RUN domains: a new family of domains involved in Ras-like GTPase signaling

RUN domains are present in several proteins that are linked particularly to the functions of GTPases in the Rap and Rab families. They could hence play an important role in multiple Ras-like GTPase signaling pathways.     

Atypical mechanism of regulation of the Wrch-1 Rho family small GTPase

Rho family GTPases are GDP/GTP-regulated molecular switches that regulate signaling pathways controlling diverse cellular processes. Wrch-1 was identified as a Wnt-1 regulated Cdc42 homolog, upregulated by Wnt1 signaling in Wnt1-transformed mouse mammary cells, and was able to promote formation of filopodia and activate the PAK serine/threonine kinase. Wrch-1 shares significant sequence and functional similarity with the Cdc42 small GTPase. However, Wrch-1 possesses a unique N-terminal 46 amino acid sequence extension that contains putative Src homology 3 (SH3) domain-interacting motifs. We determined the contribution of the N terminus to Wrch-1 regulation and activity. We observed that Wrch-1 possesses properties that distinguish it from Cdc42 and other Rho family GTPases. Unlike Cdc42, Wrch-1 possesses an extremely rapid, intrinsic guanine nucleotide exchange activity. Although the N terminus did not influence GTPase or GDP/GTP cycling activity in vitro, N-terminal truncation of Wrch-1 enhanced its ability to interact with and activate PAK and to cause growth transformation. The N terminus associated with the Grb2 SH3 domain-containing adaptor protein, and this association increased the levels of active Wrch-1 in cells. We propose that Grb2 overcomes N-terminal negative regulation to promote Wrch-1 effector interaction. Thus, Wrch-1 exhibits an atypical model of regulation not seen in other Rho family GTPases.     

A Ras-like GTPase is involved in hyphal growth guidance in the filamentous fungus Ashbya gossypii

Characteristic features of morphogenesis in filamentous fungi are sustained polar growth at tips of hyphae and frequent initiation of novel growth sites (branches) along the extending hyphae. We have begun to study regulation of this process on the molecular level by using the model fungus Ashbya gossypii. We found that the A. gossypii Ras-like GTPase Rsr1p/Bud1p localizes to the tip region and that it is involved in apical polarization of the actin cytoskeleton, a determinant of growth direction. In the absence of RSR1/BUD1, hyphal growth was severely slowed down due to frequent phases of pausing of growth at the hyphal tip. During pausing events a hyphal tip marker, encoded by the polarisome component AgSPA2, disappeared from the tip as was shown by in vivo time-lapse fluorescence microscopy of green fluorescent protein-labeled AgSpa2p. Reoccurrence of AgSpa2p was required for the resumption of hyphal growth. In the Agrsr1/bud1Delta deletion mutant, resumption of growth occurred at the hyphal tip in a frequently uncoordinated manner to the previous axis of polarity. Additionally, hyphal filaments in the mutant developed aberrant branching sites by mislocalizing AgSpa2p thus distorting hyphal morphology. These results define AgRsr1p/Bud1p as a key regulator of hyphal growth guidance.     

The small GTPase Ran: interpreting the signs

The small GTPase Ran has roles in nuclear transport, mitotic spindle assembly and nuclear envelope assembly. During the past three years, it has become clear that many of these processes rely on conserved molecular mechanisms involving Ran-GTP-binding proteins of the importin-beta superfamily. Moreover, recent experimental evidence has documented the distribution of Ran-GTP within cells and supported the notion that Ran plays a central role in the spatial and temporal organization of the eukaryotic cell.     

Cloning and characterization of a Dictyostelium gene encoding a small GTPase of the Rab11 family

Eukaryotic cells achieve complexity by compartmentalizing a subset of cellular functions into membrane-bound organelles. Maintaining this high level of cellular organization requires precise regulation of traffic between membranes. This task is accomplished, in part, by rab proteins. How these small GTPases regulate membrane traffic between cellular compartments is not clear. Here we report the characterization of a novel rab GTPase from the soil amoebae Dictyostelium discoideum. The predicted coding sequence of the new rab gene, Dictyostelium rab11b, encodes a protein of 25 kD containing all the structural hallmarks of a rab GTPase. Comparison of the sequence with the GenBank database and cladistic analysis demonstrated Dictyostelium rab11b to be a divergent member of the rab11 branch of rab proteins. Southern analysis revealed the presence of related genes in Dictyostelium. RNAse protection assays showed the Dictyostelium rab11b gene to be expressed at uniform levels throughout growth and development. Gene deletion experiments revealed that Dictyostelium rab11b was not essential for growth or development. Conceivably, the function of rab11b may be redundant with that of related genes in this organism. J. Cell. Biochem. 70:29–37, 1998. © 1998 Wiley-Liss, inc.     

Rho GTPase signaling in Dictyostelium discoideum: insights from the genome

Rho GTPases are ubiquitously expressed across the eukaryotes where they act as molecular switches participating in the regulation of many cellular processes. We present an inventory of proteins involved in Rho-regulated signaling pathways in Dictyostelium discoideum that have been identified in the completed genome sequence. In Dictyostelium the Rho family is encoded by 18 genes and one pseudogene. Some of the Rho GTPases (Rac1a/b/c, RacF1/F2 and RacB) are members of the Rac subfamily, and one, RacA, belongs to the RhoBTB subfamily. The Cdc42 and Rho subfamilies, characteristic of metazoa and fungi, are absent. The activities of these GTPases are regulated by two members of the RhoGDI family, by eight members of the Dock180/zizimin family and by a surprisingly large number of proteins carrying RhoGEF (42 genes) or RhoGAP (43 genes) domains or both (three genes). Most of these show domain compositions not found in other organisms, although some have clear homologs in metazoa and/or fungi. Among the (in many cases putative) effectors found in Dictyostelium are the CRIB domain proteins (WASP and two related proteins, eight PAK kinases and a novel gelsolin-related protein), components of the Scar/WAVE complex, 10 formins, four IQGAPs, two members of the PCH family, numerous lipid kinases and phospholipases, and components of the NADPH oxidase and the exocyst complexes. In general, the repertoire of Rho signaling components of Dictyostelium is similar to that of metazoa and fungi.     

Interaction cloning of Rabin3, a novel protein that associates with the Ras-like GTPase Rab3A.

Rab3A is a small, Ras-like GTPase expressed in neuroendocrine cells, in which it is associated with secretory vesicle membranes and regulates exocytosis. Using the yeast two-hybrid system, we have identified a rat brain cDNA encoding a novel 50-kDa protein, which we have named Rabin3, that interacts with Rab3A and Rab3D but not with other small GTPases (Rab3C, Rab2, Ran, or Ras). Several independent point mutations in the effector domain of Rab3A (F51L, V55E, and G56D) which do not alter nucleotide binding by the GTPase abolish the interaction with Rabin3, while another mutation (V52A) appears to increase the interaction. These results demonstrate that the interaction is highly specific. However, a glutathione S-transferase-Rabin3 fusion protein associates only weakly in vitro with recombinant Rab3A and possesses no detectable GTPase-activating protein or nucleotide exchange activity, and Rabin3 overexpressed in adrenal chromaffin cells has no observable effect on secretion. The protein possess a sequence characteristic of coiled-coil domains and a second small region with sequence similarity to a Saccharomyces cerevisiae protein, Sec2p, Sec2p is essential for constitutive secretion in yeast cells and interacts with Sec4p, a close relative of the Rab3A GTPase. Rabin3 mRNA and protein are widely expressed but are particularly abundant in testes.     

Local control of AMPA receptor trafficking at the postsynaptic terminal by a small GTPase of the Rab family

The delivery of neurotransmitter receptors into the synaptic membrane is essential for synaptic function and plasticity. However, the molecular mechanisms of these specialized trafficking events and their integration with the intracellular membrane transport machinery are virtually unknown. Here, we have investigated the role of the Rab family of membrane sorting proteins in the late stages of receptor trafficking into the postsynaptic membrane. We have identified Rab8, a vesicular transport protein associated with trans-Golgi network membranes, as a critical component of the cellular machinery that delivers AMPA-type glutamatergic receptors (AMPARs) into synapses. Using electron microscopic techniques, we have found that Rab8 is localized in close proximity to the synaptic membrane, including the postsynaptic density. Electrophysiological studies indicated that Rab8 is necessary for the synaptic delivery of AMPARs during plasticity (long-term potentiation) and during constitutive receptor cycling. In addition, Rab8 is required for AMPAR delivery into the spine surface, but not for receptor transport from the dendritic shaft into the spine compartment or for delivery into the dendritic surface. Therefore, Rab8 specifically drives the local delivery of AMPARs into synapses. These results demonstrate a new role for the cellular secretory machinery in the control of synaptic function and plasticity directly at the postsynaptic membrane.     

Backbone assignment and secondary structure of Rnd1, an unusual Rho family small GTPase

Rho GTPases have attracted considerable interest as signaling molecules due to their variety of functional roles in cells. Rnd1 is a relatively recently discovered Rho GTPase with no enzymatic activity against its bound GTP nucleotide, setting it apart from other family members. Research has revealed a critical role for Rnd1 not only in neurite outgrowth, dendrite development, axon guidance, but also in gastric cancer and in endothelial cells during inflammation. Structural information is crucial for understanding the mechanism that forms the basis for protein–protein interactions and functions, but until recently there were no reports of NMR studies directly on the Rnd1 protein. In this paper we report assignments for the majority of Rnd1 NMR resonances based on 2D and 3D NMR spectra. Rnd1 assignment was a challenging task, however, despite optimization strategies that have facilitated NMR studies of the protein (Cao and Buck in Small GTPase 2:295–304, 2012). Besides common triple-resonance experiments, 3D HNCA, 3D HN(CO)CA, 3D HNCO which are usually employed for sequence assignment, 3D NOESY experiments and specific labeling of 13 kinds of amino acids were also utilized to gain as many ¹H(N), ¹³C, and ¹⁵N resonances assignments as possible. For 170 cross peaks observed out of 183 possible mainchain N–H correlations in the ¹H–¹⁵N TROSY spectrum, backbone assignment was finally completed for 127 resonances. The secondary structure was then defined by chemical shifts and TALOS+ based on the assignments. The overall structure in solution compares well with that of Rnd1 in a crystal, except for two short segments, residues 77–83 and residues 127–131. Given that some features are shared among Rho GTPases, Rnd1 assignments are also compared with two other family members, Cdc42 and Rac1. The overall level of Rnd1 assignment is lower than for Cdc42 and Rac1, consistent with its lower stability and possibly increased internal dynamics. However, while the Rnd1 switch II region remained un-assigned, the switch I region could be more fully assigned compared to Cdc42 and Rac1. The NMR assignment and structure analysis reported here provides a robust basis for future study of the binding between Rnd1 and other proteins, as well as for further studies of the molecular function of this unusual GTPase.     

The Arabidopsis Rab GTPase family: another enigma variation

The Arabidopsis genome sequence reveals that gene families such as the Rab GTPase family, which encodes key determinants of vesicle-targeting specificity, are considerably more diverse in plants and mammals than in yeast. In mammals, this diversity appears to reflect the complexity of membrane trafficking. Phylogenetic analyses indicate that, despite its large size, the Arabidopsis Rab family lacks most of the Rab subclasses found in mammals. The Arabidopsis Rab family has, however, undergone a distinct 'adaptive radiation' that has given rise to proteins that may perform plant-specific functions.     

The Ras-like GTPase Gem is involved in cell shape remodelling and interacts with the novel kinesin-like protein KIF9

Gem belongs to the Rad/Gem/Kir (RGK) subfamily of Ras-related GTPases, which also comprises Rem, Rem2 and Ges. The RGK family members Ges and Rem have been shown to produce endothelial cell sprouting and reorganization of the actin cytoskeleton upon overexpression. Here we show that high intracellular Gem levels promote profound changes in cell morphology and we investigate how this phenotype arises dynamically. We also show that this effect requires intact microtubules and microfilaments, and that Gem is associated with both cytoskeletal components. In order to investigate the mechanisms of Gem recruitment to the cytoskeleton, we performed a yeast two-hybrid screen and identified a novel kinesin-like protein, termed KIF9, as a new Gem interacting partner. We further show that Gem and KIF9 interact by co-immunoprecipitation. Furthermore, Gem and KIF9 display identical patterns of gene expression in different tissues and developmental stages. The Gem- KIF9 interaction reported here is the first molecular link between RGK family members and the microtubule cytoskeleton.     

Analysis of the small GTPase gene superfamily of Arabidopsis

Small GTP-binding proteins regulate diverse processes in eukaryotic cells such as signal transduction, cell proliferation, cytoskeletal organization, and intracellular membrane trafficking. These proteins function as molecular switches that cycle between "active" and "inactive" states, and this cycle is linked to the binding and hydrolysis of GTP. The Arabidopsis genome contains 93 genes that encode small GTP-binding protein homologs. Phylogenetic analysis of these genes shows that plants contain Rab, Rho, Arf, and Ran GTPases, but no Ras GTPases. We have assembled complete lists of these small GTPases families, as well as accessory proteins that control their activity, and review what is known of the functions of individual members of these families in Arabidopsis. We also discuss the possible roles of these GTPases in relation to their similarity to orthologs with known functions and localizations in yeast and/or animal systems.     

Effects of cell tension on the small GTPase Rac

Cells in the body are subjected to mechanical stresses such as tension, compression, and shear stress. These mechanical stresses play important roles in both physiological and pathological processes; however, mechanisms transducing mechanical stresses into biochemical signals remain elusive. Here, we demonstrated that equibiaxial stretch inhibited lamellipodia formation through deactivation of Rac. Nearly maximal effects on Rac activity were obtained with 10% strain. GAP-resistant, constitutively active V12Rac reversed this inhibition, supporting a critical role for Rac inhibition in the response to stretch. In contrast, activation of endogenous Rac with a constitutively active nucleotide exchange factor did not, suggesting that regulation of GAP activity most likely mediates the inhibition. Uniaxial stretch suppressed lamellipodia along the sides lengthened by stretch and increased it at the adjacent ends. A fluorescence assay for localized Rac showed comparable changes in activity along the sides versus the ends after uniaxial stretch. Blocking polarization of Rac activity by expressing V12Rac prevented subsequent alignment of actin stress fibers. Treatment with Y-27632 or ML-7 that inhibits myosin phosphorylation and contractility increased lamellipodia through Rac activation and decreased cell polarization. We hypothesize that regulation of Rac activity by tension may be important for motility, polarization, and directionality of cell movement.     

Nerve growth factor-dependent activation of the small GTPase Rin

The Rit and Rin proteins comprise a distinct and evolutionarily conserved subfamily of Ras-related small GTPases. Although we have defined a role for Rit-mediated signal transduction in the regulation of cell proliferation and transformation, the function of Rin remains largely unknown. Because we demonstrate that Rin is developmentally regulated and expressed in adult neurons, we examined its role in neuronal signaling. In this study, we show that stimulation of PC6 cells with either epidermal growth factor or nerve growth factor (NGF) results in rapid activation of Rin. This activation correlates with the onset of Ras activation, and dominant-negative Ras completely inhibits Rin activation induced by NGF. Further examination of Ras-mediated Rin activation suggests that this process is dependent upon neuronally expressed regulatory factors. Expression of mutationally activated H-Ras fails to activate Rin in non-neuronal cells, but results in potent stimulation of Rin-GTP levels in a variety of neuronal cell lines. Furthermore, although constitutively activated Rin does not induce neurite outgrowth on its own, both NGF-induced and oncogenic Ras-induced neurite outgrowth were inhibited by the expression of dominant-negative Rin. Together, these studies indicate that Rin activation is a direct downstream effect of growth factor-dependent signaling in neuronal cells and suggest that Rin may function to transduce signals within the mature nervous system.     

Rho GTPase signaling in Dictyostelium discoideum: Insights from the genome

Rho GTPases are ubiquitously expressed across the eukaryotes where they act as molecular switches participating in the regulation of many cellular processes. We present an inventory of proteins involved in Rho-regulated signaling pathways in Dictyostelium discoideum that have been identified in the completed genome sequence. In Dictyostelium the Rho family is encoded by 18 genes and one pseudogene. Some of the Rho GTPases (Rac1a/b/c, RacF1/F2 and RacB) are members of the Rac subfamily, and one, RacA, belongs to the RhoBTB subfamily. The Cdc42 and Rho subfamilies, characteristic of metazoa and fungi, are absent. The activities of these GTPases are regulated by two members of the RhoGDI family, by eight members of the Dock180/zizimin family and by a surprisingly large number of proteins carrying RhoGEF (42 genes) or RhoGAP (43 genes) domains or both (three genes). Most of these show domain compositions not found in other organisms, although some have clear homologs in metazoa and/or fungi. Among the (in many cases putative) effectors found in Dictyostelium are the CRIB domain proteins (WASP and two related proteins, eight PAK kinases and a novel gelsolin-related protein), components of the Scar/WAVE complex, 10 formins, four IQGAPs, two members of the PCH family, numerous lipid kinases and phospholipases, and components of the NADPH oxidase and the exocyst complexes. In general, the repertoire of Rho signaling components of Dictyostelium is similar to that of metazoa and fungi.     

A family of small repeated elements with some transposon-like properties in the genome of Neisseria gonorrhoeae

A physical technique known as two-dimensional S1 nuclease heteroduplex mapping has been applied to genomic DNA from the Gram-negative coccus Neisseria gonorrhoeae. This has resulted in the detection of two novel types of repetitive sequences. The first type is a repetitive sequence family of 152 base pairs (bp), whose ends are composed of inverted repeats of 26 bp. There are approximately 20 copies of this sequence, in both N. gonorrhoeae and Neisseria meningitidis (Correia, F., Inouye, S., and Inouye, M. (1986) J. Bacteriol. 167, 1009-1011). The second type of sequence is a 1443-bp duplication in the N. gonorrhoeae genome. The two classes of sequence are linked positionally. Each copy of the long duplicated sequence is adjacent to a member of the 152-bp repetitive sequence. In one instance two copies of the 152-bp repetitive sequence are separated by a 436-bp central region and are in an inverted orientation with respect to one another, resembling a compound transposable element.     

PIWI-interacting small RNAs: the vanguard of genome defence

PIWI-interacting RNAs (piRNAs) are a distinct class of small non-coding RNAs that form the piRNA-induced silencing complex (piRISC) in the germ line of many animal species. The piRISC protects the integrity of the genome from invasion by 'genomic parasites'--transposable elements--by silencing them. Owing to their limited expression in gonads and their sequence diversity, piRNAs have been the most mysterious class of small non-coding RNAs regulating RNA silencing. Now, much progress is being made into our understanding of their biogenesis and molecular functions, including the specific subcellular compartmentalization of the piRNA pathway in granular cytoplasmic bodies.