Nitric oxide causes macrophage migration via the HIF-1-stimulated small GTPases Cdc42 and Rac1

Hypoxia-inducible factor 1 (HIF-1) is a key regulator of tumor development. Recently, the tumor microenvironment, with the presence of tumor-associated macrophages (TAMs), has gained considerable interest. The mechanisms of macrophage/TAM migration as well as the role of HIF-1 in macrophages for tumor progression are still under debate. We present evidence that under normoxic conditions, nitric oxide (NO) promotes macrophage migration. The response was impaired in macrophages from leukocyte conditional HIF-1α^−/− mice. NO production and cell migration in response to cytokines were attenuated in macrophages from iNOS^−/− mice, suggesting that iNOS-derived NO transmits cytokine signaling toward cell migration. We further identified the small GTPases Cdc42 and Rac1 as effectors of the NO–HIF axis to drive macrophage migration by modulating the actin cytoskeleton. Our observations strengthen the role of HIF-1 in macrophages as a target of NO in facilitating functional responses such as migration.     

Characterization of small GTPases Cdc42 and Rac and the relationship between Cdc42 and actin cytoskeleton in vegetative and ectomycorrhizal hyphae of Suillus bovinus

This work reports the isolation and molecular characterization of CDC42 and RAC1 cDNAs from the ectomycorrhiza forming filamentous homobasidiomycete Suillus bovinus. Previously, no RAC gene was described from filamentous fungi and no CDC42 gene was described from homobasidiomycetes. Southern hybridization with SbCDC42 and SbRAC1 cDNAs indicated that the S. bovinus genome contains only one CDC42 and one RAC1 gene. The predicted amino acid sequence of SbRaclp is 77% identical with the Rac1B protein of chick, whereas SbCdc42p is most identical with Schizosaccharomyces pombe Cdc42p, showing 88% identity. In the predicted amino acid sequences of SbRaclp and SbCdc42p, the five guanine nucleotide binding regions, switch I and II, and the effector domain are highly identical to those known in other small GTPases. These domain structures suggest that in S. bovinus, SbRac1p and SbCdc42p function as molecular switches regulating the organization of actin cytoskeleton, similar to yeasts and mammals. SbRAC1 and SbCDC42 were expressed in vegetative and ectomycorrhizal hyphae, and SbCdc42p was detected in ectomycorrhiza-forming hyphae if growth and differentiation of the symbiotic hyphae took place. Cdc42p and actin were localized at the tips of S. bovinus vegetative hyphae. Similar to yeast, in filamentous fungi Cdc42p may be necessary to maintain the actin cytoskeleton at hyphal tips, making the polarized growth of the hyphae possible. In developing ectomycorrhiza, Cdc42p and actin were visualized in association with plasma membrane in swollen cells typical to the symbiotic hyphae. The role of Cdc42p and actin in regulation of the growth pattern and morphogenesis of ectomycorrhizal hyphae is discussed.     

Pathogenicity island-dependent activation of Rho GTPases Rac1 and Cdc42 in Helicobacter pylori infection

Helicobacter pylori has been identified as the major aetiological agent in the development of chronic gastritis and duodenal ulcer, and it plays a role in the development of gastric carcinoma. Attachment of H. pylori to gastric epithelial cells leads to nuclear and cytoskeletal responses in host cells. Here, we show that Rho GTPases Rac1 and Cdc42 were activated during infection of gastric epithelial cells with either the wild-type H. pylori or the mutant strain cagA. In contrast, no activation of Rho GTPases was observed when H. pylori mutant strains (virB7 and PAI) were used that lack functional type IV secretion apparatus. We demonstrated that H. pylori-induced activation of Rac1 and Cdc42 led to the activation of p21-activated kinase 1 (PAK1) mediating nuclear responses, whereas the mutant strain PAI had no effect on PAK1 activity. Activation of Rac1, Cdc42 and PAK1 represented a very early event in colonization of gastric epithelial cells by H. pylori. Rac1 and Cdc42 were recruited to the sites of bacterial attachment and are therefore probably involved in the regulation of local and overall cytoskeleton rearrangement in host cells. Finally, actin rearrangement and epithelial cell motility in H. pylori infection depended on the presence of a functional type IV secretion system encoded by the cag pathogenicity island (PAI).     

The TRE17 oncogene encodes a component of a novel effector pathway for Rho GTPases Cdc42 and Rac1 and stimulates actin remodeling

The Rho family GTPases Cdc42 and Rac1 play fundamental roles in transformation and actin remodeling. Here, we demonstrate that the TRE17 oncogene encodes a component of a novel effector pathway for these GTPases. TRE17 coprecipitated specifically with the active forms of Cdc42 and Rac1 in vivo. Furthermore, the subcellular localization of TRE17 was dramatically regulated by these GTPases and mitogens. Under serum-starved conditions, TRE17 localized predominantly to filamentous structures within the cell. Epidermal growth factor (EGF) induced relocalization of TRE17 to the plasma membrane in a Cdc42-/Rac1-dependent manner. Coexpression of activated alleles of Cdc42 or Rac1 also caused complete redistribution of TRE17 to the plasma membrane, where it partially colocalized with the GTPases in filopodia and ruffles, respectively. Membrane recruitment of TRE17 by EGF or the GTPases was dependent on actin polymerization. Finally, we found that a C-terminal truncation mutant of TRE17 induced the accumulation of cortical actin, mimicking the effects of activated Cdc42. Together, these results identify TRE17 as part of a novel effector complex for Cdc42 and Rac1, potentially contributing to their effects on actin remodeling. The present study provides insights into the regulation and cellular function of this previously uncharacterized oncogene.     

Role of the small Rho GTPases Rac1 and Cdc42 in host cell invasion of Campylobacter jejuni

Host cell invasion of the food-borne pathogen Campylobacter jejuni is one of the primary reasons of tissue damage in humans but molecular mechanisms are widely unclear. Here, we show that C. jejuni triggers membrane ruffling in the eukaryotic cell followed by invasion in a very specific manner first with its tip followed by the flagellar end. To pinpoint important signalling events involved in the C. jejuni invasion process, we examined the role of small Rho family GTPases. Using specific GTPase-modifying toxins, inhibitors and GTPase expression constructs we show that Rac1 and Cdc42, but not RhoA, are involved in C. jejuni invasion. In agreement with these observations, we found that internalization of C. jejuni is accompanied by a time-dependent activation of both Rac1 and Cdc42. Finally, we show that the activation of these GTPases involves different host cell kinases and the bacterial fibronectin-binding protein CadF. Thus, CadF is a bifunctional protein which triggers bacterial binding to host cells as well as signalling leading to GTPase activation. Collectively, our results suggest that C. jejuni invade host target cells by a unique mechanism and the activation of the Rho GTPase members Rac1 and Cdc42 plays a crucial role in this entry process.     

The roles of Cdc42 and Rac1 in the formation of plasma membrane protrusions in cancer epithelial HeLa cells

The inducible model of clones generated from the cervical cancer epithelial HeLa cell line has shown the role of DOCK10 as a guanine-nucleotide exchange factor for Rho GTPases Cdc42 and Rac1 and as an inducer of filopodia and plasma membrane (PM) ruffles. In this model, constitutively active (CA) mutants of Cdc42 and Rac1 promote filopodia and ruffles, respectively, as in other models. DOCK9 also induces filopodia and ruffles, although ruffling activity is less prominent. By exploiting this model further, the aim of this work is to provide a more complete understanding of the role of Cdc42 and Rac1, and their interactions with DOCK10 and DOCK9, in regulation of PM protrusions. New clones have been generated from HeLa, including single clones expressing one form of wild-type (WT) or dominant negative (DN) Cdc42 or Rac1, and double clones co-expressing one of them together with either DOCK10 or DOCK9. Expression of WT- and DN-Cdc42 induced filopodia. WT-Cdc42 and, especially, DN-Cdc42 also gave rise to veil protrusions, which were neutralized by DOCK10. Moreover, DN-Cdc42 stimulated the emergence of ruffles, further increased by DOCK10, and WT-Cdc42 also augmented ruffles in presence of DOCK9 and DOCK10. WT-Rac1 greatly increased PM blebbing, as did DN-Rac1 more moderately. In both cases, blebs were enhanced by DOCK10. DN-Rac1 and CA-Rac1 moderately raised filopodia, and DOCK10 and DOCK9 had opposed effects on filopodia (up and down, respectively) in presence of WT-Rac1. As conclusions, we highlight that Cdc42 promotes filopodia regardless of its conformational state, and Rac1 induces blebs in conformations other than CA, especially WT-Rac1, in the inducible HeLa clones. The model could be useful to learn more about the mechanisms underlying PM protrusions.

     

BNIP-2 induces cell elongation and membrane protrusions by interacting with Cdc42 via a unique Cdc42-binding motif within its BNIP-2 and Cdc42GAP homology domain

The Cdc42 small GTPase regulates cytoskeletal reorganization and cell morphological changes that result in cellular extensions, migration, or cytokinesis. We previously showed that BNIP-2 interacted with Cdc42 and its cognate inactivator, p50RhoGAP/Cdc42GAP via its BNIP-2 and Cdc42GAP homology (BCH) domain, but its cellular and physiological roles still remain unclear. We report here that following transient expression of BNIP-2 in various cells, the expressed protein was located in irregular spots throughout the cytoplasm and concentrated at the leading edge of cellular extensions. The induced cell elongation and membrane protrusions required an intact BCH domain and were variously inhibited by coexpression of dominant negative mutants of Cdc42 (completely inhibited), Rac1 (partially inhibited), and RhoA (least inhibited). Presence of the Cdc42/Rac1 interactive binding (CRIB) motif alone as the dominant negative mutant of p21-activated kinase also inhibited the BNIP-2 effect. Bioinformatic analyses together with progressive deletional mutagenesis and binding studies revealed that a distal part of the BNIP-2 BCH domain contained a sequence with low homology to CRIB motif. However, in contrary to most effectors, BNIP-2 binding to Cdc42 was mediated exclusively via the unique sequence motif 285VPMEYVGI292. Cells expressing the BNIP-2 mutants devoid of this motif or/and the 34-amino acids immediately upstream to this sequence failed to elicit cell elongation and membrane protrusions despite that the protein still remained in the cytoplasm and interacted with Cdc42GAP. Evidence is presented where BNIP-2 in vivo induces cell dynamics by recruiting Cdc42 via its BCH domain, thus providing a novel mechanism for regulating Cdc42 signaling pathway.     

Rho GTPases and actin dynamics in membrane protrusions and vesicle trafficking

Rho GTPases are well known to regulate actin dynamics. They activate two types of actin nucleators, WASP/WAVE proteins and Diaphanous-related formins (DRFs), which induce different types of actin organization. Their ability to interact with membranes allows them to target actin polymerization to discrete sites on the plasma membrane and to intracellular membrane compartments and thereby induce membrane protrusions or regulate vesicle movement. Most studies have concentrated on just three of the 22 mammalian Rho proteins, RhoA, Rac1 and Cdc42. However, recent research indicates that several other members of the Rho family, including Rif, RhoD, TC10 and Wrch1, and also related Rho-of-plants proteins (ROPs) in plants, stimulate actin polymerization and affect plasma membrane protrusion and/or vesicular traffic.     

Endocytosis of E-cadherin regulated by Rac and Cdc42 small G proteins through IQGAP1 and actin filaments

E-cadherin is a key cell-cell adhesion molecule at adherens junctions (AJs) and undergoes endocytosis when AJs are disrupted by the action of extracellular signals. To elucidate the mechanism of this endocytosis, we developed here a new cell-free assay system for this reaction using the AJ-enriched fraction from rat liver. We found here that non-trans-interacting, but not trans-interacting, E-cadherin underwent endocytosis in a clathrin-dependent manner. The endocytosis of trans-interacting E-cadherin was inhibited by Rac and Cdc42 small G proteins, which were activated by trans-interacting E-cadherin or trans-interacting nectins, which are known to induce the formation of AJs in cooperation with E-cadherin. This inhibition was mediated by reorganization of the actin cytoskeleton by Rac and Cdc42 through IQGAP1, an actin filament-binding protein and a downstream target of Rac and Cdc42. These results indicate the important role of the Rac/Cdc42-IQGAP1 system in the dynamic organization and maintenance of the E-cadherin-based AJs.     

Genetic deletion of the Pten tumor suppressor gene promotes cell motility by activation of Rac1 and Cdc42 GTPases

Pten (Phosphatase and tensin homolog deleted on chromosome 10) is a recently identified tumor suppressor gene which is deleted or mutated in a variety of primary human cancers and in three cancer predisposition syndromes [1]. Pten regulates apoptosis and cell cycle progression through its phosphatase activity on phosphatidylinositol (PI) 3,4,5-trisphosphate (PI(3,4,5)P(3)), a product of PI 3-kinase [2-5]. Pten has also been implicated in controlling cell migration [6], but the exact mechanism is not very clear. Using the isogenic Pten(+/+) and Pten(-/-) mouse fibroblast lines, here we show that Pten deficiency led to increased cell motility. Reintroducing the wild-type Pten, but not the catalytically inactive Pten C124S or lipid-phosphatase-deficient Pten G129E mutant, reduced the enhanced cell motility of Pten-deficient cells. Moreover, phosphorylation of the focal adhesion kinase p125(FAK) was not changed in Pten(-/-) cells. Instead, significant increases in the endogenous activities of Rac1 and Cdc42, two small GTPases involved in regulating the actin cytoskeleton [7], were observed in Pten(-/-) cells. Overexpression of dominant-negative mutant forms of Rac1 and Cdc42 reversed the cell migration phenotype of Pten(-/-) cells. Thus, our studies suggest that Pten negatively controls cell motility through its lipid phosphatase activity by down-regulating Rac1 and Cdc42.     

Transforming growth factor-beta-induced mobilization of actin cytoskeleton requires signaling by small GTPases Cdc42 and RhoA

Transforming growth factor-beta (TGF-beta) is a potent regulator of cell growth and differentiation in many cell types. The Smad signaling pathway constitutes a main signal transduction route downstream of TGF-beta receptors. We studied TGF-beta-induced rearrangements of the actin filament system and found that TGF-beta 1 treatment of PC-3U human prostate carcinoma cells resulted in a rapid formation of lamellipodia. Interestingly, this response was shown to be independent of the Smad signaling pathway; instead, it required the activity of the Rho GTPases Cdc42 and RhoA, because ectopic expression of dominant negative mutant Cdc42 and RhoA abrogated the response. Long-term stimulation with TGF-beta 1 resulted in an assembly of stress fibers; this response required both signaling via Cdc42 and RhoA, and Smad proteins. A known downstream effector of Cdc42 is p38(MAPK); treatment of the cells with the p38(MAPK) inhibitor 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(pyridyl)1H-imidazole (SB203580), as well as ectopic expression of a kinase-inactive p38(MAPK), abrogated the TGF-beta-induced actin reorganization. Moreover, treatment of cells with the inhibitors of the RhoA target-protein Rho-associated coiled-coil kinase (+)-R-trans-4-(aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide (Y-27632) and 1-5(-isoquinolinesulfonyl)homopiperazine (HA-1077), as well as ectopic expression of kinase-inactive Rho coiled-coil kinase-1, abrogated the TGF-beta 1-induced formation of stress fibers. Collectively, these data indicate that TGF-beta-induced membrane ruffles occur via Rho GTPase-dependent pathways, whereas long-term effects require cooperation between Smad and Rho GTPase signaling pathways.     

Specific contributions of the small GTPases Rho, Rac, and Cdc42 to Dbl transformation.

Dbl is a representative prototype of a growing family of oncogene products that contain the Dbl homology/pleckstrin homology elements in their primary structures and are associated with a variety of neoplastic pathologies. Members of the Dbl family have been shown to function as physiological activators (guanine nucleotide exchange factors) of the Rho-like small GTPases. Although the expression of GTPase-defective versions of Rho proteins has been shown to induce a transformed phenotype under different conditions, their transformation capacity has been typically weak and incomplete relative to that exhibited by dbl-like oncogenes. Moreover, in some cases (e.g. NIH3T3 fibroblasts), expression of GTPase-defective Cdc42 results in growth inhibition. Thus, in attempting to reconstitute dbl-induced transformation of NIH3T3 fibroblasts, we have generated spontaneously activated ("fast-cycling") mutants of Cdc42, Rac1, and RhoA that mimic the functional effects of activation by the Dbl oncoprotein. When stably expressed in NIH3T3 cells, all three mutants caused the loss of serum dependence and showed increased saturation density. Furthermore, all three stable cell lines were tumorigenic when injected into nude mice. Our data demonstrate that all three Dbl targets need to be activated to promote the full complement of Dbl effects. More importantly, activation of each of these GTP-binding proteins contributes to a different and distinct facet of cellular transformation.     

Stimulation of phospholipase C-beta2 by the Rho GTPases Cdc42Hs and Rac1.

Neutrophils contain a soluble guanine-nucleotidebinding protein, made up of two components with molecular masses of 23 and 26 kDa, that mediates stimulation of phospholipase C-beta2 (PLCbeta2). We have identified the two components of the stimulatory heterodimer by amino acid sequencing as a Rho GTPase and the Rho guanine nucleotide dissociation inhibitor LyGDI. Using recombinant Rho GTPases and LyGDI, we demonstrate that PLCbeta2 is stimulated by guanosine 5'-O-(3-thiotriphosphate) (GTP[S])-activated Cdc42HsxLyGDI, but not by RhoAxLyGDI. Stimulation of PLCbeta2, which was also observed for GTP[S]-activated recombinant Rac1, was independent of LyGDI, but required C-terminal processing of Cdc42Hs/Rac1. Cdc42Hs/Rac1 also stimulated PLCbeta2 in a system made up of purified recombinant proteins, suggesting that this function is mediated by direct protein-protein interaction. The Cdc42Hs mutants F37A and Y40C failed to stimulate PLCbeta2, indicating that the Cdc42Hs effector site is involved in this interaction. The results identify PLCbeta2 as a novel effector of the Rho GTPases Cdc42Hs and Rac1, and as the first mammalian effector directly regulated by both heterotrimeric and low-molecular-mass GTP-binding proteins.     

Stimulation of fascin spikes by thrombospondin-1 is mediated by the GTPases Rac and Cdc42

Cell adhesion to extracellular matrix is an important physiological stimulus for organization of the actin-based cytoskeleton. Adhesion to the matrix glycoprotein thrombospondin-1 (TSP-1) triggers the sustained formation of F-actin microspikes that contain the actin-bundling protein fascin. These structures are also implicated in cell migration, which may be an important function of TSP-1 in tissue remodelling and wound repair. To further understand the function of fascin microspikes, we examined whether their assembly is regulated by Rho family GTPases. We report that expression of constitutively active mutants of Rac or Cdc42 triggered localization of fascin to lamellipodia, filopodia, and cell edges in fibroblasts or myoblasts. Biochemical assays demonstrated prolonged activation of Rac and Cdc42 in C2C12 cells adherent to TSP-1 and activation of the downstream kinase p21-activated kinase (PAK). Expression of dominant-negative Rac or Cdc42 in C2C12 myoblasts blocked spreading and formation of fascin spikes on TSP-1. Spreading and spike assembly were also blocked by pharmacological inhibition of F-actin turnover. Shear-loading of monospecific anti-fascin immunoglobulins, which block the binding of fascin to actin into cytoplasm, strongly inhibited spreading, actin cytoskeletal organization and migration on TSP-1 and also affected the motility of cells on fibronectin. We conclude that fascin is a critical component downstream of Rac and Cdc42 that is needed for actin cytoskeletal organization and cell migration responses to thrombospondin-1.     

Cdc42Hs and Rac1 GTPases induce the collapse of the vimentin intermediate filament network

In this study we show that expression of active Cdc42Hs and Rac1 GTPases, two Rho family members, leads to the reorganization of the vimentin intermediate filament (IF) network, showing a perinuclear collapse. Cdc42Hs displays a stronger effect than Rac1 as 90% versus 75% of GTPase-expressing cells show vimentin collapse. Similar vimentin IF modifications were observed when endogenous Cdc42Hs was activated by bradykinin treatment, endogenous Rac1 by platelet-derived growth factor/epidermal growth factor, or both endogenous proteins upon expression of active RhoG. This reorganization of the vimentin IF network is not associated with any significant increase in soluble vimentin. Using effector loop mutants of Cdc42Hs and Rac1, we show that the vimentin collapse is mostly independent of CRIB (Cdc42Hs or Rac-interacting binding)-mediated pathways such as JNK or PAK activation but is associated with actin reorganization. This does not result from F-actin depolymerization, because cytochalasin D treatment or Scar-WA expression have merely no effect on vimentin organization. Finally, we show that genistein treatment of Cdc42 and Rac1-expressing cells strongly reduces vimentin collapse, whereas staurosporin, wortmannin, LY-294002, R(p)-cAMP, or RII, the regulatory subunit of protein kinase A, remain ineffective. Moreover, we detected an increase in cellular tyrosine phosphorylation content after Cdc42Hs and Rac1 expression without modification of the vimentin phosphorylation status. These data indicate that Cdc42Hs and Rac1 GTPases control vimentin IF organization involving tyrosine phosphorylation events.     

Cdc42, Rac1, and Rac2 display distinct patterns of activation during phagocytosis

The small G proteins Cdc42, Rac1, and Rac2 regulate the rearrangements of actin and membrane necessary for Fcgamma receptor-mediated phagocytosis by macrophages. Activated, GTP-bound Cdc42, Rac1, and Rac2 bind to the p21-binding domain (PBD) of PAK1, and this interaction provided a basis for microscopic methods to localize activation of these G proteins inside cells. Fluorescence resonance energy transfer-based stoichiometry of fluorescent chimeras of actin, PBD, Cdc42, Rac1, and Rac2 was used to quantify G protein activation relative to actin movements during phagocytosis of IgG-opsonized erythrocytes. The activation dynamics of endogenous G proteins, localized using yellow fluorescent protein-labeled PBD, was restricted to phagocytic cups, with a prominent spike of activation over an actin-poor region at the base of the cup. Refinements of fluorescence resonance energy transfer stoichiometry allowed calculation of the fractions of activated GTPases in forming phagosomes. Cdc42 activation was restricted to the leading margin of the cell, whereas Rac1 was active throughout the phagocytic cup. During phagosome closure, activation of Rac1 and Rac2 increased uniformly and transiently in the actin-poor region of phagosomal membrane. These distinct roles for Cdc42, Rac1, and Rac2 in the component activities of phagocytosis indicate mechanisms by which their differential regulation coordinates rearrangements of actin and membranes.     

Endothelin 1 induces beta 1Pix translocation and Cdc42 activation via protein kinase A-dependent pathway

p21-activated kinase (Pak)-interacting exchange factor (Pix), a Rho family guanine nucleotide exchange factor (GEF), has been shown to co-localize with Pak and form activated Cdc42- and Rac1-driven focal complexes. In this study we have presented evidence that treatment of human mesangial cells (HMC) with endothelin 1 (ET-1) and stimulation of adenylate cyclase with either forskolin or with the cAMP analog 8-Br-cAMP activated the GTP loading of Cdc42. Transient expression of constitutively active G alpha(s) also stimulated Cdc42. In addition, overexpression of beta(1)Pix enhanced ET-1-induced Cdc42 activation, whereas the expression of beta(1)Pix SH3m(W43K), which lacks the ability to bind Pak, and beta(1)PixDHm(L238R/L239S), which lacks GEF activity, decreased ET-1-induced Cdc42 activation. Furthermore, ET-1 stimulation induced beta(1)Pix translocation to focal complexes. Interestingly, pretreatment of HMC with protein kinase A (PKA) inhibitors blocked both Cdc42 activation and beta(1)Pix translocation induced by ET-1, indicating the involvement of the PKA pathway. Through site-directed mutagenesis studies of consensus PKA phosphorylation sites and in vitro PKA kinase assay, we have shown that beta(1)Pix is phosphorylated by PKA. Using purified recombinant beta(1)Pix(wt) and beta(1)Pix mutants, we have identified Ser-516 and Thr-526 as the major phosphorylation sites by PKA. beta(1)Pix(S516A/T526A), in which both phosphorylation sites are replaced by alanine, blocks beta(1)Pix translocation and Cdc42 activation. Our results have provided evidence that stimulation of PKA pathway by ET-1 or cAMP analog results in beta(1)Pix phosphorylation, which in turn controls beta(1)Pix translocation to focal complexes and Cdc42 activation.     

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.     

Signals from the Ras, Rac, and Rho GTPases converge on the Pak protein kinase in Rat-1 fibroblasts

Ras plays a key role in regulating cellular proliferation, differentiation, and transformation. Raf is the major effector of Ras in the Ras > Raf > Mek > extracellular signal-activated kinase (ERK) cascade. A second effector is phosphoinositide 3-OH kinase (PI 3-kinase), which, in turn, activates the small G protein Rac. Rac also has multiple effectors, one of which is the serine threonine kinase Pak (p65(Pak)). Here we show that Ras, but not Raf, activates Pak1 in cotransfection assays of Rat-1 cells but not NIH 3T3 cells. We tested agents that activate or block specific components downstream of Ras and demonstrate a Ras > PI 3-kinase > Rac/Cdc42 > Pak signal. Although these studies suggest that the signal from Ras through PI 3-kinase is sufficient to activate Pak, additional studies suggested that other effectors contribute to Pak activation. RasV12S35 and RasV12G37, two effector mutant proteins which fail to activate PI 3-kinase, did not activate Pak when tested alone but activated Pak when they were cotransfected. Similarly, RacV12H40, an effector mutant that does not bind Pak, and Rho both cooperated with Raf to activate Pak. A dominant negative Rho mutant also inhibited Ras activation of Pak. All combinations of Rac/Raf and Ras/Raf and Rho/Raf effector mutants that transform cells cooperatively stimulated ERK. Cooperation was Pak dependent, since all combinations were inhibited by kinase-deficient Pak mutants in both transformation assays and ERK activation assays. These data suggest that other Ras effectors can collaborate with PI 3-kinase and with each other to activate Pak. Furthermore, the strong correlation between Pak activation and cooperative transformation suggests that Pak activation is necessary, although not sufficient, for cooperative transformation of Rat-1 fibroblasts by Ras, Rac, and Rho.