Spatial control of Rho (Rac-Rop) signaling in tip-growing plant cells

Spatially restricted signaling by Rho GTPases is essential for the polarization of eukaryotic cells, which is required for the morphogenesis, mobility and division of single cells, and for the development of multicellular organisms. Rac-Rop GTPases, which constitute a plant-specific Rho GTPase subfamily, accumulate at the apical plasma membrane of pollen tubes and root hairs, where they control rapid polar cell expansion by a process known as tip growth. Here, recent insights into the spatial control of Rac-Rop-dependent signaling in tip-growing plant cells by regulatory proteins (i.e. Rho GTPase-activating proteins, Rho guanine nucleotide dissociation inhibitors, Rho guanine nucleotide-exchange factors and phosphoinositide-specific phospholipase C) and lipids [phosphatidylinositol (4,5)-bisphosphate and diacyl glycerol] are summarized. A model is presented, which integrates the current knowledge concerning the molecular mechanisms that maintain the polarization of Rho signaling in plant cells.     

GDIs: central regulatory molecules in Rho GTPase activation

The GDP dissociation inhibitors (GDIs) are pivotal regulators of Rho GTPase function. GDIs control the access of Rho GTPases to regulatory guanine nucleotide exchange factors and GTPase-activating proteins, to effector targets and to membranes where such effectors reside. We discuss here our current understanding of how Rho GTPase-GDI complexes are regulated by various proteins, lipids and enzymes that exert GDI displacement activity. We propose that phosphorylation mediated by diverse kinases might provide a means of controlling and coordinating Rho GTPase activation.     

Off the beaten paths: alternative and crosstalk regulation of Rho GTPases

Rho proteins are small GTPases of the Ras superfamily that regulate a wide variety of biological processes, ranging from gene expression to cell migration. Mechanistically, the major Rho GTPases function as molecular switches cycling between an inactive GDP-bound and an active GTP-bound conformation, although several Rho proteins spontaneously exchange nucleotides or are simply devoid of GTPase activity. For over a decade, RhoGEFs and RhoGAPs have been established as the mainstream regulators of Rho proteins, respectively flipping the switch on or off. However, regulation by GEFs and GAPs leaves several fundamental questions on the operation of the Rho switch unanswered, indicating that the regulation of Rho proteins does not rely exclusively on RhoGEFs and RhoGAPs. Recent evidence indeed suggests that Rho GTPases are finely tuned by multiple alternative regulatory mechanisms, including post-translational modifications and protein degradation, as well as crosstalk mechanisms between Rho proteins. Here we review these alternative mechanisms and discuss how they alter Rho protein function and signaling. We also envision how the classic binary Rho switch may indeed function more like a switchboard with multiple switches and dials that can all contribute to the regulation of Rho protein function.     

Bacterial protein toxins targeting rho GTPases

Several bacterial protein toxins target eukaryotic cells by modulating the functions of Rho GTPases that are involved in various signal processes and in the regulation of the actin cytoskeleton. The toxins inhibit Rho functions by ADP-ribosylation or glucosylation and activate them by deamidation and transglutamination. New findings indicate that the GTPases are also targeted by various 'injected' toxins which are introduced into the eukaryotic cells by the type-III secretion system. The injected toxins do not covalently modify Rho GTPases, but manipulate their regulatory GTPase cycle by acting as GTPase-activating proteins or guanine nucleotide exchange factors.     

Protein–protein‐interactions in a multiplexed,miniaturized format a functional analysis of Rho GTPase activation and inhibition

A miniaturized, bead‐based protein–protein‐interaction assay was developed to study the interaction of Rho GTPases with regulatory proteins. The setup, which uses only minute amounts of sample, was used to analyze small molecules that inhibit the interaction between Rho GTPases and RhoGDIα. Prenylcysteine analogues and the replacement of GDP by non‐hydrolysable GTP analogues prevented the formation of Rho GTPase‐RhoGDIα complexes in a concentration‐dependent manner.     

Detection of Rho GEF and GAP activity through a sensitive split luciferase assay system

Rho GTPases regulate the assembly of cellular actin structures and are activated by GEFs (guanine-nucleotide-exchange factors) and rendered inactive by GAPs (GTPase-activating proteins). Using the Rho GTPases Cdc42, Rac1 and RhoA, and the GTPase-binding portions of the effector proteins p21-activated kinase and Rhophilin1, we have developed split luciferase assays for detecting both GEF and GAP regulation of these GTPases. The system relies on purifying split luciferase fusion proteins of the GTPases and effectors from bacteria, and our results show that the assays replicate GEF and GAP specificities at nanomolar concentrations for several previously characterized Rho family GEFs (Dbl, Vav2, Trio and Asef) and GAPs [p190, Cdc42 GAP and PTPL1-associated RhoGAP]. The assay detected activities associated with purified recombinant GEFs and GAPs, cell lysates expressing exogenous proteins, and immunoprecipitates of endogenous Vav1 and p190. The results demonstrate that the split luciferase system provides an effective sensitive alternative to radioactivity-based assays for detecting GTPase regulatory protein activities and is adaptable to a variety of assay conditions.     

How important are Rho GTPases in neurosecretion?

Rho GTPases are small GTP binding proteins belonging to the Ras superfamily which act as molecular switches that regulate many cellular function including cell morphology, cell to cell interaction, cell migration and adhesion. In neuronal cells, Rho GTPases have been proposed to regulate neuronal development and synaptic plasticity. However, the role of Rho GTPases in neurosecretion is poorly documented. In this review, we discuss data that highlight the importance of Rho GTPases and their regulators into the control of neurotransmitter and hormone release in neurons and neuroendocrine cells, respectively.     

Rho GTPases are involved in the regulation of NF-kappaB by genotoxic stress

A common cellular response to genotoxic agents and inflammatory cytokines is the activation of NF-kappaB. Here, we addressed the question of whether small GTPases of the Rho family are involved in the stimulation of NF-kappaB signaling by genotoxic agents or TNFalpha in HeLa cells. Inhibition of isoprenylation of Rho proteins by use of the HMG-CoA reductase inhibitor lovastatin attenuated UV-, doxorubicin-, and TNFalpha-induced degradation of IkappaBalpha as well as drug-stimulated DNA binding activity of NF-kappaB. Furthermore, NF-kappaB-regulated gene expression stimulated by either UV irradiation or treatment with TNFalpha was abrogated by lovastatin pretreatment. This indicates that isoprenylated regulatory proteins participate in the regulation of NF-kappaB by DNA-damaging agents as well as by TNFalpha. Specific blockage of Rho signaling by Clostridium difficile toxin B attenuated UV- and doxorubicin-induced activation of NF-kappaB, but did not affect stimulation of NF-kappaB by TNFalpha. Obviously, signaling to NF-kappaB by genotoxic and nongenotoxic stimuli occurs via different molecular mechanisms, either involving Rho GTPases or not. Based on the data, we suggest Rho GTPases to be essentially required for genotoxic stress-induced signaling to NF-kappaB.     

The RGK family of GTP-binding proteins: regulators of voltage-dependent calcium channels and cytoskeleton remodeling

RGK proteins constitute a novel subfamily of small Ras-related proteins that function as potent inhibitors of voltage-dependent (VDCC) Ca(2+) channels and regulators of actin cytoskeletal dynamics. Within the larger Ras superfamily, RGK proteins have distinct regulatory and structural characteristics, including nonconservative amino acid substitutions within regions known to participate in nucleotide binding and hydrolysis and a C-terminal extension that contains conserved regulatory sites which control both subcellular localization and function. RGK GTPases interact with the VDCC beta-subunit (Ca(V)beta) and inhibit Rho/Rho kinase signaling to regulate VDCC activity and the cytoskeleton respectively. Binding of both calmodulin and 14-3-3 to RGK proteins, and regulation by phosphorylation controls cellular trafficking and the downstream signaling of RGK proteins, suggesting that a complex interplay between interacting protein factors and trafficking contribute to their regulation.     

Controlling the switches: Rho GTPase regulation during animal cell mitosis

Animal cell division is a fundamental process that requires complex changes in cytoskeletal organization and function. Aberrant cell division often has disastrous consequences for the cell and can lead to cell senescence, neoplastic transformation or death. As important regulators of the actin cytoskeleton, Rho GTPases play major roles in regulating many aspects of mitosis and cytokinesis. These include centrosome duplication and separation, generation of cortical rigidity, microtubule–kinetochore stabilization, cleavage furrow formation, contractile ring formation and constriction, and abscission. The ability of Rho proteins to function as regulators of cell division depends on their ability to cycle between their active, GTP-bound and inactive, GDP-bound states. However, Rho proteins are inherently inefficient at fulfilling this cycle and require the actions of regulatory proteins that enhance GTP binding (RhoGEFs), stimulate GTPase activity (RhoGAPs), and sequester inactive Rho proteins in the cytosol (RhoGDIs). The roles of these regulatory proteins in controlling cell division are an area of active investigation. In this review we will delineate the current state of knowledge of how specific RhoGEFs, RhoGAPs and RhoGDIs control mitosis and cytokinesis, and highlight the mechanisms by which their functions are controlled.     

Rho GTPases和细胞凋亡

细胞凋亡涉及细胞骨架的形态学改变,Rho GTPases在细胞骨架改变中起着至关重要的作用。近年来的研究揭示了Rho蛋白家族在肌动蛋白(actin)聚合、解聚及actin-myosin的分子调节机制。同时越来越多的研究表明,Rho GTPases在巨噬细胞吞噬凋亡小体中也发挥了关键作用。本综述就Rho GTPases信号途径在细胞凋亡中细胞骨架的结构改变及凋亡小体被吞噬过程中的作用进行具体讨论。     

Hematopoietic-specific Rho GTPases Rac2 and RhoH and human blood disorders

The small guanosine triphosphotases (GTPases) Rho proteins are members of the Ras-like superfamily. Similar to Ras, most Rho GTPases cycle between active GTP-bound, and inactive GDP-bound conformations and act as molecular switches that control multiple cellular functions. While most Rho GTPases are expressed widely, the expression of Rac2 and RhoH are restricted to hematopoietic cells. RhoH is an atypical GTPase that lacks GTPase activity and remains in the active conformation. The generation of mouse knock-out lines has led to new understanding of the functions of both of these proteins in blood cells. The phenotype of these mice also led to the identification of mutations in human RAC2 and RHOH genes and the role of these proteins in immunodeficiency diseases. This review outlines the basic biology of Rho GTPases, focusing on Rac and RhoH and summarizes human diseases associated with mutations of these genes.     

Novel Split-Luciferase-Based Genetically Encoded Biosensors for Noninvasive Visualization of Rho GTPases

Rho family GTPases are critical regulators of many important cellular processes and the dysregulation of their activities is implicated in a variety of human diseases including oncogenesis and propagation of malignancy. The traditional methods, such as “pull-down” or two-hybrid procedures, are poorly suited to dynamically evaluate the activity of Rho GTPases, especially in living mammalian cells. To provide a novel alternative approach to analyzing Rho GTPase-associated signaling pathways in vivo, we developed a series of bioluminescent biosensors based on the genetically engineered firefly luciferase. These split-luciferase-based biosensors enable non-invasive visualization and quantification of the activity of Rho GTPases in living subjects. The strategy is to reasonably split the gene of firefly luciferase protein into two inactive fragments and then respectively fuse the two fragments to Rho GTPase and the GTPase-binding domain (GBD) of the specific effector. Upon Rho GTPase interacting with the binding domain in a GTP-dependent manner, these two luciferase fragments are brought into close proximity, leading to luciferase reconstitution and photon production in the presence of the substrate. Using these bimolecular luminescence complementation (BiLC) biosensors, we successfully visualized and quantified the activities of the three best characterized Rho GTPases by measuring the luminescence in living cells. We also experimentally investigated the sensitivity of these Rho GTPase biosensors to upstream regulatory proteins and extracellular ligands without lysing cells and doing labor-intensive works. By virtue of the unique functional characteristics of bioluminescence imaging, the BiLC-based biosensors provide an enormous potential for in vivo imaging of Rho GTPase signaling pathways and high-throughput screening of therapeutic drugs targeted to Rho GTPases and (or) upstream molecules in the near future.     

The role of Rho GTPases and associated kinases in regulating neurite outgrowth

Neurones are highly specialised cells that can extend over great distances, enabling the complex networking of the nervous system. We are beginning to understand in detail the molecular mechanisms that control the shape of neurones during development. One family of proteins that are clearly essential are the Rho GTPases which have a pivotal role in regulating the actin cytoskeleton in all cell types. The Rho GTPases are responsible for the activation and downregulation of many downstream kinases. This review discusses individual kinases that are regulated by three members of the Rho GTPases, Rac, Rho and Cdc42 and their function during neurite outgrowth and remodelling.     

Direct interaction of p21-activated kinase 4 with PDZ-RhoGEF, a G protein-linked Rho guanine exchange factor

Rho GTPases regulate a wide variety of cellular processes, ranging from actin cytoskeleton remodeling to cell cycle progression and gene expression. Cell surface receptors act through a complex regulatory molecular network that includes guanine exchange factors (GEFs), GTPase activating proteins, and guanine dissociation inhibitors to achieve the coordinated activation and deactivation of Rho proteins, thereby controlling cell motility and ultimately cell fate. Here we found that a member of the RGL-containing family of Rho guanine exchange factors, PDZ RhoGEF, which, together with LARG and p115RhoGEF, links the G(12/13) family of heterotrimeric G proteins to Rho activation, binds through its C-terminal region to the serine-threonine kinase p21-activated kinase 4 (PAK4), an effector for Cdc42. This interaction results in the phosphorylation of PDZ RhoGEF and abolishes its ability to mediate the accumulation of Rho-GTP by Galpha13. Moreover, when overexpressed, active PAK4 was able to dramatically decrease Rho-GTP loading in vivo and the formation of actin stress fibers in response to serum or LPA stimulation. Together, these results provide evidence that PAK4 can negatively regulate the activation of Rho through a direct protein-protein interaction with G protein-linked Rho GEFs, thus providing a novel potential mechanism for cross-talk among Rho GTPases.     

Structural basis of the Rho GTPase signaling

Small GTPases of the Rho family serve as conformational switches in a wide variety of signal transduction pathways that regulate diverse cellular functions. The GTP-bound forms of Rho GTPases are capable of interacting with downstream effectors that control cytoskeletal rearrangements. Regulators that stimulate nucleotide exchange, the hydrolytic cycle and distribution between the membrane and cytosol control the switch. Detailed pictures of Rho GTPase switching, effector recognition and regulation by regulators have emerged from recent structural investigations. These include the most extensively studied Rho GTPases, RhoA, Rac1, 2 and Cdc42, and their complexes with effectors and regulators. These studies have revealed the general diversity of effector and regulator structures, and in particular the structural features concerning the specific interactions involved in Rho effector recognition and regulator interactions with Rho GTPase. These findings provide a critical insight into the nature of Rho GTPase activity and consequently allow for a detailed manipulation of signaling pathways mediated by these proteins.     

Clostridial toxins: Molecular probes of Rho-dependent signaling and apoptosis

The Rho family small GTPases are members of the Ras superfamily of small GTPases. Rho proteins were first determined to act as key regulators of many types of actin cytoskeletal-dependent cellular functions. Recent work by several investigators indicates that Rho GTPases are also critical modulators of several important intracellular and nuclear signal transduction pathways. Certain clostridial toxins and exoenzymes covalently modify, and thereby inactivate, specific types of Rho family GTPases. As such, these microbial enzymes have proven invaluable in helping to identify structural and functional attributes of Rho GTPases.