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PubMed1996

Ras, Rap, and Rac small GTP-binding proteins are targets for Clostridium sordellii lethal toxin glucosylation.

Lethal toxin (LT) from Clostridium sordellii is one of the high molecular mass clostridial cytotoxins. On cultured cells, it causes a rounding of cell bodies and a disruption of actin stress fibers. We demonstrate that LT is a glucosyltransferase that uses UDP-Glc as a cofactor to covalently modify 21-kDa proteins both in vitro and in vivo. LT glucosylates Ras, Rap, and Rac. In Ras, threonine at position 35 was identified as the target amino acid glucosylated by LT. Other related members of the Ras GTPase superfamily, including RhoA, Cdc42, and Rab6, were not modified by LT. Incubation of serum-starved Swiss 3T3 cells with LT prevents the epidermal growth factor-induced phosphorylation of mitogen-activated protein kinases ERK1 and ERK2, indicating that the toxin blocks Ras function in vivo. We also demonstrate that LT acts inside the cell and that the glucosylation reaction is required to observe its dramatic effect on cell morphology. LT is thus a powerful tool to inhibit Ras function in vivo.

3T3 Cells↗

PubMed1999

Molecular dynamics simulations of the Ras:Raf and Rap:Raf complexes.

The protein Raf is an immediate downstream target of Ras in the MAP kinase signalling pathway. The complex of Ras with the Ras-binding domain (RBD) of Raf has been modelled by homology to the (E30D,K31E)-Rap1A:RBD complex, and both have been subjected to multiple molecular dynamics simulations in solution. While both complexes are stable, several rearrangements occur in the Ras:RBD simulations: the RBD loop 100-109 moves closer to Ras, Arg73 in the RBD moves towards Ras to form a salt bridge with Ras-Asp33, and Loop 4 of the Ras switch II region shifts upwards toward the RBD. The Ras:RBD interactions (including the RBD-Arg73 interaction) are consistent with available NMR and mutagenesis data on the Ras: RBD complex in solution. The Ras switch II region does not interact directly with the RBD, although indirect interactions exist through the effector domain and bridging water molecules. No large-scale RBD motion is seen in the Ras:RBD complex, compared to the Rap:RBD complex, to suggest an allosteric activation of Raf by Ras. This may be because the Raf kinase domain (whose structure is unknown) is not included in the model.

Amino Acid Sequence↗

PubMed2005

XEpac, a guanine nucleotide-exchange factor for Rap GTPase, is a novel hatching gland specific marker during the Xenopus embryogenesis.

cAMP is a second messenger controlling various cellular processes through cAMP-dependent protein kinase (cAPK, PKA) and cyclic nucleotide-gated ion channels. Recently, the PKA-independent-cAMP-mediated signaling pathway by means of exchange protein directly activated by cAMP (Epac) has been demonstrated. Epac is a guanine nucleotide-exchange factor (GEF) for Rap, a Ras-like small GTPase. To investigate this new target for cAMP in development, we have isolated Xepac, the Xenopus laevis homologue of Epac by cDNA library screening. Xepac (Xepac1) encodes 890 amino acids, which have 57% identity with human Epac1 and 59% with that of rat Epac1 in amino acids. Whole-mount in situ hybridization and reverse transcriptase-polymerase chain reaction analysis show that XEpac is expressed both maternally and zygotically and is restricted within the developing hatching gland. Intriguingly, overexpression of XEpac induces the anterior markers XAG-1 and XOtx2 and can convert ectoderm into cement- and hatching gland-expressing cells. These results suggest that XEpac contains anterior positional information.

Animals↗

PubMed1997

Mapping of the gene (NRAP) encoding N-RAP in the mouse and human genomes.

N-RAP is a nebulin-related actin-binding protein found at the myotendon junction in skeletal muscle and at the intercalated disks in cardiac muscle. We mapped the NRAP gene to mouse chromosome 19 using interspecific crosses and to human chromosome 10 using radiation hybrid panels. Comparative analysis of the mouse and human genomes indicates that the NRAP gene is located in regions of conserved synteny between the two species.

Animals↗

PubMed1994

Concerted evolution and RAPping in mitochondrial VNTRs and the molecular geography of cricket populations.

In recent years, many animal species have been shown to exhibit a variable number of tandem repeats (VNTR) in the control region of their mitochondrial DNA. To characterize the nucleotide polymorphism of a VNTR system, this region was sequenced in individuals from two species of crickets (genus Gryllus) collected at a mainland locality and an island locality within each species. The data reveal a clear pattern of concerted evolution: homogeneity among repeats within individuals and populations, but heterogeneity among the tandem arrays from divergent populations, and between species. The patterns of nucleotide polymorphisms within the arrays show several instances of partial homogenization where derived nucleotides have not swept through all the repeats in the array. These serve as examples of concerted evolution "caught in the act". Additional repeat associated polymorphisms (RAPs) shed light on the molecular basis of the insertion and deletion of repeat units and, with the concerted evolution of VNTRs, provide phylogeographic resolution of island and mainland populations within and between species. The polymorphisms within VNTRs also provide a simple system in which to examine the balance of mutation and drift and offer a unique view of the histories of genomic and organismal phenomena in the patterns and processes of molecular evolution.

Animals↗

PubMed1988

Immunocytochemical localization of ras p21 product in thyroid follicular cells of normal rats using monoclonal antibody RAP-5.

The ultrastructural localization of ras p21 product was studied immunocytochemically in thyroid follicular cells of normal rats using pre-embedded peroxidase-labelled antibody techniques and a monoclonal antibody, RAP-5, which had been raised against a synthetic peptide corresponding to amino acid positions 10-17 of the ras p21 protein. The ras p21 product was detected in cisternae of rough endoplasmic reticulum, Golgi apparatus and the subapical portion of apical plasma membrane, in which it was most concentrated. This study indicated that the p21 product may be synthesized in the rough endoplasmic reticulum and finally localized at the subapical portion of the thyroid follicular cells, and also that the apical plasma membrane may be a major site for the reception of environmental stimuli.

Animals↗

PubMed2006

Epac- and Rap- independent ERK1/2 phosphorylation induced by Gs-coupled receptor stimulation in HEK293 cells.

Serotonin activates Ras and Ras-dependent ERK1/2 phosphorylation in HEK293 cells expressing G(s)-coupled 5-HT(4) or 5-HT(7) serotonin receptors through unknown mechanisms. Both Epac/Rap-dependent and -independent pathways for Ras-dependent ERK1/2 activation have been suggested. Epac overexpression or Epac-specific 8-CPT-2'-O-Me-cAMP did not cause ERK1/2 phosphorylation, despite Rap activation. The data did not support a role for PLCepsilon or DAG-dependent Ras GEFs of the Ras-GRP family in Ras-dependent ERK1/2 phosphorylation. However, serotonin stimulated phosphorylation of endogenous and recombinant Ras-GRF1, increased [Ca(2+)](i) and caused Ca(2+)- and calmodulin-dependent ERK1/2 phosphorylation. Different signalling pathways seem to be utilised by G(s)-coupled receptors in various isolates of HEK293 cells.

Calcium↗

PubMed2006

Mycobacterium bovis BCG as a delivery system for the RAP-1 antigen from Babesia bovis.

Babesia bovis is the causative agent of babesiosis, a tick-borne disease that is a major cause of loss to livestock production in Latin America. Vaccination against Babesia species represents a major challenge against cattle morbidity and mortality in enzootic areas. The aim of this study was to evaluate the capacity of Bacille Calmette-Guerin (BCG) to deliver the rhoptry associated protein (RAP-1) antigen of B. bovis and to stimulate specific cellular and humoral immune responses in mice. Two of five mycobacterial expression vectors efficiently expressed the antigen. These constructs were subsequently studied in vivo following three immunization protocols. The construct with the greatest in vivo stability proved to be the one that induced the strongest immune responses. Our data support the hypothesis that specific T lymphocyte priming by rBCG can be employed as a component of a combined vaccine strategy to induce long-lasting humoral and cellular immune responsiveness towards B. bovis and encourage further work on the application of rBCG to the development of Babesia vaccines.

Animals↗

PubMed2006

Biochemistry of the Rap-specific guanine nucleotide exchange factors PDZ-GEF1 and -2.

PDZ-GEFs represent one of four types of highly conserved Rap-specific guanine nucleotide exchange factors. They contain a number of well-known protein domains, including a "related to cyclic nucleotide binding domain" (RCBD), a PDZ-domain, a Ras-associating domain (RA), and, of course, a catalytic domain required for their exchange activity. Since their cloning more than 5 years ago, relatively little has been learned about their mode of regulation. Although their activity may in part depend on regulated membrane localization by means of the RA and/or PDZ domain, it seems highly likely that PDZ-GEFs can be modified by additional mechanisms as well. Based on analogy of the regulatory mechanisms of the cAMP-responsive GEF Epac, in the past we postulated a role for the RCBD domain in this. In this chapter, we give a detailed description of the methods that were used to unravel this mechanism in vitro and in vivo.

Animals↗

PubMed1999

A novel gene encoding a ras-like GTP-binding protein from Trypanosoma brucei: an evolutionary ancestor of the ras and rap genes of higher eukaryotes?

The ras superfamily of GTP binding proteins encompasses a wide range of family members, related by conserved amino-acid motifs, and act as molecular binary switches that play key roles in cellular processes. Gene duplication and divergence has been postulated as the mechanism by which such family members have evolved their specific functions. We have cloned and sequenced a ras-like gene, tbrlp, from the primitive eukaryote Trypanosoma brucei. The gene encodes a protein of 227 amino acids and contains the six conserved subdomains that designate it as a ras/rap subfamily member. However, the presence of key diagnostic residues characteristic of both the ras and rap families of GTP confuse the familial classification of this gene. Phylogenetic analysis of the GTP binding domain places its origins at the divergence point of the ras/rap families and suggests that tbrlp is an ancestral gene to the ras/rap genes of higher eukaryotes.

Amino Acid Sequence↗

PubMed1996

RNA polymerase II-associated protein (RAP) 74 binds transcription factor (TF) IIB and blocks TFIIB-RAP30 binding.

A set of deletion mutants of human RNA polymerase II-associated protein (RAP) 30, the small subunit of transcription factor IIF (TFIIF; RAP30/74), was constructed to map functional domains. Mutants were tested for accurate transcriptional activity, RAP74 binding, and TFIIB binding. Transcription assays indicate the importance of both N- and C-terminal sequences for RAP30 function. RAP74 binds to the N-terminal region of RAP30 between amino acids 1 and 98. TFIIB binds to an overlapping region of RAP30, localized to amino acids 1-176 (amino acids 27-152 comprise a minimal binding region). The C-terminal region of RAP74 (amino acids 358-517) binds directly and independently to TFIIB. Interestingly, RAP74 blocks TFIIB-RAP30 binding, both by binding TFIIB and by binding RAP30. When the TFIIF complex is intact, therefore, TFIIB-TFIIF contact is maintained through RAP74. If the TFIIB-RAP30 interaction is physiologically important, the TFIIF complex must dissociate within some transcription complexes.

Amino Acid Sequence↗

PubMed2002

Rap-specific GTPase activating protein follows an alternative mechanism.

Rap1 is a small GTPase that is involved in signal transduction cascades. It is highly homologous to Ras but it is down-regulated by its own set of GTPase activating proteins (GAPs). To investigate the mechanism of the GTP-hydrolysis reaction catalyzed by Rap1GAP, a catalytically active fragment was expressed in Escherichia coli and characterized by kinetic and mutagenesis studies. The GTPase reaction of Rap1 is stimulated 10(5)-fold by Rap1GAP and has a k(cat) of 6 s(-1) at 25 degrees C. The catalytic effect of GAPs from Ras, Rho, and Rabs depends on a crucial arginine which is inserted into the active site. However, all seven highly conserved arginines of Rap1GAP can be mutated without dramatically reducing V(max) of the GTP-hydrolysis reaction. We found instead two lysines whose mutations reduce catalysis 25- and 100-fold, most likely by an affinity effect. Rap1GAP does also not supply the crucial glutamine that is missing in Rap proteins at position 61. The Rap1(G12V) mutant which in Ras reduces catalysis 10(6)-fold is shown to be efficiently down-regulated by Rap1GAP. As an alternative, Rap1(F64A) is shown by kinetic and cell biological studies to be a Rap1GAP-resistant mutant. This study supports the notion of a completely different mechanism of the Rap1GAP-catalyzed GTP-hydrolysis reaction on Rap1.

Amino Acid Sequence↗

PubMed2002

The delta subunit of retinal rod cGMP phosphodiesterase regulates the membrane association of Ras and Rap GTPases.

Post-translational modifications of GTPases from the Ras superfamily enable them to associate with membrane compartments where they exert their biological activities. However, no protein acting like Rho and Rab dissociation inhibitor (GDI) that regulate the membrane association of Rho and Rab GTPases has been described for Ras and closely related proteins. We report here that the delta subunit of retinal rod phosphodiesterase (PDEdelta) is able to interact with prenylated Ras and Rap proteins, and to solubilize them from membranes, independently of their nucleotide-bound (GDP or GTP) state. We show that PDEdelta exhibits striking structural similarities with RhoGDI, namely conservation of the Ig-like fold and presence of a series of hydrophobic residues which could act as in RhoGDI to sequester the prenyl group of its target proteins, thereby providing structural support for the biochemical activity of PDEdelta. We observe that the overexpression of PDEdelta interferes with Ras trafficking and propose that it may play a role in the process that delivers prenylated proteins from endomembranes, once they have undergone proteolysis and carboxymethylation, to the structures that ensure trafficking to their respective resident compartments.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed2004

RAP: a new computer program for de novo identification of repeated sequences in whole genomes.

MOTIVATION: DNA repeats are a common feature of most genomic sequences. Their de novo identification is still difficult despite being a crucial step in genomic analysis and oligonucleotides design. Several efficient algorithms based on word counting are available, but too short words decrease specificity while long words decrease sensitivity, particularly in degenerated repeats. RESULTS: The Repeat Analysis Program (RAP) is based on a new word-counting algorithm optimized for high resolution repeat identification using gapped words. Many different overlapping gapped words can be counted at the same genomic position, thus producing a better signal than the single ungapped word. This results in better specificity both in terms of low-frequency detection, being able to identify sequences repeated only once, and highly divergent detection, producing a generally high score in most intron sequences. AVAILABILITY: The program is freely available for non-profit organizations, upon request to the authors. CONTACT: giorgio.valle@unipd.it SUPPLEMENTARY INFORMATION: The program has been tested on the Caenorhabditis elegans genome using word lengths of 12, 14 and 16 bases. The full analysis has been implemented in the UCSC Genome Browser and is accessible at http://genome.cribi.unipd.it.

Algorithms↗

PubMed1990

A ras-related gene from the lower eukaryote Dictyostelium that is highly conserved relative to the human rap genes.

The cellular slime mold Dictyostelium discoideum contains two ras genes, DdrasG and Ddras that are differentially expressed during development. We have characterized a gene that hybridized to both Ddras and DdrasG under low, but not under high stringency conditions. The deduced amino acid sequence is highly conserved with respect to the human rap (Krev-1, smg21) proteins and the corresponding gene has been designated Ddrap1. The Ddrap1 gene is expressed at all stages during development but is expressed maximally during the aggregation and culmination periods when the expression of Ddras and DdrasG is declining. During vegetative growth and early development Ddrap1 cDNA hybridizes to a single mRNA of 1.1 kb. As development progresses the level of this mRNA declines and messages of 1.0 and 1.3 kb appear.

Amino Acid Sequence↗

PubMed1977

Rapping with a chaplain.

Group discussions for senior physical therapy students, led by the hospital chaplain, were held at the University of Florida Medical Center. The idea for these "rap sessions" originated with the Department of Physical Therapy at Shands Teaching Hospital. The content of the discussions grew out of the students' experiences during their clinical assignments. The experience was deemed beneficial to the learning process by students, clinic staff, and faculty and helped to create a more relaxed atmosphere for learning in the clinic.

Group Processes↗

PubMed2004

Alcohol use disorders among emergency department-treated older adolescents: a new brief screen (RUFT-Cut) using the AUDIT, CAGE, CRAFFT, and RAPS-QF.

BACKGROUND: Early identification of alcohol use disorders (AUD) among emergency department (ED)-treated patients is important for facilitating intervention and further evaluation outside EDs. A number of brief screening instruments have been developed for identifying patients with AUD, but it is not clear whether they are practical and perform well with older adolescents in an ED setting. This study contrasted four brief screening instruments for detecting DSM-IV-defined AUD and tested a newly developed brief screen for use among ED-treated older adolescents. METHODS: The Alcohol Use Disorders Identification Test (AUDIT), the CAGE, the CRAFFT, and a modified RAPS-QF were given to 93 alcohol-using older adolescents (55% men; aged 18-20 years) in an ED. Receiver operator characteristic analyses were used to evaluate the performance of brief screens against the criterion of a lifetime DSM-IV alcohol abuse or dependence diagnosis. RESULTS: Of existing instruments, the AUDIT had the best overall performance in identifying AUD (sensitivity, 82%; specificity, 78%). A new, shorter screening instrument composed of two AUDIT items, two CRAFFT items, and one CAGE item (RUFT-Cut) performed as well as the AUDIT (sensitivity, 82%; specificity, 78%). CONCLUSIONS: Among existing alcohol screening instruments, the AUDIT performed best for identifying ED-treated older adolescents with alcohol use disorders. The RUFT-Cut is a brief screening instrument for AUD that shows promise for identifying ED-treated older adolescents who are in need of intervention or further evaluation. Future research should focus on use of the RUFT-Cut in other settings with larger, more diverse samples of adolescents.

Adolescent↗

PubMed2000

Differential processing of propeptide inhibitors of Rap phosphatases in Bacillus subtilis.

In the phosphorelay signal transduction system for sporulation initiation in Bacillus subtilis, the opposing activities of histidine kinases and aspartyl phosphate phosphatases determine the cell's decision whether to continue with vegetative growth or to initiate the differentiation process. Regulated dephosphorylation of the Spo0A and Spo0F response regulators allows a variety of negative signals from physiological processes that are antithetical to sporulation to impact on the activation level of the phosphorelay. Spo0F approximately P is the known target of two related phosphatases, RapA and RapB. In addition to RapA and RapB, a third member of the Rap family of phosphatases, RapE, specifically dephosphorylated the Spo0F approximately P intermediate in response to competence development. RapE phosphatase activity was found to be controlled by a pentapeptide (SRNVT) generated from within the carboxy-terminal domain of the phrE gene product. A synthetic PhrE pentapeptide could (i) complement the sporulation deficiency caused by deregulated RapE activity of a phrE mutant and (ii) inhibit RapE-dependent dephosphorylation of Spo0F approximately P in in vitro experiments. The PhrE pentapeptide did not inhibit the phosphatase activity of RapA and RapB. These results confirm previous conclusions that the specificity for recognition of the target phosphatase is contained within the amino acid sequence of the pentapeptide inhibitor.

Amino Acid Sequence↗