Small GTPase Signaling Research Articles

Protein array analysis of oligomerization-induced changes in alpha-synuclein protein–protein interactions points to an interference with Cdc42 effector proteins

Aggregation of α-synuclein may contribute to neuropathology in Parkinson's disease patients and in transgenic animal models. Natively unfolded α-synuclein binds to various proteins and conformational changes due to α-synuclein misfolding may alter physiological interactions. In the present study, we used protein arrays spotted with 5000 recombinant human proteins for a large scale interaction analysis of monomeric versus oligomeric α-synuclein. Monomeric α-synuclein bound to arrayed cAMP regulated phosphoprotein 19 and binding appears to be disrupted by α-synuclein oligomerization. Incubation with recombinant α-synuclein oligomers lead to the identification of several GTPase activating proteins and Cdc42 effector proteins as binding partners. Protein database searches revealed a Cdc42/Rac interactive binding domain in some interactors. To demonstrate in vivo relevance, we analyzed brainstem protein extracts from α-synuclein(A30P) transgenic mice. Pull-down assays using beads conjugated with a Cdc42/Rac interactive binding domain lead to an enrichment of endogenous α-synuclein oligomers. Cdc42 effector proteins were also co-immunoprecipitated with α-synuclein from brainstem lysates and were colocalized with α-synuclein aggregates in brain sections by double immunostaining. By two-dimensional gel electrophoretic analysis of synaptosomal fractions from transgenic mouse brains we detected additional isoforms of septin 6, a downstream target of Cdc42 effector proteins.Small GTPases have recently been identified in a genetic modifier screen to suppress α-synuclein toxicity in yeast. Our data indicate that components of small GTPase signal transduction pathways may be directly targeted by α-synuclein oligomers which potentially leads to signaling deficits and neurodegeneration.

Cell growth and differentiation in Arabidopsis epidermal cells

Plant epidermal cells are morphologically diverse, differing in size, shape, and function. Their unique morphologies reflect the integral function each cell performs in the organ to which it belongs. Cell morphogenesis involves multiple cellular processes acting in concert to create specialized shapes. The Arabidopsis epidermis contains numerous cell types greatly differing in shape, size, and function. Work on three types of epidermal cells, namely trichomes, root hairs, and pavement cells, has made significant progress towards understanding how plant cells reach their final morphology. These three cell types have highly distinct morphologies and each has become a model cell for the study of morphological processes. A growing body of knowledge is creating a picture of how endoreduplication, cytoskeletal dynamics, vesicle transport, and small GTPase signalling, work in concert to create specialized shapes. Similar mechanisms that determine cell shape and polarity are shared between these cell types, while certain mechanisms remain specific to each.

The Molecular Mechanisms of Action of Bisphosphonates

BPs can be grouped into two general classes according to their chemical structure and the molecular mechanism by which they inhibit osteoclast-mediated bone resorption. The simple BPs can be metabolically incorporated into non-hydrolysable analogues of ATP that accumulate intracellularly in osteoclasts, causing osteoclast cell death by apoptosis. By contrast, the more potent N-BPs inhibit FPP synthase, an enzyme in the mevalonate pathway. Inhibition of this enzyme in osteoclasts prevents the biosynthesis of isoprenoid lipids that are required for the prenylation of small GTPase signalling proteins necessary for osteoclast function. Inhibition of FPP synthase in cells other than osteoclasts also appears to account for the adverse effects of N-BPs in vivo (including the acute phase reaction) and for the anti-tumour effects of N-BPs in vitro.

Actin polymerization modulates CD44 surface expression, MMP‐9 activation, and osteoclast function

CD44 and MMP-9 are implicated in cell migration. In the current study, we tested the hypothesis that actin polymerization is critical for CD44 surface expression and MMP-9 activity on the cell surface. To understand the underlying molecular mechanisms involved in CD44 surface expression and MMP-9 activity on the cell surface, osteoclasts were treated with bisphosphonate (BP) alendronate, cytochalasin D (Cyt D), and a broad-spectrum MMP inhibitor (GM6001). BP has been reported to block the mevalonate pathway, thereby preventing prenylation of small GTPase signaling required for actin cytoskeleton modulation. We show in this study that osteoclasts secrete CD44 and MMP-9 into the resorption bay during migration and bone resorption. Results indicate that actin polymerization is critical for CD44 surface expression and osteoclast function. In particular, the surface expression of CD44 and the membrane activity of MMP-9 are reduced in osteoclasts treated with alendronate and Cyt D despite the membrane levels of MMP-9 being unaffected. Although GM6001 blocked MMP-9 activity, osteoclast migration, and bone resorption, the surface levels of CD44 were unaffected. We suggest that the surface expression of CD44 requires actin polymerization. Disruption of podosome and actin ring structures by Cyt D and alendronate not only resulted in reduced localization of MMP-9 in these structures but also in osteoclast migration and bone resorption. These results suggest that inhibition of actin polymerization by alendronate and Cyt D is effective in blocking CD44/MMP-9 complex formation on the cell surface, secretion of active form of MMP-9, and osteoclast migration. CD44/MMP-9 complex formation may signify a unique motility-enhancing signal in osteoclast function.

Involvement of PLEKHM1 in osteoclastic vesicular transport and osteopetrosis in incisors absent rats and humans

This study illustrates that Plekhm1 is an essential protein for bone resorption, as loss-of-function mutations were found to underlie the osteopetrotic phenotype of the incisors absent rat as well as an intermediate type of human osteopetrosis. Electron and confocal microscopic analysis demonstrated that monocytes from a patient homozygous for the mutation differentiated into osteoclasts normally, but when cultured on dentine discs, the osteoclasts failed to form ruffled borders and showed little evidence of bone resorption. The presence of both RUN and pleckstrin homology domains suggests that Plekhm1 may be linked to small GTPase signaling. We found that Plekhm1 colocalized with Rab7 to late endosomal/lysosomal vesicles in HEK293 and osteoclast-like cells, an effect that was dependent on the prenylation of Rab7. In conclusion, we believe PLEKHM1 to be a novel gene implicated in the development of osteopetrosis, with a putative critical function in vesicular transport in the osteoclast.

Big roles for small GTPases in the control of directed cell movement

Small GTPases are involved in the control of diverse cellular behaviours, including cellular growth, differentiation and motility. In addition, recent studies have revealed new roles for small GTPases in the regulation of eukaryotic chemotaxis. Efficient chemotaxis results from co-ordinated chemoattractant gradient sensing, cell polarization and cellular motility, and accumulating data suggest that small GTPase signalling plays a central role in each of these processes as well as in signal relay. The present review summarizes these recent findings, which shed light on the molecular mechanisms by which small GTPases control directed cell migration.

Defective Adhesion, Migration and Homing Are Associated with Altered Rho GTPase Activity in Cells from Fanconi Anemia Patients.

Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure and predisposition to malignancy. One of the potential therapeutic options for patients with FA is collection of autologous multipotent hematopoietic progenitors prior to the development of severe pancytopenia for autologous transplantation and gene therapy. However, poor engraftment of FA hematopoietic cells represents a major obstacle for effective transplantation. Our current study attempted to investigate the mechanism underlying defective engraftment of FA bone marrow (BM) cells. Using BM cells from patients carrying mutations in the FA complementation group A (FA-A), we demonstrate that SDF (Stromal cell-derived factor)-1alpha – and integrin-mediated migration and adhesion, respectively, is defective in FA primary BM cells compared to those from normal donors (more than 2-fold decrease in both migration and adhesion compared to normal BM cells). Complementation of the FA-A defect by retrovirus gene transfer of FANCA gene almost completely restores the ability of the BM cells to migrate towards the chemokine SDF-1alpha. Similar results are obtained with primary fibroblast cells derived from a FA-A patient, which show 3-fold and 35% decrease in adhesion and migration, respectively, compared to FANCA-corrected cells. Furthermore, when transplanted into immunodeficient Nod/scid recipient mice, the FA-A BM cells exhibited significantly impaired homing function whereas normal cells were efficiently homed in the bone marrow. A significant decrease in the activity of the Rho GTPase Cdc42 in FA-A cells is found associated with the patient cell defective functions. Taken together, these data suggest that the FA proteins play a role in the regulation of cell adhesion and migration in addition to maintaining genomic stability, influencing homing and engraftment, possibly via the small GTPase signaling pathway. These findings may have implications in development of strategies for restoring engraftment function of FA hematopoietic cells.

Molecular Mechanisms of Action of Bisphosphonates: Current Status

Bisphosphonates are currently the most important class of antiresorptive agents used in the treatment of metabolic bone diseases, including tumor-associated osteolysis and hypercalcemia. These compounds have high affinity for calcium ions and therefore target bone mineral, where they are internalized by bone-resorbing osteoclasts and inhibit osteoclast function. This article reviews the pharmacology of bisphosphonates and the relationship between chemical structure and antiresorptive potency. We also describe new insights into their intracellular molecular mechanisms of action, methods for assessing the effects of bisphosphonates on protein prenylation, and their potential as direct antitumor agents. Nitrogen-containing bisphosphonates act intracellularly by inhibiting farnesyl diphosphate synthase, an enzyme of the mevalonate pathway, thereby preventing prenylation of small GTPase signaling proteins required for normal cellular function. Inhibition of farnesyl diphosphate synthase also seems to account for their antitumor effects observed in vitro and for the activation of gamma,delta T cells, a feature of the acute-phase response to bisphosphonate treatment in humans. Bisphosphonates that lack a nitrogen in the chemical structure do not inhibit protein prenylation and have a different mode of action that seems to involve primarily the formation of cytotoxic metabolites in osteoclasts. Bisphosphonates are highly effective inhibitors of bone resorption that selectively affect osteoclasts in vivo but could also have direct effects on other cell types, such as tumor cells. After >30 years of clinical use, their molecular mechanisms of action on osteoclasts are finally becoming clear but their exact antitumor properties remain to be clarified.

The GAP1 family of GTPase-activating proteins: spatial and temporal regulators of small GTPase signalling

Ras proteins are binary switches that, by cycling between inactive GDP-bound and active GTP-bound conformations, regulate multiple cellular signalling pathways including those that control cell growth, differentiation and survival. Approximately 30% of all human tumours express Ras-containing oncogenic mutations that lock the protein into a constitutively active conformation. The activation status of Ras is regulated by two groups of proteins: GEFs (guanine nucleotide-exchange factors) bind to Ras and enhance the exchange of GDP for GTP, thereby activating it, whereas GAPs (GTPase-activating proteins) inactivate Ras by binding to the GTP-bound form and enhancing the hydrolysis of the bound nucleotide back to GDP. In this review, we focus on a group of key regulators of Ras inactivation, the GAP1 family of Ras-GAPs. The members of this family are GAP1m, GAP1IP4BP, CAPRI (Ca2+-promoted Ras inactivator) and RASAL (Ras-GTPase-activating-like protein) and, as we will discuss, they are emerging as important modulators of Ras and small GTPase signalling that are subject to regulation by a diverse array of events and second messenger signals.

A comprehensive map of the toll‐like receptor signaling network

Recognition of pathogen-associated molecular signatures is critically important in proper activation of the immune system. The toll-like receptor (TLR) signaling network is responsible for innate immune response. In mammalians, there are 11 TLRs that recognize a variety of ligands from pathogens to trigger immunological responses. In this paper, we present a comprehensive map of TLRs and interleukin 1 receptor signaling networks based on papers published so far. The map illustrates the possible existence of a main network subsystem that has a bow-tie structure in which myeloid differentiation primary response gene 88 (MyD88) is a nonredundant core element, two collateral subsystems with small GTPase and phosphatidylinositol signaling, and MyD88-independent pathway. There is extensive crosstalk between the main bow-tie network and subsystems, as well as feedback and feedforward controls. One obvious feature of this network is the fragility against removal of the nonredundant core element, which is MyD88, and involvement of collateral subsystems for generating different reactions and gene expressions for different stimuli.

Regulation of cancer cell motility through actin reorganization

Cell migration is a critical step in tumor invasion and metastasis, and regulation of this process will lead to appropriate therapies for treating cancer. Cancer cells migrate in various ways, according to cell type and degree of differentiation. The different types of cell migration are regulated by different mechanisms. Reorganization of the actin cytoskeleton is the primary mechanism of cell motility and is essential for most types of cell migration. Actin reorganization is regulated by Rho family small GTPases such as Rho, Rac, and Cdc42. These small GTPases transmit extracellular chemotactic signals to downstream effectors. Of these downstream effectors, Wiskott-Aldrich syndrome protein (WASP) family proteins are key regulators of cell migration. Activated WASP family proteins induce the formation of protrusive membrane structures involved in cell migration and degradation of the extracellular matrix. Inhibition of Rho family small GTPase signaling suppresses the migration and invasion of cancer cells. Thus, control of cell migration via the actin cytoskeleton provides the possibility of regulating cancer cell invasion and metastasis.

Lysophosphatidic Acid, a Disintegrin and Metalloprotease-17 and Heparin-Binding Epidermal Growth Factor–Like Growth Factor in Ovarian Cancer: The First Word, Not the Last

Cancer of the ovary is the leading cause of death for women with gynecologic malignancies. Over 90% of ovarian cancers derive from malignant transformation of the ovarian surface epithelium (OSE), from which cells disseminate into the peritoneal cavity, invade locally or spread via lymphatics,

Elucidating effects of long-term expression of HIV-1 Nef on astrocytes by microarray, promoter, and literature analyses

The challenge of microarray analysis is to unveil the biological mechanisms behind the chip data. Due to the sometimes counteracting influences of de novo transcription, RNA processing and degradation, the discovery of any particular mechanism is difficult. Therefore, a combination of data- and knowledge-driven analysis appears to be the best way to attack the problem. We analyzed human astrocytes stably expressing the HIV-1 nef gene by microarray analyses to elucidate the effects of constitutive HIV-1 Nef expression on the transcriptome of astrocytes. Statistical evaluation of microarray results revealed small clusters of genes specifically up-regulated by native Nef protein in contrast to astrocytes expressing a non-myristoylated Nef variant. At least three significantly overrepresented gene ontology groups (small GTPase signaling, regulation of apoptosis and lipid metabolism) were detected. The JAK/STAT pathway was clearly associated with those genes. This finding agreed well with a literature-based approach, where a network was derived by combined literature and promoter sequence analysis. Promoter organization suggested potentially coordinated transcriptional regulation of some of these genes. Both results were in line with previously reported phenotypic changes.

An inducible translocation strategy to rapidly activate and inhibit small GTPase signaling pathways

We made substantial advances in the implementation of a rapamycin-triggered heterodimerization strategy. Using molecular engineering of different targeting and enzymatic fusion constructs and a new rapamycin analog, Rho GTPases were directly activated or inactivated on a timescale of seconds, which was followed by pronounced cell morphological changes. As signaling processes often occur within minutes, such rapid perturbations provide a powerful tool to investigate the role, selectivity and timing of Rho GTPase-mediated signaling processes.

Activin Type II Receptor Restoration in ACVR2 -Deficient Colon Cancer Cells Induces Transforming Growth Factor-β Response Pathway Genes

The activin type II receptor (ACVR2) gene is a putative tumor suppressor gene that is frequently mutated in microsatellite-unstable colon cancers (MSI-H colon cancers). ACVR2 is a member of the transforming growth factor (TGF)-beta type II receptor (TGFBR2) family and controls cell growth and differentiation. SMAD proteins are major intracellular effectors shared by ACVR2 and TGFBR2 signaling; however, additional shared effector mechanisms remain to be explored. To discover novel mechanisms transmitting the ACVR2 signal, we restored ACVR2 function by transfecting wild-type ACVR2 (wt-ACVR2) into a MSI-H colon cancer cell line carrying an ACVR2 frameshift mutation. The effect of ACVR2 restoration on cell growth, SMAD phosphorylation, and global molecular phenotype was then evaluated. Decreased cell growth was observed in wt-ACVR2 transfectants relative to ACVR2-deficient vector-transfected controls. Western blotting revealed higher expression of phosphorylated SMAD2 in wt-ACVR2 transfectants versus controls, suggesting cells deficient in ACVR2 had impaired SMAD signaling. Microarray-based differential expression analysis revealed substantial ACVR2-induced overexpression of genes implicated in the control of cell growth and tumorigenesis, including the activator protein (AP)-1 complex genes JUND, JUN, and FOSB, as well as the small GTPase signal transduction family members, RHOB, ARHE, and ARHGDIA. Overexpression of these genes is shared with TGFBR2 activation. This observed similarity between the activin and TGF-beta signaling systems suggests that activin may serve as an alternative activator of TGF-beta effectors, including SMADs, and that frameshift mutation of ACVR2 may contribute to MSI-H colon tumorigenesis via disruption of alternate TGF-beta effector pathways.

New insights into the molecular mechanisms of action of bisphosphonates.

Bisphosphonates are currently the most important and effective class of anti-resorptive drugs available, but the exact molecular mechanisms by which they inhibit osteoclast-mediated bone resorption have only recently been identified. Due to the targeting of bisphosphonates to bone mineral and the ability of osteoclasts to release bone-bound bisphosphonate, a direct effect on mature osteoclasts appears to be the most important route of action. As a result of recent discoveries concerning their molecular mechanism of action, bisphosphonates can be grouped into two classes. The simple bisphosphonates that closely resemble PPi (such as clodronate, etidronate and tiludronate) can be metabolically incorporated into non-hydrolysable analogues of ATP that accumulate intracellularly in osteoclasts, resulting in induction of osteoclast apoptosis. By contrast, the more potent, nitrogen-containing bisphosphonates (such as pamidronate, alendronate, risedronate, ibandronate and zoledronate) appear to act as analogues of isoprenoid diphosphate lipids, thereby inhibiting FPP synthase, an enzyme in the mevalonate pathway. Inhibition of this enzyme in osteoclasts prevents the biosynthesis of isoprenoid lipids (FPP and GGPP) that are essential for the post-translational farnesylation and geranylgeranylation of small GTPase signalling proteins. Loss of bone-resorptive activity and osteoclast apoptosis is due primarily to loss of geranylgeranylated small GTPases. Identification of FPP synthase as the target of nitrogen-containing bisphosphonates has also helped explain the molecular basis for the adverse effects of these agents in the GI tract and on the immune system.

PLC- : A Shared Effector Protein in Ras-, Rho-, and G -Mediated Signaling

The conceptual segregation of G protein-stimulated cell signaling responses into those mediated by heterotrimeric G proteins versus those promoted by small GTPases of the Ras superfamily is no longer vogue. PLC-epsilon, an isozyme of the phospholipase C (PLC) family, has been identified recently and dramatically extends our understanding of the crosstalk that occurs between heterotrimeric and small monomeric GTPases. Like the widely studied PLC-beta isozymes, PLC-epsilon is activated by Gbetagamma released upon activation of heterotrimeric G proteins. However, PLC-epsilon markedly differs from the PLC-beta isozymes in its capacity for activation by Galpha(12/13) - but not Galpha(q) -coupled receptors. PLC-epsilon contains two Ras-associating domains located near the C terminus, and H-Ras regulates PLC-epsilon as a downstream effector. Rho also activates PLC-epsilon, but in a mechanism independent of the C-terminal Ras-associating domains. Therefore, Ca(2+) mobilization and activation of protein kinase C are signaling responses associated with activation of both H-Ras and Rho. A guanine nucleotide exchange domain conserved in the N terminus of PLC-epsilon potentially confers a capacity for activators of this isozyme to cast signals into additional signaling pathways mediated by GTPases of the Ras superfamily. Thus, PLC-epsilon is a multifunctional nexus protein that senses and mediates crosstalk between heterotrimeric and small GTPase signaling pathways.

Cdc42 and RhoA are differentially regulated during arachidonate-mediated HeLa cell adhesion.

Cell adhesion to extracellular matrix requires stimulation of an eicosanoid signaling pathway through the metabolism of arachidonate by 5-lipoxygenase to leukotrienes and cyclooxygenase-1/2 to prostaglandins, as well as activation of the small GTPase signaling pathway involving Cdc42 and Rho. These signaling pathways direct remodeling of the actin cytoskeleton during the adhesion process, specifically the polymerization of actin during cell spreading and the bundling of actin filaments when cells migrate. However, few studies linking these signaling pathways have been described in the literature. We have previously shown that HeLa cell adhesion to collagen requires oxidation of arachidonic acid (AA) by lipoxygenase for actin polymerization and cell spreading, and cyclooxygenase for bundling actin filaments during cell migration. We demonstrate that small GTPase activity is required for HeLa cell spreading upon gelatin, and that Cdc42 is activated while Rho is downregulated during the spreading process. Using constitutively active and dominant negative expression studies, we show that Cdc42 is required for HeLa cell spreading and migration, while activated RhoA is antagonistic towards spreading. Constitutively active RhoA promotes cell migration and increases the degree of actin bundling in HeLa cells. Further, we demonstrate that activation of either the AA oxidation pathway or the small GTPase pathway cannot rescue inhibition of spreading when the alternate pathway is blocked. Our results suggest (1) both the eicosanoid signaling pathway and small GTPase activation are required during HeLa cell adhesion, and (2) these signaling pathways converge to properly direct remodeling of the actin cytoskeleton during HeLa cell spreading and migration upon collagen.

Disruption of the Diaphanous-Related Formin Drf1 Gene Encoding mDia1 Reveals a Role for Drf3 as an Effector for Cdc42

Background: Mammalian Diaphanous-related formins (Drfs) act as Rho small GTPase effectors during growth factor-induced cytoskeletal remodeling and cell division. While both p140 mDia1 (herein called Drf1) and p134 mDia2 (Drf3) have been shown to bind in vitro to activated RhoA-C, and Drf3 has also been shown to bind to Cdc42, little is known about the cellular function of these GTPase effector pairs. Thus, we have begun targeting the murine Drf genes to address their various contributions to small GTPase signaling in cytoskeletal remodeling and development.Results:Drf1 +/+, +/−, and −/− cell lines were derived from embryonic stem cells. While some Drf1 +/− lines had fewer actin stress fibers, several Drf1 +/− and −/− cells were more motile and had more abundant lamella and filopodia. Because the apparent “gain-of-function” corresponded with elevated levels of Drf3 protein expression, we hypothesized that the effects on the actin cytoskeleton were due to Cdc42 utilization of Drf3 as an effector. In this study, we found that inactive Drf3 variants and microinjected Drf3 antibodies interfered with Cdc42-induced filopodia. In addition, we observed that Drf3 contains a previously unidentified CRIB-like motif within its GTPase binding domain (GBD). By fluorescent resonance energy transfer (FRET) analysis, we demonstrate that this motif is required for Cdc42 binding and Drf3 recruitment to the leading edge and, surprisingly, to the microtubule organizing center (MTOC) of migrating fibroblasts.Conclusions: Our observations extend the role of the mammalian Drfs in cell signaling and demonstrate that Cdc42 not only activates Drf3, but guides the effector to sites at the cell cortex where it remodels the actin cytoskeleton.

A new Wave of complexes

Actin reorganization is crucial in many biological processes such as cell locomotion and maintenance of cell shape. The Cdc42/Rac1 small GTPase signaling pathway has emerged as a major pathway mediating de novo assembly of actin. Upon activation by external signals, Cdc42/Rac1 switch into their active forms and activate WASP proteins, which in turn bind to the Arp2/3 complex and activate its actin-nucleation activity.In order to avoid constitutive activation of the Arp2/3 complex, its interaction with WASP proteins is tightly regulated. All WASP family proteins (WASP, N-WASP, WAVE1, WAVE2 and WAVE3) contain a C-terminal WA domain, which binds to and activates the Arp2/3 complex, and various N-terminal regulatory domains [1xSpatial and temporal regulation of actin polymerization for cytoskeleton formation through Arp2/3 complex and WASP/WAVE proteins. Suetsugu, S. et al. Cell Motil. Cytoskeleton. 2002; 51: 113–122Crossref | PubMed | Scopus (47)See all References][1]. Isolated WASP and N-WASP exist in an autoinhibited conformation, owing to the interaction between the WA domain and an N-terminal basic motif. Binding of Cdc42 to WASP/N-WASP dissociates this intramolecular interaction and unmasks the WA domain, allowing its interaction with the Arp2/3 complex. Unlike WASP/N-WASP, isolated WAVE proteins exist in the activated form. Although WAVE proteins act downstream of Rac, they do not interact directly with Rac. Until recently, the mechanism of regulation of WAVE proteins was unknown. IRSp53, an insulin receptor substrate, was identified as a link between WAVE2 and Rac [2xIRSp53 is an essential intermediate between Rac and WAVE in the regulation of membrane ruffling. Miki, H. et al. Nature. 2000; 408: 732–735Crossref | PubMed | Scopus (390)See all References][2]. However, it is unclear how IRSp53 couples Rac activation to WAVE2. Now, Kirschner and colleagues have found a novel complex that acts as a WAVE1 trans-inhibitory complex [3xThe mechanism of regulation of WAVE1-induced actin nucleation by Rac1 and Nck. Eden, S. et al. Nature. 2002; 418: 790–793Crossref | PubMed | Scopus (492)See all References][3].In a search for WAVE1-binding proteins in bovine brain extracts, the authors identified a 500-kDa complex comprising WAVE1, PIR121 (a Rac1-binding protein), Nap125 (an Nck-binding protein), Abi2 (‘Abl-interactor 2’ – homologous to hNap1-binding protein) and a novel 9-kDa protein, HSPC300. In this complex, WAVE1 is unable to activate Arp2/3 complexes. Both RacGTPγS and Nck can relieve this inhibition. RacGTPγS and Nck dissociate the complex into two subcomplexes, the first comprising PIR121, Nap125 and probably Rac or Nck and a second subcomplex comprising HSPC300 and WAVE1. The HSPC300–WAVE1 subcomplex is able to activate the Arp2/3 complex. Very little is known about PIR121 and Nap125. PIR121 has been described as a potential mediator of p53-dependent apoptosis and as interacting with fragile-X mental retardation protein (FMRP), an RNA-binding protein associated with a messenger ribonucleoprotein complex. This suggests that, after dissociation from the WAVE1 complex, the PIR121–Nap125 subcomplex might participate in a currently uncharacterized translational/transcriptional processes. Thus, the complex reported in this work gives us a first glimpse into how WAVE proteins are regulated and provides a nice tool for understanding how the cellular machinery coordinates different cellular processes.