Binding Of Rac Research Articles

The intellectual disability protein PAK3 regulates oligodendrocyte precursor cell differentiation

Oligodendrocyte and myelin deficits have been reported in mental/psychiatric diseases. The p21-activated kinase 3 (PAK3), a serine/threonine kinase, whose activity is stimulated by the binding of active Rac and Cdc42 GTPases is affected in these pathologies. Indeed, many mutations of Pak3 gene have been described in non-syndromic intellectual disability diseases. Pak3 is expressed mainly in the brain where its role has been investigated in neurons but not in glial cells. Here, we showed that PAK3 is highly expressed in oligodendrocyte precursors (OPCs) and its expression decreases in mature oligodendrocytes. In the developing white matter of the Pak3 knockout mice, we found defects of oligodendrocyte differentiation in the corpus callosum and to a lesser extent in the anterior commissure, which were compensated at the adult stage. In vitro experiments in OPC cultures, derived from Pak3 knockout and wild type brains, support a developmental and cell-autonomous role for PAK3 in regulating OPC differentiation into mature oligodendrocytes. Moreover, we did not detect any obvious alterations of the proliferation or migration of Pak3 null OPCs compared to wild type. Overall, our data highlight PAK3 as a new regulator of OPC differentiation.

Comparative effect of lidocaine, bupivacaine and RAC 109 on myocardial conduction and contractility in the rabbit

Local anesthetic toxicity includes a decrease in ventricular conduction velocity and a decrease in myocardial contractile force. We tested the hypothesis that, like conduction, contraction was stereoselectively impaired by bupivacaine. We compared R(+) and S(−) bupivacaine to S(+) and R(−) RAC 109 in order to study the effects of hydrophobicity and ionization. We measured the changes in QRS duration and developed pressure in isolated perfused rabbit hearts. Binding of bupivacaine and RAC 109 to the ryanodine receptor was measured. The effect on cell shortening and relenghtening was measured on isolated rabbit cardiomyocytes. A mixed-effect pharmacodynamic model was used. The decrease in conduction velocity induced by the molecules was markedly stereospecific. All local anesthetics decreased ventricular velocity in a stereospecific manner with a RAC I(+)/II(−) and bupivacaine R(+)/S(−) potency ratio of maximum effect of 1.7 and 2.25 respectively. Contractility decreased in a dose dependent manner but this negative inotropic effect was not stereospecific with a C50 (concentration leading to half maximum effect) of 30 and 23μM for RAC and bupivacaine respectively. The two drugs exhibited two-site binding to ryanodyne that may partly explain the observed effect. An effect on relaxation was observed only at very high concentrations. In conclusion, bupivacaine, a long acting local anesthetic, decreases ventricular conduction in a stereospecific manner, and decreases contractility non-stereospecifically.

The Fer tyrosine kinase regulates interactions of Rho GDP-Dissociation Inhibitor α with the small GTPase Rac

BackgroundRhoGDI proteins are important regulators of the small GTPase Rac, because they shuttle Rac from the cytoplasm to membranes and also protect Rac from activation, deactivation and degradation. How the binding and release of Rac from RhoGDI is regulated is not precisely understood.ResultsWe report that the non-receptor tyrosine kinase Fer is able to phosphorylate RhoGDIα and form a direct protein complex with it. This interaction is mediated by the C-terminal end of RhoGDIα. Activation of Fer by reactive oxygen species caused increased phosphorylation of RhoGDIα and pervanadate treatment further augmented this. Tyrosine phosphorylation of RhoGDIα by Fer prevented subsequent binding of Rac to RhoGDIα, but once a RhoGDIα-Rac complex was formed, the Fer kinase was not able to cause Rac release through tyrosine phosphorylation of preformed RhoGDIα-Rac complexes.ConclusionsThese results identify tyrosine phosphorylation of RhoGDIα by Fer as a mechanism to regulate binding of RhoGDIα to Rac.

Phospholipase C isozymes as effectors of Ras superfamily GTPases

The physiological effects of many extracellular stimuli are initiated through receptor-promoted activation of phospholipase C and inositol lipid signaling pathways. The historical view that phospholipase C-promoted signaling primarily occurs through activation of heterotrimeric G proteins or tyrosine kinases has expanded in recent years with the realization that at least three different mammalian phospholipase C isozymes are directly activated by members of the Ras superfamily of GTPases. Thus, Ras, Rap, Rac, and Rho GTPases all specifically regulate certain phospholipase C isozymes, and insight into the physiological significance of these signaling responses is beginning to accrue. High resolution three-dimensional structures of phospholipase C isozymes also are beginning to shed light on their mechanism of activation.

Characterization of Ser338 Phosphorylation for Raf-1 Activation

Raf kinases are essential for regulating cell proliferation, survival, and tumorigenesis. However, the mechanisms by which Raf is activated are still incompletely understood. Phosphorylation plays a critical role in Raf activation in response to mitogens. The present study characterizes phosphorylation of Ser338, a crucial event for Raf-1 activation. Here we report that mutation of Lys375 to Met diminishes phosphorylation of Ser338 on both wild type Raf-1 in cells treated with epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA) and a constitutively active mutant in which Tyr340/Tyr341 are replaced by 2 aspartic acids, a conserved substitution present in natural B-Raf. The loss of Ser338 phosphorylation in these Raf mutants is not engendered by a mutation-induced conformational change, inasmuch as mutation of another site (Ser471 to Ala) in the activation segment also abolishes Ser338 phosphorylation, whereas both the kinase-dead mutants of Raf-1 are phosphorylated well by active Pak1. Furthermore, our data demonstrate that EGF-stimulated phosphorylation of Ser338 is inhibited by Sorafenib, a Raf kinase inhibitor, but not by the MEK inhibitor U0126. Interestingly, a kinase-dead mutation and Sorafenib also markedly reduce phosphorylation of Ser445 on B-Raf, a site equivalent to Raf-1 Ser338. Finally, our data reveal that Ser338 is phosphorylated on inactive Raf-1 by an active mutant of Raf-1 when they are dimerized in cells and that artificial dimerization of Raf-1 causes Ser338 phosphorylation, accompanied by activation of ERK1/2. Altogether, our data suggest that Ser338 on Raf-1 is autophosphorylated in response to mitogens.

ErbB-2 and Met Reciprocally Regulate Cellular Signaling via Plexin-B1

Sema4D-induced activation of plexin-B1 has been reported to evoke different and sometimes opposing cellular responses. The mechanisms underlying the versatility of plexin-B1-mediated effects are not clear. Plexin-B1 can associate with the receptor tyrosine kinases ErbB-2 and Met. Here we show that Sema4D-induced activation and inactivation of RhoA require ErbB-2 and Met, respectively. In breast carcinoma cells, Sema4D can have pro- and anti-migratory effects depending on the presence of ErbB-2 and Met, and the exchange of the two receptor tyrosine kinases is sufficient to convert the cellular response to Sema4D from pro- to anti-migratory and vice versa. This work identifies a novel mechanism by which plexin-mediated signaling can be regulated and explains how Sema4D can exert different biological activities through the differential association of its receptor with ErbB-2 and Met.

A Prodigiosin Analogue Inactivates NADPH Oxidase in Macrophage Cells by Inhibiting Assembly of p47phox and Rac

Prodigiosins are natural red pigments that have multi-biological activities. Recently, we discovered a marine bacterial strain, which produces a red pigment. Extensive two-dimensional nuclear magnetic resonance and mass spectrometry analysis showed that the pigment is a prodigiosin analogue (PG-L-1). Here, we investigated the effect of PG-L-1 on NADPH oxidase activity in macrophage cells. PG-L-1 significantly inhibited superoxide anion (O(2)(-)) production by phorbol 12-myristate 13-acetate (PMA)-stimulated RAW 264.7 cells, a mouse macrophage cell line. The ED(50) value was estimated to be approximately 0.3 microM. PG-L-1 had no direct scavenging effect on O(2)(-) generated by hypoxanthine/xanthine oxidase system in electron spin resonance-spin trapping determinations, suggesting that this compound directly acts on the O(2)(-) production system, NADPH oxidase, in macrophage cells. We further investigated the effect of PG-L-1 on the behaviour of the cytosolic components of the NADPH oxidase, p67(phox), p47(phox), p40(phox), Rac and protein kinase C (PKC), in PMA-stimulated RAW 264.7 cells. Although PG-L-1 showed no effect on the activation of PKC, the immunoblotting analysis using specific antibodies showed that PG-L-1 strongly inhibits the association of p47(phox) and Rac in the plasma membrane of PMA-stimulated RAW 264.7 cells. These results suggest that PG-L-1 inactivates NADPH oxidase through the inhibition of the binding of p47(phox) and Rac to the membrane components of NADPH oxidase.

Gabapentin-Lactam Induces Dendritic Filopodia and Motility in Cultured Hippocampal Neurons

Gabapentin is currently used as a therapeutic agent against epilepsy as well as neuropathic pain. In contrast to gabapentin, its derivative gabapentin-lactam has a pronounced neuroprotective activity. We have studied in cultured hippocampal neurons whether gabapentin-lactam has also neurotrophic effects. Gabapentin-lactam enhanced the formation of dendritic filopodia, which are necessary for synapse formation. It also induced a network of F-actin-containing neurites. In studies with time lapse microscopy, gabapentin-lactam increased the addition but also the elimination of new branches. Affinity precipitation assays showed that gabapentin-lactam increased the GTP binding of the small GTPases Rac and Cdc42, which facilitate branch addition. Gabapentin-lactam also activated RhoA and phosphatidylinositol 3-kinases. In neurons transfected with dominant-negative RhoA or treated with the RhoA-inactivating C3 toxin, gabapentin-lactam increased the number of dendrites and branches. In the presence of Y-27632, which inhibits Rho kinase, newly added branches induced by gabapentin-lactam were no longer eliminated so that gabapentin-lactam increased the number of branches. Y-27632 [(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide] also prevented the gabapentin-lactam induced activation of phosphatidylinositol 3-kinases. The phosphatidylinositol 3-kinase inhibitor LY294002 [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride] reduced the elimination of newly added branches caused by gabapentin-lactam and thus facilitated branch formation. In contrast to gabapentin-lactam, gabapentin had no effect on dendritic filopodia or motility. The effects exerted by gabapentin-lactam on dendritic arborization may be of potential therapeutic interest.

Chronic Ethanol Treatment Impairs Rac and Cdc42 Activation in Rat Hepatocytes

The effects of chronic ethanol feeding on rat hepatocytes have been shown to include impaired cell-extracellular matrix (ECM) adhesion events, such as decreased attachment and spreading as well as increased integrin-actin cytoskeleton association. These results, observed previously by this laboratory, are highly suggestive of impaired actin cytoskeleton reorganization, an event mediated by differential activation of the Rho family GTPases Rac, Cdc42, and RhoA. Therefore, the purpose of this study was to examine the effects of chronic ethanol administration on these GTPases. Male Wistar rats were pair-fed 4 to 5 weeks with a liquid diet containing either ethanol (as 36% of total calories) or isocaloric carbohydrate. Hepatocytes were isolated and plated on collagen IV up to 24 hours. At specific times, the hepatocytes were lysed and these lysates were analyzed for RhoA, Cdc42, and Rac activation. In freshly isolated hepatocytes from ethanol-fed rats, the GTP-bound (active) forms of Rac and Cdc42 were significantly decreased compared with pair-fed control rats, while the GTP-bound form of RhoA was not significantly altered. These ethanol-induced impairments in Rac and Cdc42 activation persisted even after plating the hepatocytes on collagen IV. Additionally, chronic ethanol treatment did not directly affect GTP binding of Cdc42 and Rac, as incorporation of GTPgammaS was not affected. Chronic ethanol administration selectively impairs Rac and Cdc42 activation in rat hepatocytes. As activation of these 2 GTPases is crucial for efficient cell attachment and spreading on ECM substrates, the results from this study suggest that the ethanol-induced impairments in Rac and Cdc42 activation are responsible for the impaired hepatocyte-ECM adhesion events observed previously by our laboratory. Furthermore, these results raise the intriguing possibility that these GTPases are involved in other ethanol-induced functional impairments, such as protein trafficking and receptor-mediated endocytosis.

In Vivo Dynamics of Rac-Membrane Interactions

The small GTPase Rac cycles between the membrane and the cytosol as it is activated by nucleotide exchange factors (GEFs) and inactivated by GTPase-activating proteins (GAPs). Solubility in the cytosol is conferred by binding of Rac to guanine-nucleotide dissociation inhibitors (GDIs). To analyze the in vivo dynamics of Rac, we developed a photobleaching method to measure the dissociation rate constant (k(off)) of membrane-bound GFP-Rac. We find that k(off) is 0.048 s(-1) for wtRac and approximately 10-fold less (0.004 s(-1)) for G12VRac. Thus, the major route for dissociation is conversion of membrane-bound GTP-Rac to GDP-Rac; however, dissociation of GTP-Rac occurs at a detectable rate. Overexpression of the GEF Tiam1 unexpectedly decreased k(off) for wtRac, most likely by converting membrane-bound GDP-Rac back to GTP-Rac. Both overexpression and small hairpin RNA-mediated suppression of RhoGDI strongly affected the amount of membrane-bound Rac but surprisingly had only slight effects on k(off). These results indicate that RhoGDI controls Rac function mainly through effects on activation and/or membrane association.

The regulation of mDia1 by autoinhibition and its release by Rho•GTP

Formins induce the nucleation and polymerisation of unbranched actin filaments via the formin-homology domains 1 and 2. Diaphanous-related formins (Drfs) are regulated by a RhoGTPase-binding domain situated in the amino-terminal (N-terminal) region and a carboxy-terminal Diaphanous-autoregulatory domain (DAD), whose interaction stabilises an autoinhibited inactive conformation. Binding of active Rho releases DAD and activates the catalytic activity of mDia. Here, we report on the interaction of DAD with the regulatory N-terminus of mDia1 (mDia(N)) and its release by Rho*GTP. We have defined the elements required for tight binding and solved the three-dimensional structure of a complex between an mDia(N) construct and DAD by X-ray crystallography. The core DAD region is an alpha-helical peptide, which binds in the most highly conserved region of mDia(N) using mainly hydrophobic interactions. The structure suggests a two-step mechanism for release of autoinhibition whereby Rho*GTP, although having a partially nonoverlapping binding site, displaces DAD by ionic repulsion and steric clashes. We show that Rho*GTP accelerates the dissociation of DAD from the mDia(N)*DAD complex.

Substrate Specificity and Recognition Is Conferred by the Pleckstrin Homology Domain of the Dbl Family Guanine Nucleotide Exchange Factor P-Rex2

Dbl family guanine nucleotide exchange factors (GEFs) are characterized by the presence of a catalytic Dbl homology domain followed invariably by a lipid-binding pleckstrin homology (PH) domain. To date, substrate recognition and specificity of this family of GEFs has been reported to be mediated exclusively via the Dbl homology domain. Here we report the novel and unexpected finding that, in the Dbl family Rac-specific GEF P-Rex2, it is the PH domain that confers substrate specificity and recognition. Moreover, the beta3beta4 loop of the PH domain of P-Rex2 is the determinant for Rac1 recognition, as substitution of the beta3beta4 loop of the PH domain of Dbs (a RhoA- and Cdc42-specific GEF) with that of P-Rex2 confers Rac1-specific binding capability to the PH domain of Dbs. The contact interface between the PH domain of P-Rex2 and Rac1 involves the switch loop and helix 3 of Rac1. Moreover, substitution of helix 3 of Cdc42 with that of Rac1 now enables the PH domain of P-Rex2 to bind this Cdc42 chimera. Despite having the ability to recognize this chimeric Cdc42, P-Rex2 is unable to catalyze nucleotide exchange on Cdc42, suggesting that recognition of substrate and catalysis are two distinct events. Thus substrate recognition can now be added to the growing list of functions that are being attributed to the PH domain of Dbl family GEFs.

Phylogenetic and structural analysis of the Drosophila melanogaster p21-activated kinase DmPAK3

P21-activated kinases (PAKs) are a family of serine/threonine kinases whose diverse cellular functions in cytoskeletal reorganisation, cell motility, transformation and cell death are regulated both by the binding of the small RhoGTPases RAC and CDC42 and by RhoGTPase independent mechanisms. The genome of Drosophila melanogaster encodes three different PAK proteins: DmPAK1, DmMBT (DmPAK2) and DmPAK3. Although structurally related, DmPAK and DmMBT control different developmental processes and also differ in the regulation of their kinase activity through binding of RAC or CDC42. Here, we report the characterisation of DmPAK3. The phylogenetic analysis provides evidence that DmPAK3 and the related proteins from Drosophila pseudoobscura, Anopheles gambiae and Apis mellifera make up a distinct subgroup within the PAK protein family, which might be confined to insects. The structural differences of this PAK subgroup are also reflected by the selective binding of DmPAK3 to RAC-like RhoGTPases. Our biochemical analysis supports a model in which DmPAK3 can form homodimers where the N-terminal regulatory domain of one DmPAK3 protein can bind to and thereby inhibit the catalytic domain of the other DmPAK3 protein. Binding of activated RAC to the regulatory domain or mutation of the RAC-binding site in DmPAK3 relieves the inhibitory effect on kinase activity. Furthermore, our data indicate a function of DmPAK3 in reorganisation of the actin cytoskeleton.

The Effect of Anionic Amphiphiles on the Recruitment of Rac in Neutrophils

Anionic amphiphiles have been shown to influence the NADPH oxidase system. Although one target of the amphiphile action is p47(phox), the cell-free activation of the enzyme in the absence of p47(phox) is also influenced. In the present study, we examined the actions of sodium dodecyl sulfate (SDS) on the NADPH oxidase system in vivo. Treatment of guinea pig neutrophils with the amphiphile caused the translocation of Rac to a membrane fraction and its conversion to the GTP-bound form. Because SDS had little effect on p47(phox), it increased the superoxide production only when p47(phox) was otherwise activated. Inhibitors of phosphoinositide 3-kinases had no effect on the SDS-induced translocation of Rac to the membrane. However, the inhibitors prevented the conversion of Rac to its GTP-bound form, indicating that these two processes can be controlled separately. In a cell-free system, SDS induced the binding of p47(phox) and Rac to the membrane preparation. The SDS concentration inducing the Rac binding was lower than that inducing the p47(phox) binding. Thus we observed that Rac is more sensitive to SDS than p47(phox) both in vivo and in vitro. The results suggest a role of natural amphiphiles such as unsaturated fatty acids in regulation of Rac activation.

Mechanism of p21-activated Kinase 6-mediated Inhibition of Androgen Receptor Signaling

PAK6 was first identified as an androgen receptor (AR)-interacting protein able to inhibit AR-mediated transcriptional responses. PAK6 is a serine/threonine kinase belonging to the p21-activated kinase (PAK) family implicated in actin reorganization and cell motility, gene transcription, apoptosis, and cell transformation. We investigated the biochemical basis for inhibition of AR signaling by PAK6. We compared the kinase activity of PAK6 with two other well characterized members of the PAK family, PAK1 and PAK4. Like PAK4, PAK6 possesses a constitutive basal kinase activity that, unlike PAK1, is not modulated by the binding of active Rac or Cdc42 GTPases. In order to test the involvement of PAK6 kinase activity in suppression of AR-mediated transcription, we generated kinase-dead (K436A) and kinase-active (S531N) mutants of PAK6. We show that PAK6 kinase activity is required for effective PAK6-induced repression of AR signaling. Suppression does not depend upon GTPase binding to PAK6 and is not mimicked by the closely related PAK1 and PAK4 isoforms. Kinase-dependent inhibition by PAK6 extended to the enhanced AR-mediated transcription seen in the presence of coactivating molecules and to the action of AR coinhibitors. Active PAK6 inhibited nuclear translocation of the stimulated AR, suggesting a possible mechanism for inhibition of AR responsiveness. Finally, we observe that autophosphorylated, active PAK6 protein is differently expressed among prostate cancer cell lines. Modulation of PAK6 activity may be responsible for regulation of AR signaling in various forms of prostate cancer.

Biology of the p21-activated kinases.

The p21-activated kinases (PAKs) 1-3 are serine/threonine protein kinases whose activity is stimulated by the binding of active Rac and Cdc42 GTPases. Our understanding of the regulation and biology of these important signaling proteins has increased tremendously since their discovery in the mid-1990s. PAKs 1-3 are activated by a variety of GTPase-dependent and -independent mechanisms. This complexity reflects the contributions of PAK function in many cellular signaling pathways and the need to carefully control PAK action in a highly localized manner. PAKs serve as important regulators of cytoskeletal dynamics and cell motility, transcription through MAP kinase cascades, death and survival signaling, and cell-cycle progression. Consequently, PAKs have also been implicated in a number of pathological conditions and in cell transformation. We propose here a key role for PAK action in coordinating the dynamics of the actin and microtubule cytoskeletons during directional motility of cells, as well as in other functions requiring cytoskeletal polarization.

Interaction between p21-activated protein kinase and Rac during differentiation of HL-60 human promyelocytic leukemia cell induced by all-trans-retinoic acid.

Undifferentiated human promyelocytic leukemia HL-60 cells show little or no superoxide production, but generate a very low O(2)(-) concentration upon incubation with all-trans-retinoic acid (ATRA). Its production reaches a maximum within 20 h, and thereafter is maintained at an almost constant level. The differentiated cells show phorbol 12-myristate 13-acetate (PMA)-stimulated NADPH oxidase activity consistent with the amount of gp91phox (phagocytic oxidase) expressed in the plasma membrane. Three isoforms of p21-activated serine/threonine kinases, PAK68, PAK65 and PAK62, were found in both cytosolic and membrane fractions, and their contents were significantly increased during induced differentiation. The amount of Rac identified in the two fractions was also markedly enhanced by ATRA- induced differentiation. In contrast, neither PAK nor Rac was seen in the plasma membrane of undifferentiated HL-60 or human neutrophil, but they were abundant in the cytoplasmic fraction. Binding of Rac with PAK isoforms was shown in the membrane upon induced differentiation of HL-60 cells. Direct binding of purified Rac1 to PAK68 was quantified using a fluorescent analog of GTP (methylanthraniloyl guanosine-5'-[beta,gamma-imido]triphosphate) bound to Rac as a reporter group. Rac1 bound to PAK68 with a 1 : 1 stoichiometry and with a K(d) value of 6.7 nm.

Calmodulin-binding and Autoinhibitory Domains ofAcanthamoeba Myosin I Heavy Chain Kinase, a p21-activated Kinase (PAK)

The sequence homology between Acanthamoeba myosin I heavy chain kinase (MIHCK) and other p21-activated kinases (PAKs) is relatively low, including only the catalytic domain and a short PAK N-terminal motif (PAN), and even these regions are not highly homologous. In this paper, we report the expression in insect cells of full-length, fully regulated Acanthamoeba MIHCK and further characterize the regulation of this PAK by Rac, calmodulin, and autoinhibition. We map the autoinhibitory region of MIHCK to its PAN region and show that the PAN region inhibits autophosphorylation and kinase activity of unphosphorylated full-length MIHCK and its expressed catalytic domain but has very little effect on either when they are phosphorylated. These properties are similar to those reported for mammalian PAK1. Unlike PAK1, MIHCK is activated by Rac only in the presence of phospholipid. However, peptides containing the PAN region of MIHCK bind Rac in the absence of lipid, and Rac binding reverses the inhibition of the MIHCK catalytic domain by PAN peptides. Our data suggest that a region N-terminal to PAN is required for optimal binding of Rac. Also unlike mammalian PAK, phospholipid stimulation of Acanthamoeba MIHCK and Dictyostelium MIHCK) (which is also a PAK) is inhibited by Ca(2+)-calmodulin. In contrast to Dictyostelium MIHCK, however, Ca(2+)-calmodulin also inhibits Rac-induced activity of Acanthamoeba MIHCK. The basic region N-terminal to PAN is essential for calmodulin binding.

The power of belief

l-Dopa and apomorphine are active drugs used in the treatment of patients suffering from Parkinson's disease (PD), a neurodegenerative disease in which the dopaminergic pathway involved in the regulation of behavioral and sensorimotor functions is severely impaired. The mechanisms involved are still poorly understood; however, a recent study has demonstrated that the level of expectation of benefit from treatment might influence and enhance the endogenous release of dopamine from the nigralstriatal pathway independently of the efficacy of the drug. In order to evaluate the power of the placebo effect in PD, researchers have studied the binding of the competitive dopamine receptor ligand [11C] raclopride by PET scanning. Patients receiving placebo or active drug showed comparably less binding of RAC in the striatum, indicating endogenous release of dopamine. The results published in Science (293, 1164–1166) highlight the importance of placebo controls in evaluating therapeutical intervention. SS

Structure of the TPR Domain of p67 phox in Complex with Rac·GTP

p67 phox is an essential part of the NADPH oxidase, a multiprotein enzyme complex that produces superoxide ions in response to microbial infection. Binding of the small GTPase Rac to p67 phox is a key step in the assembly of the active enzyme complex. The structure of Rac·GTP bound to the N-terminal TPR (tetratrico-peptide repeat) domain of p67 phox reveals a novel mode of Rho family/effector interaction and explains the basis of GTPase specificity. Complex formation is largely mediated by an insertion between two TPR motifs, suggesting an unsuspected versatility of TPR domains in target recognition and in their more general role as scaffolds for the assembly of multiprotein complexes.