Laminin-1 activates Cdc42 in the mechanism of laminin-1-mediated neurite outgrowth

Here, we investigated the role of the small Rho GTPases Rac, Cdc42, and Rho in the mechanism of laminin-1-mediated neurite outgrowth in PC12 cells. PC12 cells were transfected with plasmids expressing wild-type and dominant-negative mutants of Rac (RacN17), Cdc42 (Cdc42N17), or Rho (RhoN19). Over 90% of the dominant-negative Rho- and Rac-transfected cells extended neurites when plated on laminin-1; however, none of the PC12 cells transfected with the dominant-negative Cdc42 mutant extended neurites. In cells cotransfected with plasmids expressing c-Jun N-terminal kinase and wild-type Cdc42, laminin-1 treatment stimulated detectable levels of c-Jun phosphorylation. Further, cotransfection with c-Jun N-terminal kinase and the dominant-negative Cdc42 mutant blocked laminin-1-mediated c-Jun phosphorylation. Transfection with either wild-type Rac or the dominant-negative Rac did not effect c-Jun phosphorylation. These data demonstrate that Cdc42 is activated by laminin-1 and that Cdc42 activation is required in the mechanism of laminin-1-mediated neurite outgrowth.     

Characterization of EHop-016, novel small molecule inhibitor of Rac GTPase

The Rho GTPase Rac regulates actin cytoskeleton reorganization to form cell surface extensions (lamellipodia) required for cell migration/invasion during cancer metastasis. Rac hyperactivation and overexpression are associated with aggressive cancers; thus, interference of the interaction of Rac with its direct upstream activators, guanine nucleotide exchange factors (GEFs), is a viable strategy for inhibiting Rac activity. We synthesized EHop-016, a novel inhibitor of Rac activity, based on the structure of the established Rac/Rac GEF inhibitor NSC23766. Herein, we demonstrate that EHop-016 inhibits Rac activity in the MDA-MB-435 metastatic cancer cells that overexpress Rac and exhibits high endogenous Rac activity. The IC(50) of 1.1 μM for Rac inhibition by EHop-016 is ～100-fold lower than for NSC23766. EHop-016 is specific for Rac1 and Rac3 at concentrations of ≤5 μM. At higher concentrations, EHop-016 inhibits the close homolog Cdc42. In MDA-MB-435 cells that demonstrate high active levels of the Rac GEF Vav2, EHop-016 inhibits the association of Vav2 with a nucleotide-free Rac1(G15A), which has a high affinity for activated GEFs. EHop-016 also inhibits the Rac activity of MDA-MB-231 metastatic breast cancer cells and reduces Rac-directed lamellipodia formation in both cell lines. EHop-016 decreases Rac downstream effects of PAK1 (p21-activated kinase 1) activity and directed migration of metastatic cancer cells. Moreover, at effective concentrations (<5 μM), EHop-016 does not affect the viability of transformed mammary epithelial cells (MCF-10A) and reduces viability of MDA-MB-435 cells by only 20%. Therefore, EHop-016 holds promise as a targeted therapeutic agent for the treatment of metastatic cancers with high Rac activity.     

Synergistic Activation of p21-activated Kinase 1 by Phosphatidylinositol 4,5-Bisphosphate and Rho GTPases

Autoinhibited p21-activated kinase 1 (Pak1) can be activated in vitro by the plasma membrane-bound Rho GTPases Rac1 and Cdc42 as well as by the lipid phosphatidylinositol (4,5)-bisphosphate (PIP₂). Activator binding is mediated by a GTPase-binding motif and an adjacent phosphoinositide-binding motif. Whether these two classes of activators play alternative, additive, or synergistic roles in Pak1 activation is unknown, as is their contributions to Pak1 activation in vivo. To address these questions, we developed a system to mimic the membrane anchoring of Rho GTPases by creating liposomes containing both PIP₂ and a Ni²⁺-NTA modified lipid capable of binding hexahistidine-tagged Cdc42. We find that among all biologically relevant phosphoinositides, only PIP₂ is able to synergistically activate Pak1 in concert with Cdc42. Membrane binding of the kinase was highly sensitive to the spatial density of PIP₂ and Pak1 demonstrated dramatically enhanced affinity for Cdc42 anchored in a PIP₂ environment. To validate these findings in vivo, we utilized an inducible recruitment system to drive the ectopic synthesis of PIP₂ on Golgi membranes, which normally have active Cdc42 but lack significant concentrations of PIP₂. Pak1 was recruited to PIP₂-containing membranes in a manner dependent on the ability of Pak1 to bind to both PIP₂ and Cdc42. These findings provide a mechanistic explanation for the essential role of both phosphoinositides and GTPases in Pak1 recruitment and activation. In contrast, Ack, another Cdc42 effector kinase that lacks an analogous phosphoinositide-binding motif, fails to show the same enhancement of membrane binding and activation by PIP₂, thus indicating that regulation by PIP₂ and Cdc42 could provide a combinatorial code for activation of different GTPase effectors in different subcellular locations.     

Rho GAPs and GEFs: Controling switches in endothelial cell adhesion

Within blood vessels, endothelial cell–cell and cell–matrix adhesions are crucial to preserve barrier function, and these adhesions are tightly controlled during vascular development, angiogenesis, and transendothelial migration of inflammatory cells. Endothelial cellular signaling that occurs via the family of Rho GTPases coordinates these cell adhesion structures through cytoskeletal remodelling. In turn, Rho GTPases are regulated by GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). To understand how endothelial cells initiate changes in the activity of Rho GTPases, and thereby regulate cell adhesion, we will discuss the role of Rho GAPs and GEFs in vascular biology. Many potentially important Rho regulators have not been studied in detail in endothelial cells. We therefore will first overview which GAPs and GEFs are highly expressed in endothelium, based on comparative gene expression analysis of human endothelial cells compared with other tissue cell types. Subsequently, we discuss the relevance of Rho GAPs and GEFs for endothelial cell adhesion in vascular homeostasis and disease.     

Yeast Cdc42 functions at a late step in exocytosis, specifically during polarized growth of the emerging bud.

The Rho family GTPase Cdc42 is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells. In yeast, the role of Cdc42 in polarization of cell growth includes polarization of the actin cytoskeleton, which delivers secretory vesicles to growth sites at the plasma membrane. We now describe a novel temperature-sensitive mutant, cdc42-6, that reveals a role for Cdc42 in docking and fusion of secretory vesicles that is independent of its role in actin polarization. cdc42-6 mutants can polarize actin and deliver secretory vesicles to the bud, but fail to fuse those vesicles with the plasma membrane. This defect is manifested only during the early stages of bud formation when growth is most highly polarized, and appears to reflect a requirement for Cdc42 to maintain maximally active exocytic machinery at sites of high vesicle throughput. Extensive genetic interactions between cdc42-6 and mutations in exocytic components support this hypothesis, and indicate a functional overlap with Rho3, which also regulates both actin organization and exocytosis. Localization data suggest that the defect in cdc42-6 cells is not at the level of the localization of the exocytic apparatus. Rather, we suggest that Cdc42 acts as an allosteric regulator of the vesicle docking and fusion apparatus to provide maximal function at sites of polarized growth.     

Rab35 regulates neurite outgrowth and cell shape

Recent studies have identified Rab35 in the endocytic pathway and as a regulator of cytokinesis; however its molecular mechanisms are currently unknown. Here, we find that Rab35 colocalizes with actin filaments and with Cdc42, Rac1 and RhoA, and that Rab35 can activate Cdc42 both in vivo and in vitro. We find activated Rab35 stimulates neurite outgrowth in PC12 and N1E-115 cells via a Cdc42-dependent pathway and that siRNA knockdown of Rab35 activity abolishes neurite outgrowth in these cell lines. We conclude that one function of Rab35 is to regulate Rho-family GTPases and that this role has consequences for neurite outgrowth.

Structured summary

MINT-7012081: Rac1(uniprotkb:P63000) and Rab 35 (uniprotkb:Q15286) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7012070: actin (uniprotkb:P60709) and Rab 35 (uniprotkb:Q15286) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7012095: cdc42 (uniprotkb:P60953) and Rab 35 (uniprotkb:Q15286) colocalize (MI:0403) by fluorescence microscopy (MI:0416)     

Mathematical Model for Spatial Segregation of the Rho-Family GTPases Based on Inhibitory Crosstalk

Cdc42, Rac, and Rho are small GTPases known to play a central role in signal transduction to the actin cytoskeleton. These proteins regulate cell motility, by affecting nucleation, uncapping, and depolymerization of actin filaments, and acto-myosin contractility. Studies of crosstalk and mutual feedbacks in these three proteins have led to a number of proposals for their interaction. At the same time, observations of the spatio-temporal dynamics of Rho-family proteins give evidence of spatial polarization and mutual exclusion between Cdc42/Rac and Rho. In this paper, we formulate a mathematical model to account for such observations, based on the known underlying biology of these proteins. We first investigate which of the crosstalk schemes proposed in the literature is consistent with observed dynamics, and then derive a simple model that can correctly describe these dynamics (assuming crosstalk is mediated via Rho GEFs). We show that cooperativity is an essential ingredient in the interactions of the proteins. The co-occurrence of a stable rest state with the possibility of fast spatial segregation can be related to bistability in a set of underlying ODEs in which the inactive forms of these proteins are fixed at a constant level. We show that the fast diffusion of the inactive forms is essential for stabilizing the transition fronts in the PDE formulation of the model, leading to robust spatial polarization, rather than traveling waves.     

The diaphanous-related formin DAAM1 collaborates with the Rho GTPases RhoA and Cdc42, CIP4 and Src in regulating cell morphogenesis and actin dynamics

Binding partners for the Cdc42 effector CIP4 were identified by the yeast two-hybrid system, as well as by testing potential CIP4-binding proteins in coimmunoprecipitation experiments. One of the CIP4-binding proteins, DAAM1, was characterised in more detail. DAAM1 is a ubiquitously expressed member of the mammalian diaphanous-related formins, which include proteins such as mDia1 and mDia2. DAAM1 was shown to bind to the SH3 domain of CIP4 in vivo. Ectopically expressed DAAM1 localised in dotted pattern at the dorsal side of transfected cells and the protein was accumulated in the proximity to the microtubule organising centre. Moreover, ectopic expression of DAAM1 induced a marked alteration of the cell morphology, seen as rounding up of the cells, the formation of branched protrusions as well as a reduction of stress-fibres in the transfected cells. Coimmunoprecipitation experiments demonstrated that DAAM1 bound to RhoA and Cdc42 in a GTP-dependent manner. Moreover, DAAM1 was found to interact and collaborate with the non-receptor tyrosine kinase Src in the formation of branched protrusions. Taken together, our data indicate that DAAM1 communicates with Rho GTPases, CIP4 and Src in the regulation of the signalling pathways that co-ordinate the dynamics of the actin filament system.     

Hijacking of Rho GTPases during bacterial infection

Highly pathogenic bacteria, including Yersinia, Salmonella, E. coli and Clostridia, produce an amazing array of virulence factors that target Rho proteins. These pathogens exploit and/or impair many aspects of Rho protein activities by activating or inhibiting these key molecular switches. Here, we describe examples illustrating how modulation of Rho protein activity is the underlying molecular mechanism used by pathogens to disrupt host epithelial/endothelial barriers, paralyze immune cell migration and phagocytic functions, invade epithelial cells, replicate, and form reservoirs or disseminate in epithelia. Remarkably, emerging evidence points to the capacity of target cells to not only perceive the imbalance of Rho activity induced by virulence factors but also to respond by stimulating the production of anti-microbial responses that alert the host to the pathogenic threat. Furthermore, toxins that activate Rho proteins have been extremely useful in revealing the exquisite cellular regulations of these GTPases, notably by the ubiquitin and proteasome system. Finally, a number of studies indicate that toxins targeting Rho proteins have great potential in the development of new therapeutic tools.     

Binding of Cdc42 to phospholipase D1 is important in neurite outgrowth of neural stem cells

We previously demonstrated that phospholipase D (PLD) expression and PLD activity are upregulated during neuronal differentiation. In the present study, employing neural stem cells from the brain cortex of E14 rat embryos, we investigated the role of Rho family GTPases in PLD activation and in neurite outgrowth of neural stem cells during differentiation. As neuronal differentiation progressed, the expression levels of Cdc42 and RhoA increased. Furthermore, Cdc42 and PLD1 were mainly localized in neurite, whereas RhoA was localized in cytosol. Co-immunoprecipitation revealed that Cdc42 was bound to PLD1 during differentiation, whereas RhoA was associated with PLD1 during both proliferation and differentiation. These results indicate that the association between Cdc42 and PLD1 is related to neuronal differentiation. To examine the effect of Cdc42 on PLD activation and neurite outgrowth, we transfected dominant negative Cdc42 (Cdc42N17) and constitutively active Cdc42 (Cdc42V12) into neural stem cells, respectively. Overexpression of Cdc42N17 decreased both PLD activity and neurite outgrowth, whereas co-transfection with Cdc42N17 and PLD1 restored them. On the other hand, Cdc42V12 increased both PLD activity and neurite outgrowth, suggesting that active state of Cdc42 is important in upregulation of PLD activity which is responsible for the increase of neurite outgrowth.     

Dock6, a Dock-C subfamily guanine nucleotide exchanger, has the dual specificity for Rac1 and Cdc42 and regulates neurite outgrowth

Small GTPases of the Rho family, Rho, Rac, and Cdc42, are critical regulators of the changes in the actin cytoskeleton. Rho GTPases are typically activated by Dbl-homology (DH)-domain-containing guanine nucleotide exchange factors (GEFs). Recent genetic and biochemical studies revealed a new type of GEF for the Rho GTPases. This family is composed of 11 genes, designated as Dock1 to Dock11, and is structurally divided into four classes Dock-A, -B, -C, and -D. Dock-A and -B subfamilies are typically GEFs specific for Rac1, while the Dock-D subfamily is specific for Cdc42. Here we show that Dock6, a member of the Dock-C subfamily, exchanges GDP for GTP for Rac1 and Cdc42 in vitro and in vivo. Furthermore, we find that, in mouse N1E-115 neuroblastoma cells, expression of Dock6 is increased following differentiation. Transfection of the catalytic Dock Homology Region-2 (DHR-2) domain of Dock6 promotes neurite outgrowth mediated by Rac1 and Cdc42. Conversely, knockdown of endogenous Dock6 by small interference RNA reduces activation of Rac1 and Cdc42 and neurite outgrowth. Taken together, these results suggest that Dock6 differs from all of the identified Dock180-related proteins, in that it is the GEF specific for both Rac1 and Cdc42 and may be one of physiological regulators of neurite outgrowth.     

ARHGAP30 is a Wrch-1-interacting protein involved in actin dynamics and cell adhesion

The atypical Rho GTPase Wrch-1 has been proposed roles in cell migration, focal adhesion dissolution, stress fibre break down and tight junction heterogeneity. A screen for Wrch-1 binding-partners identified the novel RhoGAP protein, ARHGAP30, as a Wrch-1 interactor. ARHGAP30 is related to the Cdc42- and Rac1-specific RhoGAP CdGAP, which was likewise found to bind Wrch-1. In contrast to CdGAP, ARHGAP30 serves as a Rac1- and RhoA-specific RhoGAP. Ectopic expression of ARHGAP30 results in membrane blebbing and dissolution of stress-fibres and focal adhesions. Our data suggest roles for ARHGAP30 and CdGAP in regulation of cell adhesion downstream of Wrch-1.     

Identification and characterization of the Cdc42-binding site of IQGAP1

IQGAP1 is a multi-domained protein that integrates signaling of the Rho family GTPase Cdc42 with regulation of the cytoskeleton. Using SPOT analysis and in vitro peptide competition assays we have identified a 24 amino acid region of IQGAP1 that is necessary for Cdc42 binding. Both in vitro and in vivo analyses reveal that deletion of this sequence abolishes binding of IQGAP1 to Cdc42. In addition, the ability of IQGAP1 to increase the amount of active Cdc42 in cells is abrogated upon removal of this region. An IQGAP1 mutant lacking the Cdc42 binding site mislocalizes to the cell periphery. These observations specifically define a short sequence of IQGAP1 that is required for its interaction with Cdc42 and demonstrate that Cdc42 binding is necessary for the normal subcellular distribution of IQGAP1.     

JNK phosphorylation of paxillin, acting through the Rac1 and Cdc42 signaling cascade, mediates neurite extension in N1E-115 cells

Neurons extend neurites from the cell body before formation of the polarized processes of an axon and dendrites. Neurite outgrowth involves remodeling of the cytoskeletal components, which are initially regulated by small GTPases of the Rho family. Here we show that c-Jun N-terminal kinase (JNK), which is controlled by Rho GTPases Rac1 and Cdc42, is activated following neurite extension in mouse N1E-115 neuroblastoma cells as a model. The extension is inhibited by JNK inhibitors (SP600125 and the small JNK-binding peptide) and Clostridium difficile Toxin B, the inhibitor for Rho GTPases. Additionally, paxillin, the multifunctional focal adhesion protein, is phosphorylated at Ser 178 by upregulation of the Rac1/Cdc42/JNK cascade. Conversely, transfection of the paxillin construct harboring the Ser 178-to-Ala mutation into cells inhibits neurite extension. Taken together, these results suggest the novel role of the Rac1/Cdc42/JNK signaling cascade in neurite extension and indicate that the downstream target paxillin may be one of the convergent points of various signaling pathways underlying neurite extension.     

E3 ubiquitin ligases in regulating stress fiber,lamellipodium, and focal adhesion dynamics

Recent discoveries have unveiled the roles of a complicated network of E3 ubiquitin ligases in regulating cell migration machineries. The E3 ubiquitin ligases Smurf1 and Cul/BACURD ubiquitinate RhoA to regulate stress fiber formation and cell polarity, and ASB2α ubiquitinates filamins to modulate cytoskeletal stiffness, thus regulating cell spreading and cell migration. HACE1, XIAP, and Skp1-Cul1-F-box bind to Rac1 and cause its ubiquitination and degradation, thus suppressing lamellipodium protrusions, while PIAS3, a SUMO ligase, activates Rac1 to promote lamellipodium dynamics. Smurf1 also enhances Rac1 activation but it does not ubiquitinate Rac1. Both Smurf1 and HECTD1 regulate focal adhesion (FA) assembly and (or) disassembly through ubiquitinating the talin head domain and phosphatidylinositol 4 phosphate 5-kinase type I γ (PIPKIγ90), respectively. Thus, E3 ubiquitin ligases regulate stress fiber formation, cell polarity, lamellipodium protrusions, and FA dynamics through ubiquitinating the key proteins that control these processes.