its signaling-on and -off states in order to positively influence cell growth and highlight how the Cdc42GAP can play an important role in regulating cell proliferation.     

Antiapoptotic Cdc42 mutants are potent activators of cellular transformation

Cdc42 is a small GTP-binding protein which has been implicated in a number of cellular activities, including cell morphology, motility, cell-cycle progression, and malignant transformation. While GTPase-defective forms of Cdc42 inhibit cell growth, a mutation [Cdc42(F28L)] that allows the constitutive exchange of GDP for GTP and is GTPase-competent induces cellular transformation. These results suggest that Cdc42 must cycle between its GTP- and GDP-bound states to stimulate cell growth. In attempting to design Cdc42 molecules with more potent transforming activity, we set out to generate other types of Cdc42 mutants capable of constitutive GDP-GTP exchange. Here, we describe one such mutant, generated by changing a conserved aspartic acid residue at position 118 to an asparagine. The Cdc42(D118N) protein exchanges GDP for GTP more rapidly than wild-type Cdc42, but significantly more slowly than the Cdc42(F28L) mutant. Despite its slower rate of activation, the Cdc42(D118N) mutant is more potent at inducing cellular transformation than the Cdc42(F28L) protein, and causes a significant loss in actin stress fibers, reminiscent of what is observed with fibroblasts transformed by oncogenic Ras mutants. Effector-loop mutations made within the D118N background inhibit Cdc42-induced transformation and Cdc42-mediated antiapoptotic (survival) activity to similar extents. In addition, mutating aspartic acid 121 (to asparagine), which forms part of a caspase cleavage site (DLRD, residues 118-121 of Cdc42), in combination with the F28L mutation generates a Cdc42 molecule [Cdc42(F28L/D121N)] with transforming activity significantly stronger than that of Cdc42(F28L). Thus, mutations that combine some capacity for cycling between the GTP- and GDP-bound states with increased survival against apoptotic signals yield Cdc42 molecules with the maximum capability for inducing cellular transformation.     

Cdc42-dependent mediation of UV-induced p38 activation by G protein betagamma subunits

The beta and gamma subunits of heterotrimeric GTP-binding proteins (Gbetagamma) were found to bi-directionally regulate the UV-induced activation of p38 and c-Jun NH(2)-terminal kinase, and the UV-induced activation of p38 was reported to enhance the resistance of normal keratinocytes to apoptosis. However, the signaling pathway downstream of Gbetagamma for this UV-induced p38 activation is not known. Thus, we examined the role of the Rho GTPase family in the regulation of UV-induced p38 activation by Gbetagamma. We found that overexpression of Gbetagamma increased the UV-induced activation of Cdc42 and that overexpression of constitutively active V12 Cdc42 increased the UV-induced p38 activation. Transfection of dominant negative N17 Cdc42 or small interfering RNA for Cdc42 blocked UV-induced p38 activation mediated by Gbetagamma in COS-1 and HaCaT cells. UV-induced p38 activation by Gbetagamma was blocked by overexpression of dominant negative p21-activated kinase (PAK)-interacting exchange factor beta (betaPix), and wild type betaPix stimulated the UV-induced p38 activation, which was blocked by N17 Cdc42. Gbetagamma increased the UV-induced activation of Ras, and the overexpression of V12 Ras increased UV-induced p38 activation, which was blocked by dominant negative betaPix. UV-induced p38 activation was inhibited by N17 Ras and a farnesyltransferase inhibitor, manumycin A. Gbetagamma also increased the UV-induced phosphorylation of the epidermal growth factor receptor (EGFR), and the UV-induced p38 activation was blocked by an EGFR kinase inhibitor, AG1478. From these results, we conclude that Gbetagamma mediates UV-induced activation of p38 in a Cdc42-dependent way and that EGFR, Ras, and betaPix act sequentially upstream of Cdc42 in COS-1 and HaCaT cells.     

Guanine exchange-dependent and -independent effects of Vav1 on integrin-induced T cell spreading

Vav1 is a 95-kDa member of the Dbl family of guanine exchange factors and a prominent hemopoietic cell-specific protein tyrosine kinase substrate, the involvement of which in cytoskeletal rearrangements has been linked to its ability to activate Rho family small GTPases. Beta1 integrin ligation by fibronectin induced Vav1 phosphorylation in peripheral blood lymphocytes and in two different T cell lines. Vav1 overexpression led to massive T cell spreading on beta1 integrin ligands, and, conversely, two dominant negative mutants blocked integrin-induced spreading. Vav1 and beta1 integrin ligation synergistically activated Pak, but not Rac, Cdc42, or c-Jun N-terminal kinase. In addition, Vav1 cooperated with constitutively active V12Rac mutant, but not with V12Cdc42, to induce T cell spreading after integrin occupancy. More importantly, a Vav1 mutant that lacked guanine exchange factor activity still cooperated with V12Rac. In contrast, a point mutation in the SH2 domain of Vav1 abolished this synergistic effect. Therefore, our results suggest a new regulatory effect of Vav1 in T cell spreading, which is independent of its guanine exchange factor activity.     

Identification of Rho GTPase-dependent sites in the Dbl homology domain of oncogenic Dbl that are required for transformation

The Dbl family guanine-nucleotide exchange factors (GEFs) for Rho GTPases share the structural array of a Dbl homology (DH) domain in tandem with a Pleckstrin homology (PH) domain. For oncogenic Dbl, the DH domain is responsible for the GEF activity, and the DH-PH module constitutes the minimum structural unit required for cellular transformation. To understand the structure-function relationship of the DH domain, we have investigated the role of specific residues of the DH domain of Dbl in interaction with Rho GTPases and in Dbl-induced transformation. Alanine substitution mutagenesis identified a panel of DH mutants made in the alpha1, alpha6, and alpha9 regions and the PH junction site that suffer complete or partial loss of GEF activity toward Cdc42 and RhoA. Kinetic and binding analysis of these mutants revealed that although most displayed decreased k(cat) values in the GEF reaction, the substrate binding activities of T506A and R634A were significantly reduced. E502A, Q633A, and N673A/D674A, on the other hand, retained the binding capability to the Rho GTPases but lost the GEF catalytic activity. In general, the in vitro GEF activity of the DH mutants correlated with the in vivo Cdc42- and RhoA-activating potential, and the GEF catalytic efficiency mirrored the transforming activity in NIH 3T3 cells. Moreover, the N673A/D674A mutant exhibited a potent dominant-negative effect on serum-induced cell growth and caused retraction of actin structures. These studies identify important sites of the DH domain involved in binding or catalysis of Rho proteins and demonstrate that maintaining a threshold of GEF catalytic activity, in addition to the Rho GTPase binding activity, is essential for efficient transformation by oncogenic Dbl.     

Epidermal growth factor stimulation of the ACK1/Dbl pathway in a Cdc42 and Grb2-dependent manner

The tyrosine kinase ACK1 phosphorylates and activates the guanine nucleotide exchange factor Dbl, which in turn directs the Rho family GTP-binding proteins. However, the regulatory mechanism of ACK1/Dbl signaling in response to extracellular stimuli remains poorly understood. Here we describe that epidermal growth factor stimulates the ACK1/Dbl pathway, leading to actin cytoskeletal rearrangements. The role of the two ACK1-binding proteins Cdc42 and Grb2 was assessed by overexpression of the Cdc42/Rac interactive binding domain and a dominant-negative Grb2 mutant, respectively. Specific inhibition of the interaction of ACK1 with Cdc42 or Grb2 by the use of these constructs diminished tyrosine phosphorylation of both ACK1 and Dbl in response to EGF. Therefore, the activation of ACK1 and subsequent downstream signaling require both Cdc42-dependent and Grb2-dependent processes within the cell. In addition, we show that EGF transiently induces formation of the focal complex and stress fibers when ACK1 was ectopically expressed. The induction of these structures was totally sensitive to the action of botulinum toxin C from Clostridium botulinum, suggesting a pivotal role of Rho. These results provide evidence that ACK1 acts as a mediator of EGF signals to Rho family GTP-binding proteins through phosphorylation and activation of GEFs such as Dbl.