p210(Bcr-Abl) desensitizes Cdc42 GTPase signaling for SDF-1alpha-directed migration in chronic myeloid leukemia cells.

Chronic myeloid leukemia (CML) is a lethal hematological disorder caused by the p210(Bcr-Abl) oncogene. Previous studies have suggested that p210(Bcr-Abl) transformation contributes to homing and retention defects, typical of immature myeloid cells in CML, by attenuating chemotactic response to stromal-derived factor-1alpha (SDF-1alpha). As Rho family GTPases are key regulators of the cytoskeleton and have been previously found to interact with p210(Bcr-Abl), this study aimed to determine whether p210(Bcr-Abl) signaling affects SDF-1alpha chemotaxis through Rho GTPase signaling. We found that SDF-1alpha stimulated Cdc42 GTPase activation in myeloid progenitor 32D, but not in p210(Bcr-Abl)-transformed (32Dp210) cells. In fact, the basal level of active Cdc42 was elevated in 32Dp210 cells and mononuclear cells isolated from bone marrow of CML patients. Inhibition of p210(Bcr-Abl) kinase activity decreased basal Cdc42 activity and restored SDF-1alpha-induced Cdc42 and migration responses. Transduction of active Tat-Cdc42V12 abolished this reconstituted chemotactic response. As Cdc42 is particularly important in cytoskeletal remodeling and directional sensing, these results suggest that sustained activation of Cdc42 GTPase through p210(Bcr-Abl) tyrosine kinase signaling in CML cells contributes to defects in SDF-1alpha-chemotactic response due to desensitization of the actin polarization signal required for directional migration.

Contribution of guanine exchange factor H1 in phorbol ester-induced apoptosis.

Phorbol-12-myristate-13-acetate (PMA) treatment induces erythroblastoma D2 cells kept in suspension to undergo RhoA-dependent contraction and to become proapoptotic, while attached cells are induced to differentiate accompanied by the reduction of RhoA activity. In this study, we found that guanine exchange factor H1 (GEF-H1) is highly expressed in D2 cells. Depletion of GEF-H1 expression in D2 cells decreased RhoA activity and prevented PMA-induced contraction and apoptosis. Upon PMA stimulation, GEF-H1 became associated with microtubules in cells that were induced to differentiate. As a contrast, in the proapoptotic population of cells GEF-H1 stayed in the cytoplasm without showing PMA-responsive microtubule translocation. Given that GEF-H1 is inactivated when associated with microtubules and its release into cytosol due to depolymerization of microtubules activates RhoA, our results demonstrated that nonmicrotubule-associated GEF-H1 in D2 cells contributes to the sustained activation of RhoA/ROCK signaling in suspension cells, making cells susceptible to PMA-induced apoptosis.

Rho GTPases and the control of the oxidative burst in polymorphonuclear leukocytes.

Stimulation of quiescent leukocytes activates the NADPH oxidase, a membrane-associated enzyme system that generates superoxide and other reactive oxygen species (ROS) that are used to kill bacteria within the phagosome. This chapter describes this multicomponent NADPH oxidase system, one of the first cellular systems shown to be directly regulated by Rac GTPases. We present current models of NADPH oxidase regulation by Rac2 and describe how Rac2 activation controls the timing of ROS production in adherent neutrophils. The antagonistic role that Cdc42 plays as a competitor of Rac2 for binding to the cytochrome component of the NADPH oxidase is discussed as a possible mechanism for tonic regulation of ROS production during the formation of the phagosome. Finally, we briefly depict mechanisms by which invasive bacteria can alter (inhibit) NADPH oxidase function, focusing on the effects of invasive bacteria on components and assembly of the NADPH oxidase.

Mechanisms of human neutrophil oxidant production after severe injury.

BACKGROUND: The purpose of this study was to determine the mechanisms of enhanced oxidant production after severe injury. METHODS: Neutrophils were harvested from patients within 24 hours of admission who had an injury severity score greater than 16. Nonadherent and adherent neutrophil oxidant production was measured after N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation. Translocation of cytochrome b558 and cytosolic components p47phox and p67phox were determined by oxidation-reduction spectroscopy and immunoblotting, respectively. Flow cytometry measured integrin expression. Integrin and p47phox colocalization was examined by confocal microscopy. RESULTS: Eighteen patients were studied within 15 +/- 1.4 hours. Four women and 14 men suffered a blunt injury and had a mean injury severity score of 22 (range, 16 to 34). Nonadherent patient neutrophils showed a decrease in fMLP-stimulated oxidant production, whereas adherent neutrophil oxidant production was increased in both the vehicle control and fMLP-stimulated groups. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase components p47phox and cytochrome b558 were mobilized to the plasma membrane, whereas p67phox showed minimal change. Integrin CD11b a chain showed a significant increase in expression. Confocal microscopy showed colocalization of p47phox and a chain CD11b on the plasma membrane of patient neutrophils. CONCLUSIONS: Colocalization of NADPH oxidase components and integrins may regulate the enhanced oxidant production in human neutrophils after severe injury.

De novo formation of focal complex-like structures in host cells by invading Streptococci.

Group A streptococcus (GAS) induces its own entry into eukaryotic cells in vitro and in vivo. Fibronectin (Fn) bound to protein F1, a GAS surface protein, acts as a bridge connecting the bacterium to host cell integrins. This triggers clustering of integrins, which acquire a polar pattern of distribution similar to that of protein F1 on the GAS surface. A unique and transient adhesion complex is formed at the site of GAS entry, which does not contain alpha-actinin. Vinculin is recruited to the site of GAS entry but is not required for uptake. The invading GAS recruits focal adhesion kinase (FAK), which is required for uptake and is tyrosine phosphorylated. The Src kinases, Src, Yes and Fyn, enhance the efficiency of GAS uptake but are not absolutely required for GAS entry. In addition, Rac and Cdc42, but not Rho, are required for the entry process. We suggest a model in which integrin engagement by Fn-occupied protein F1 triggers two independent signalling pathways. One is initiated by FAK recruitment and tyrosine phosphorylation, whereas the other is initiated by the recruitment and activation of Rac. The two pathways subsequently converge to trigger actin rearrangement leading to bacterial uptake.

A novel cardioprotective role of RhoA: new signaling mechanism for adenosine.

Adenosine exerts a potent cardioprotective effect that is mediated by adenosine A1 and A3 receptors. The signaling pathways activated by the A1 and A3 receptors are distinct and involve selective coupling to phospholipases C and D, respectively. The objective of our study was to elucidate the signaling mechanism that mediates the coupling of each receptor to its respective phospholipase and to test the role of RhoA as a novel mediator leading from adenosine receptors to cardioprotection. C3 transferase and dominant negative RhoA (RhoAT19N) blocked the A3 receptor-mediated phospholipase D activation and cardioprotection but had no effect on A1 receptor-mediated phospholipase C activation or cardioprotection. In contrast, pertussis toxin treatment caused a greater inhibition of the diacylglycerol accumulation induced by the A1 agonist than by the A3 agonist, and it completely abrogated the A1 agonist-mediated cardioprotection. Thus, adenosine A1 and A3 receptors are linked to different G-proteins. The A3 receptor is coupled via RhoA to activate phospholipase D in exerting its cardioprotective effect, whereas the A1 receptor is linked via Gi to phospholipase C to produce cardioprotective responses. The present study identifies a novel role for RhoA and further suggests its importance in regulating cardiac cellular function.

Structural consequences of site-directed mutagenesis in flexible protein domains: NMR characterization of the L(55,56)S mutant of RhoGDI.

The guanine dissociation inhibitor RhoGDI consists of a folded C-terminal domain and a highly flexible N-terminal region, both of which are essential for biological activity, that is, inhibition of GDP dissociation from Rho GTPases, and regulation of their partitioning between membrane and cytosol. It was shown previously that the double mutation L55S/L56S in the flexible region of RhoGDI drastically decreases its affinity for Rac1. In the present work we study the effect of this double mutation on the conformational and dynamic properties of RhoGDI, and describe the weak interaction of the mutant with Rac1 using chemical shift mapping. We show that the helical content of the region 45-56 of RhoGDI is greatly reduced upon mutation, thus increasing the entropic penalty for the immobilization of the helix, and contributing to the loss of binding. In contrast to wild-type RhoGDI, no interaction with Rac1 could be identified for amino-acid residues of the flexible domain of the mutant RhoGDI and only very weak binding was observed for the folded domain of the mutant. The origins of the effect of the L55S/L56S mutation on the binding constant (decreased by at least three orders of magnitude relative to wild-type) are discussed with particular reference to the flexibility of this part of the protein.

Interferon-gamma listericidal action is mediated by novel Rab5a functions at the phagosomal environment.

Control and clearance of Listeria monocytogenes infection is an interferon-gamma-dependent process. The listericidal mechanism of action involves activation of NADPH oxidase and inducible nitric-oxide synthase to produce reactive oxygen and nitrogen intermediate radicals, respectively. Recently, we have described in a nonpathogenic model of L. monocytogenes (hemolysin negative mutant strain) that the interferon-gamma-inducible GTPase Rab5a contributed to Listeria destruction in resting macrophages. Here, we report in a pathogenic model of L. monocytogenes (hemolysin-positive strain) that Rab5a plays a central role in Listeria destruction induced by interferon-gamma and within the phagosomal environment. These findings reveal the importance of Rab5a as the responsible factor mediating the listericidal action of interferon-gamma. Active Rab5a causes remodeling of the phagosomal environment, facilitates the translocation of Rac2 to LM phagosomes, and regulates the activity of this GTPase. Rac2 activation and translocation governs the phagocyte NADPH oxidase activity and the consequent reactive oxygen intermediate production that leads to killing of the pathogen.

Molecular basis for Rac2 regulation of phagocyte NADPH oxidase.

A Rac GTPase-regulated multiprotein NADPH oxidase is critical for the formation of reactive oxygen species (ROS) in phagocytic leukocytes and other nonphagocytic cells. NADPH oxidase reduces molecular oxygen to form superoxide anion in a two-step process. Electrons are initially transferred from NADPH to cytochrome b-associated FAD, then to cytochrome b heme and finally to molecular oxygen. We show here that Rac is required for both electron-transfer reactions. Mutational and biophysical analysis shows that Rac and p67phox independently regulate cytochrome b to catalyze the transfer of electrons from NADPH to FAD. However, they must interact with each other to induce the subsequent transfer of electrons from FAD to cytochrome b heme and molecular oxygen. This two-step model of regulation by Rac GTPase may provide a means of more effectively controlling the inflammatory responses of phagocytic leukocytes.

Structure-activity relationships in flexible protein domains: regulation of rho GTPases by RhoGDI and D4 GDI.

The guanine dissociation inhibitors RhoGDI and D4GDI inhibit guanosine 5'-diphosphate dissociation from Rho GTPases, keeping these small GTPases in an inactive state. The GDIs are made up of two domains: a flexible N-terminal domain of about 70 amino acid residues and a folded 134-residue C-terminal domain. Here, we characterize the conformation of the N-terminal regions of both RhoGDI and D4GDI using a series of NMR experiments which include (15)N relaxation and amide solvent accessibility measurements. In each protein, two regions with tendencies to form helices are identified: residues 36 to 58 and 9 to 20 in RhoGDI, and residues 36 to 57 and 20 to 25 in D4GDI. To examine the functional roles of the N-terminal domain of RhoGDI, in vitro and in vivo functional assays have been carried out with N-terminally truncated proteins. These studies show that the first 30 amino acid residues are not required for inhibition of GDP dissociation but appear to be important for GTP hydrolysis, whilst removal of the first 41 residues completely abolish the ability of RhoGDI to inhibit GDP dissociation. The combination of structural and functional studies allows us to explain why RhoGDI and D4GDI are able to interact in similar ways with the guanosine 5'-diphosphate-bound GTPase, but differ in their ability to regulate GTP-bound forms; these functional differences are attributed to the conformational differences of the N-terminal domains of the guanosine 5'-diphosphate dissociation inhibitors. Therefore, the two transient helices, appear to be associated with different biological effects of RhoGDI, providing a clear example of structure-activity relationships in a flexible protein domain.

Activation of Rac1 and the p38 mitogen-activated protein kinase pathway in response to all-trans-retinoic acid.

Several signaling pathways are activated by all-trans-retinoic acid (RA) to mediate induction of differentiation and apoptosis of malignant cells. In the present study we provide evidence that the p38 MAP kinase pathway is activated in a RA-dependent manner in the NB-4, acute pro-myelocytic leukemia, and the MCF-7, breast carcinoma, cell lines. RA treatment of cells induces a time- and dose-dependent phosphorylation of p38, and such phosphorylation results in activation of its catalytic domain. p38 activation is not inducible by RA in a variant NB-4 cell line, NB-4.007/6, which is resistant to the effects of RA, suggesting a role for this pathway in the induction of RA responses. Our data also demonstrate that the small G-protein Rac1 is activated by RA and functions as an upstream regulator of p38 activation, whereas the MAPKAPK-2 serine kinase is a downstream effector for the RA-activated p38. To obtain information on the functional role of the Rac1/p38/MAPKAPK-2 pathway in RA signaling, the effects of pharmacological inhibition of p38 on RA-induced gene transcription and cell differentiation were determined. Our results indicate that treatment of cells with the SB203580 inhibitor does not inhibit RA-dependent gene transcription via retinoic acid response elements or induction of Stat1 protein expression. However, treatment with SB203580 or SB202190 strongly enhances RA-dependent induction of cell differentiation and RA-regulated growth inhibitory responses. Altogether, our findings demonstrate that the Rac1/p38 MAP kinase pathway is activated in a RA-dependent manner and exhibits negative regulatory effects on the induction of differentiation.

Regulation of cell function by Rho GTPases.

Small GTPases of the Rho family regulate a wide variety of cell functions. In this review, we briefly describe the biological activities of Rho GTPases. Using the Rac-regulated NADPH oxidase as an example, we discuss possible regulatory points that might be exploited for drug development. Finally, we explore strategies for specific targeting of Rho GTPase-regulated signaling pathways.

Regulation of macropinocytosis by p21-activated kinase-1.

The process of macropinocytosis is an essential aspect of normal cell function, contributing to both growth and motile processes of cells. p21-activated kinases (PAKs) are targets for activated Rac and Cdc42 guanosine 5'-triphosphatases and have been shown to regulate the actin-myosin cytoskeleton. In fibroblasts PAK1 localizes to areas of membrane ruffling, as well as to amiloride-sensitive pinocytic vesicles. Expression of a PAK1 kinase autoinhibitory domain blocked both platelet-derived growth factor- and RacQ61L-stimulated uptake of 70-kDa dextran particles, whereas an inactive version of this domain did not, indicating that PAK kinase activity is required for normal growth factor-induced macropinocytosis. The mechanisms by which PAK modulate macropinocytosis were examined in NIH3T3 cell lines expressing various PAK1 constructs under the control of a tetracycline-responsive transactivator. Cells expressing PAK1 (H83,86L), a mutant that dramatically stimulates formation of dorsal membrane ruffles, exhibited increased macropinocytic uptake of 70-kDa dextran particles in the absence of additional stimulation. This effect was not antagonized by coexpression of dominant-negative Rac1-T17N. In the presence of platelet-derived growth factor, both PAK1 (H83,86L) and a highly kinase active PAK1 (T423E) mutant dramatically enhanced the uptake of 70-kDa dextran. Neither wild-type PAK1 nor vector controls exhibited enhanced macropinocytosis, nor did PAK1 (H83,86L) affect clathrin-dependent endocytic mechanisms. Active versions of PAK1 enhanced both growth factor-stimulated 70-kDa dextran uptake and efflux, suggesting that PAK1 activity modulated pinocytic vesicle cycling. These data indicate that PAK1 plays an important regulatory role in the process of macropinocytosis, perhaps related to the requirement for PAK in directed cell motility.

Localized Rac activation dynamics visualized in living cells.

Signaling proteins are thought to be tightly regulated spatially and temporally in order to generate specific and localized effects. For Rac and other small guanosine triphosphatases, binding to guanosine triphosphate leads to interaction with downstream targets and regulates subcellular localization. A method called FLAIR (fluorescence activation indicator for Rho proteins) was developed to quantify the spatio-temporal dynamics of the Rac1 nucleotide state in living cells. FLAIR revealed precise spatial control of growth factor-induced Rac activation, in membrane ruffles and in a gradient of activation at the leading edge of motile cells. FLAIR exemplifies a generally applicable approach for examining spatio-temporal control of protein activity.

p21-activated kinase (PAK1) is phosphorylated and activated by 3-phosphoinositide-dependent kinase-1 (PDK1).

In this study, we show that phosphorylated 3-phosphoinositide-dependent kinase 1 (PDK1) phosphorylates p21-activated kinase 1 (PAK1) in the presence of sphingosine. We identify threonine 423, a conserved threonine in the activation loop of kinase subdomain VIII, as the PDK1 phosphorylation site on PAK1. Threonine 423 is a previously identified PAK1 autophosphorylation site that lies within a PAK consensus phosphorylation sequence. After pretreatment with phosphatases, autophosphorylation of PAK1 occurred at all major sites except threonine 423. A phosphothreonine 423-specific antibody detected phosphorylation of recombinant, catalytically inactive PAK1 after incubation with wild-type PAK1, indicating phosphorylation of threonine 423 occurs by an intermolecular mechanism. The biological significance of PDK1 phosphorylation of PAK1 at threonine 423 in vitro is supported by the observation that these two proteins interact in vivo and that PDK1-phosphorylated PAK1 has an increased activity toward substrate. An increase of phosphorylation of catalytically inactive PAK1 was observed in COS-7 cells expressing wild-type, but not catalytically inactive, PDK1 upon elevation of intracellular sphingosine levels. PDK1 phosphorylation of PAK1 was not blocked by pretreatment with wortmannin or when PDK1 was mutated to prevent phosphatidylinositol binding, indicating this process is independent of phosphatidylinositol 3-kinase activity. The data presented here provide evidence for a novel mechanism for PAK1 regulation and activation.

The Rac1/p38 mitogen-activated protein kinase pathway is required for interferon alpha-dependent transcriptional activation but not serine phosphorylation of Stat proteins.

The p38 mitogen-activated protein (MAP) kinase is activated during engagement of the type I interferon (IFN) receptor and mediates signals essential for IFNalpha-dependent transcriptional activation via interferon-stimulated response elements without affecting formation of the ISGF3 complex. In the present study, we provide evidence that the small GTPase Rac1 is activated in a type I IFN-dependent manner and that its function is required for downstream engagement of the p38 MAP kinase pathway. We also demonstrate that p38 is required for IFNalpha-dependent gene transcription via GAS elements and regulates activation of the promoter of the PML gene that mediates growth inhibitory responses. In studies to determine whether the regulatory effects of p38 are mediated by serine phosphorylation of Stat1 or Stat3, we found that the p38 kinase inhibitors SB203580 or SB202190 or overexpression of a dominant negative p38 mutant do not inhibit phosphorylation of Stat1 or Stat3 on Ser-727 in several IFNalpha-sensitive cell lines. Altogether these data demonstrate that the Rac1/p38 MAP kinase signaling cascade plays a critical role in type I IFN signaling, functioning in cooperation with the Stat-pathway, to regulate transcriptional regulation of IFNalpha-sensitive genes and generation of growth inhibitory responses.

Regulation of cell function by Rho family GTPases.

Rho GTPases act as molecular switches to control many basic cellular activities that are also critical to the specialized functions of phagocytic leukocytes. Our laboratory has studied the regulation of Rho GTPase function, how these GTPases interact with specific effectors to modulate cell function, and how these events are coordinated in the stimulated cell. Areas of major interest include NADPH oxidase regulation by Rac2, Rac- and Cdc42-mediated control of the actin-myosin cytoskeleton via p21-activated kinase (PAK), and modulation of the apoptotic program by Rho GTPases and PAK.

Sphingosine is a novel activator of 3-phosphoinositide-dependent kinase 1.

3-Phosphoinositide-dependent kinase 1 (PDK1) has previously been shown to phosphorylate the activation loop of several AGC kinase family members. In this study, we show that p21-activated kinase 1, the activity of which is regulated by the GTP-bound form of Cdc42 and Rac and by sphingosine, is phosphorylated by PDK1. Phosphorylation of p21-activated kinase 1 by PDK1 occurred only in the presence of sphingosine, which increased PDK1 autophosphorylation 25-fold. Sphingosine increased PDK1 autophosphorylation in a concentration-dependent manner and significantly increased phosphate incorporation into known PDK1 substrates. Studies on the lipid requirement for PDK1 activation found that both sphingosine isoforms and stearylamine also increased PDK1 autophosphorylation. However, C(10)-sphingosine, octylamine, and stearic acid were unable to increase PDK1 autophosphorylation, indicating that both a positive charge and a lipid tail containing at least a C(10)-carbon backbone were required for PDK1 activation. Three PDK1 autophosphorylation sites were identified after stimulation with sphingosine in a serine-rich region located between the kinase domain and the pleckstrin homology domain using two-dimensional phosphopeptide maps and matrix assisted laser desorption/ionization mass spectroscopy. Increased phosphorylation of endogenous Akt at threonine 308 was observed in COS-7 cells expressing wild type PDK1, but not catalytically inactive PDK1, when cellular sphingosine levels were elevated by treatment with sphingomyelinase. Sphingosine thus appears to be a true PDK1 activator.

Rho family proteins modulate rapid apoptosis induced by cytotoxic T lymphocytes and Fas.

Little is known about the role of Rho proteins in apoptosis produced by stimuli evolved specifically to produce apoptosis, such as granzymes from cytotoxic T lymphocytes (CTLs) and Fas. Here we demonstrate that all three Rho family members are involved in CTL- and Fas-induced killing. Dominant-negative mutants of each Rho family member and Clostridium difficile toxin B, an inhibitor of all family members, strongly inhibited the susceptibility of cells to CTL- and Fas-induced apoptosis. Fas-induced caspase-3 activation was inhibited by C. difficile toxin. Activated mutants of each GTPase increased susceptibility to apoptosis, and activation of Cdc42 increased within 5 min of Fas stimulation. In contrast, during the time required for CTL and Fas killing, no apoptosis was produced by dominant-negative or activated mutants or by C. difficile toxin alone. Inhibition of actin polymerization using latrunculin A reduced the ability of constitutively active GTPase mutants to stimulate apoptosis and blocked Fas-induced activation of caspase-3. Furthermore, the ability of Rac to enhance apoptosis was decreased by point mutations reported to block Rac induction of actin polymerization. Rho family proteins may regulate apoptosis through their effects on the actin cytoskeleton.