Anti-(Raf-1) RNA aptamers that inhibit Ras-induced Raf-1 activation.

RNA aptamers with affinity for the Ras-binding domain (RBD) of Raf-1 were isolated from a degenerate pool by in vitro selection. These aptamers efficiently inhibited the Ras interaction with the Raf-1 RBD, and also inhibited Ras-induced Raf-1 activation in a cell-free system. The RNA aptamer with the most potent inhibitory effect specifically inhibited the Ras-Raf-1 interaction and had no affinity for the RBD of the RGL protein, a homolog of the Ral GDP dissociation stimulator. Although the aptamer was capable of binding to the B-Raf RBD, the RNA did not inhibit the interaction between Ras and the B-Raf RBD. Enzymatic and chemical probing experiments indicated that the aptamer was folded into a pseudoknot structure, and some loop regions of the pseudoknot were located at the binding interface for the Raf-1 RBD.     

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.     

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein’s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.     

Point mutants of c-raf-1 RBD with elevated binding to v-Ha-Ras

A mutational analysis of the Ras-binding domain (RBD) of c-Raf-1 identified three amino acid positions (Asn(64), Ala(85), and Val(88)) where amino acid substitution with basic residues increases the binding of RBD to recombinant v-Ha-Ras. The greatest increase in binding (6-9-fold) was observed with the A85K-RBD mutant. The elevated binding for the A85K-RBD and V88R-RBD mutants was also detected with Ras expressed in cultured mammalian cells, namely NIH-3T3 and BAF cells. None of the wild type residues in RBD positions Asn(64), Ala(85), and Val(88) have been previously implicated in the interaction with Ras (Block, C., Janknecht, R., Herrmann, C., Nassar, N., and Wittinghofer, A. (1996) Nat. Struct. Biol. 3, 244-251; Nassar, N., Horn, G., Herrmann, C., Scherer, A., McCormick, F., and Wittinghofer, A. (1995) Nature 375, 554-560). The discovery of elevated binding among the mutants in these positions implies that additional RBD residues can be used to generate the Ras. RBD complex. These findings are of particular significance in the design of Ras antagonists based on the RBD prototype. The A85K-RBD mutant can be used to develop an assay for measuring the level of activated Ras in cultured cells; Sepharose-linked A85K-RBD.GST fusion protein served as an activation-specific probe to precipitate Ras.GTP but not Ras.GDP from epidermal growth factor-stimulated cells. A85K-RBD precipitates up to 5-fold more Ras.GTP from mammalian cells than wild type RBD.     

Critical function of the Ras-associating domain as a primary Ras-binding site for regulation of Saccharomyces cerevisiae adenylyl cyclase.

Mammalian candidate effectors of the small GTPase Ras, such as RalGDS, afadin/AF-6, Rin1, and phospholipase Cepsilon, have been shown to share structurally conserved modules termed Ras-associating (RA) domains at their Ras-binding sites. The Ras-binding domains of Raf-1 and phosphoinositide 3-kinase gamma (other Ras effectors) also share a similar tertiary structure with the RA domains. On the other hand, the primary Ras-binding site of Saccharomyces cerevisiae adenylyl cyclase, the best characterized Ras effector, has been mapped by mutational studies to the leucine-rich repeats (LRR) domain (amino acids 674-1300), whose structure apparently bears no resemblance to the RA domains. By a computer algorithm-based search we have unexpectedly found an RA domain in the N-terminal 81 amino acid residues (676) of the LRR domain. The purified RA-domain polypeptide exhibits an ability to bind directly to Ras in a GTP-dependent manner and to competitively inhibit Ras-dependent activation of adenylyl cyclase in vitro, with an affinity comparable with that of the whole LRR domain. The specificity of binding of the RA domain to various Ras effector region mutants is indistinguishable from that of the full-length adenylyl cyclase. The activated RAS2 (RAS2(Val-19))-dependent heat shock sensitivity of yeast cells is suppressed by overexpression of the RA domain polypeptide. Further, mutations of the RA domain abolish its Ras binding activity, and adenylyl cyclase molecules carrying these mutations are rendered unactivatable by Ras in vitro. This RA domain bears highest similarity to the Ras-binding domain of Raf-1 based on comparison of its primary and predicted secondary structures with those of other Ras effectors. These results indicate that the RA domain is a primary Ras-binding site for activation of adenylyl cyclase, implicating RA domains as universal modules for interaction of effectors with Ras, conserved from yeast to mammals.     

Live-cell imaging of endogenous Ras-GTP illustrates predominant Ras activation at the plasma membrane

Ras-GTP imaging studies using the Ras-binding domain (RBD) of the Ras effector c-Raf as a reporter for overexpressed Ras have produced discrepant results about the possible activation of Ras at the Golgi apparatus. We report that RBD oligomerization provides probes for visualization of endogenous Ras-GTP, obviating Ras overexpression and the side effects derived thereof. RBD oligomerization results in tenacious binding to Ras-GTP and interruption of Ras signalling. Trimeric RBD probes fused to green fluorescent protein report agonist-induced endogenous Ras activation at the plasma membrane (PM) of COS-7, PC12 and Jurkat cells, but do not accumulate at the Golgi. PM illumination is exacerbated by Ras overexpression and its sensitivity to dominant-negative RasS17N and pharmacological manipulations matches Ras-GTP formation assessed biochemically. Our data illustrate that endogenous Golgi-located Ras is not under the control of growth factors and argue for the PM as the predominant site of agonist-induced Ras activation.     

Human Sin1 contains Ras-binding and pleckstrin homology domains and suppresses Ras signalling

Human Sin1 (SAPK-interacting protein 1) is a member of a conserved family of orthologous proteins that have an essential role in signal transduction in yeast and Dictyostelium. This study demonstrates that most Sin1 orthologues contain both a Raf-like Ras-binding domain (RBD) and a pleckstrin homology (PH) domain. These domains are functional in the human Sin1 protein, with the PH domain involved in lipid and membrane binding by Sin1, and the RBD able to bind activated H-and K-Ras. Sin1 and Ras co-immunoprecipitated and co-localised, showing that these proteins associate with each other in vivo. Overexpression of Sin1 inhibited the activation of ERK, Akt and JNK signalling pathways by Ras. In contrast, siRNA knockdown of endogenous Sin1 protein expression in HEK293 cells enhanced the activation of ERK1/2 by Ras. These data suggest that Sin1 is a mammalian Ras-inhibitor.     

Cell-free synthesis of the Ras-binding domain of c-Raf-1: binding studies to fluorescently labelled H-ras.

It has previously been shown that the transient kinetics of the interaction between the Ras-binding domain of c-Raf-1 and the proto-oncoprotein Ras can be followed by stopped-flow measurements using the 2',3'-(N-methylanthraniloyl) fluorescence of 2',3'-(N-methylanthraniloyl) guanyl-5'-yl-imidodiphosphate-labelled Ras. In continuation of this work, we demonstrate that the His-tagged Ras-binding domain of c-Raf-1 can also be synthesized in a cell-free expression system. After purification by Ni2+ affinity chromatography, His-tagged Ras-binding domain of c-Raf-1 could be isolated in sufficient amounts for biochemical and biophysical investigations. The results obtained describe the first example of a cell-free synthesized protein which has been used for stopped-flow measurements to determine the transient kinetics of protein-protein interactions with an effector.     

Input from Ras is required for maximal PI(3)K signalling in Drosophila

Class I phosphoinositide 3-kinases (PI(3)Ks) are activated through associated adaptor molecules in response to G protein-coupled and tyrosine kinase receptor signalling. They contain Ras-binding domains (RBDs) and can also be activated through direct association with active GTP-bound Ras. The ability of Ras to activate PI(3)K has been established in vitro and by overexpression analysis, but its relevance for normal PI(3)K function in vivo is unknown. The Drosophila class I PI(3)K, Dp110, is activated by nutrient-responsive insulin signalling and modulates growth, oogenesis and metabolism. To investigate the importance of Ras-mediated PI(3)K activation for normal PI(3)K function, we replaced Dp110 with Dp110(RBD), which is unable to bind to Ras but otherwise biochemically normal. We found that Ras-mediated Dp110 regulation is dispensable for viability. However, egg production, which requires large amounts of growth, is dramatically lowered in Dp110(RBD) flies. Furthermore, insulin cannot maximally activate PI(3)K signalling in Dp110(RBD) imaginal discs and Dp110(RBD) flies are small. Thus, Dp110 integrates inputs from its phosphotyrosine-binding adaptor and Ras to achieve maximal PI(3)K signalling in specific biological situations.     

Double-mutant analysis of the interaction of Ras with the Ras-binding domain of RGL

RalGDS is a guanine nucleotide dissociation stimulator for Ral, and one of its homologues is RGL (RalGDS-like). In this study, the effects of mutations of Ras and the Ras-binding domains (RBDs) of RalGDS and RGL on their binding have been systematically examined. The D33A mutation of Ras reduces the abilities to bind RGL-RBD and RalGDS-RBD. To identify the RGL residue interacting with Asp33 of Ras, double-mutant analyses between Ras and RGL-RBD were conducted. For example, the K685A mutation of RGL-RBD has a much smaller effect on the RGL-RBD binding ability of the D33A mutant than on those of other mutants of Ras. Accordingly, it is indicated that the attractive interaction of Asp33 in Ras with Lys685 in RGL-RBD (Lys816 in RalGDS-RBD) contributes to the Ras.RBD association. This interaction is consistent with the crystal structure of the complex of RalGDS-RBD and the E31K Ras mutant [Huang, L., Hofer, F., Martin, G. S., and Kim, S.-H. (1998) Nat. Struct. Biol. 5, 422-426]. This crystal structure exhibits interactions of the mutation-derived Lys31 side chain with three RalGDS residues. Glu31 of Ras discriminates Ras from a Ras-homologue, Rap1, with Lys31, with respect to RalGDS and RGL binding; the E31K mutation of Ras potentiates the abilities to bind RGL-RBD and RalGDS-RBD. To examine the role of Glu31 of the wild-type Ras in the interaction with RGL and RalGDS, double-mutant analyses were conducted. The Ras binding ability of the E689A mutant of RGL-RBD is much stronger than that of the wild-type RGL-RBD, and the E31K mutation of Ras no longer potentiates the Ras binding ability of the E689A mutant. Therefore, the repulsive interaction between Glu31 in Ras and Glu689 in RGL-RBD (Asp820 in RalGDS-RBD) may keep the Ras.RBD association weaker than the Rap1.RBD association, which might be relevant to the regulation of the signaling network.     

Association of yeast adenylyl cyclase with cyclase-associated protein CAP forms a second Ras-binding site which mediates its Ras-dependent activation

Posttranslational modification, in particular farnesylation, of Ras is crucial for activation of Saccharomyces cerevisiae adenylyl cyclase (CYR1). Based on the previous observation that association of CYR1 with cyclase-associated protein (CAP) is essential for its activation by posttranslationally modified Ras, we postulated that the associated CAP might contribute to the formation of a Ras-binding site of CYR1, which mediates CYR1 activation, other than the primary Ras-binding site, the leucine-rich repeat domain. Here, we observed a posttranslational modification-dependent association of Ras with a complex between CAP and CYR1 C-terminal region. When CAP mutants defective in Ras signaling but retaining the CYR1-binding activity were isolated by screening of a pool of randomly mutagenized CAP, CYR1 complexed with two of the obtained three mutants failed to be activated efficiently by modified Ras and exhibited a severely impaired ability to bind Ras, providing a genetic evidence for the importance of the physical association with Ras at the second Ras-binding site. On the other hand, CYR1, complexed with the other CAP mutant, failed to be activated by Ras but exhibited a greatly enhanced binding to Ras. Conversely, a Ras mutant E31K, which exhibits a greatly enhanced binding to the CYR1-CAP complex, failed to activate CYR1 efficiently. Thus, the strength of interaction at the second Ras-binding site appears to be a critical determinant of CYR1 regulation by Ras: too-weak and too-strong interactions are both detrimental to CYR1 activation. These results, taken together with those obtained with mammalian Raf, suggest the importance of the second Ras-binding site in effector regulation.     

A genome-wide Ras-effector interaction network

Here using structural information and protein design tools we have drawn the network of interactions between 20 Ras subfamily proteins with 50 putative Ras binding domains. To validate this network we have cloned six poorly characterized Ras binding domains (RBD) and two Ras proteins (RERG, DiRas1). These, together with previously described RBD domains, Ras and Rap proteins have been analyzed in 70 pull-down experiments. Comparing our interaction network with these and previous pull-down experiments (total of 150 cases) shows a very high accuracy for distinguishing between binders and non-binders ( approximately 0.80). Bioinformatics information was integrated to distinguish those in vitro interactions that are more likely to be relevant in vivo. We proposed several new interactions between Ras family members and effector domains that are of relevance in understanding the physiological role of these proteins. More broadly our results demonstrate that (domain-domain) interaction specificities between members of protein families can be accurately predicted using structural information.     

Structural and biochemical analysis of Ras-effector signaling via RalGDS.

The structure of the complex of Ras with the Ras-binding domain of its effector RalGDS (RGS-RBD), the first genuine Ras-effector complex, has been solved by X-ray crystallography. As with the Rap-RafRBD complex (Nasser et al., 1995), the interaction is via an inter-protein beta-sheet between the switch I region of Ras and the second strand of the RGS-RBD sheet, but the details of the interactions in the interface are remarkably different. Mutational studies were performed to investigate the contribution of selected interface residues to the binding affinity. Gel filtration experiments show that the Ras x RGS-RBD complex is a monomer. The results are compared to a recently determined structure of a similar complex using a Ras mutant (Huang et al., 1998) and are discussed in relation to partial loss-of-function mutations and the specificity of Ras versus Rap binding.     

Conditional control of chimeric antigen receptor T-cell activity through a destabilizing domain switch and its chemical ligand

Background aimsDespite the impressive efficacy of chimeric antigen receptor (CAR) T-cell therapy, adverse effects, including cytokine release syndrome and neurotoxicity, impede its therapeutic application, thus making the modulation of CAR T-cell activity a priority. The destabilizing domain mutated from Escherichia coli dihydrofolate reductase (DHFR) is inherently unstable and degraded by proteasomes unless it is stabilized by its chemical ligand trimethoprim (TMP), a Food and Drug Administration-approved drug. Here the authors reveal a strategy to modulate CAR T-cell activity at the protein level by employing DHFR and TMP as a chemical switch system.MethodsFirst, the system was demonstrated to work in human primary T cells. To introduce the system to CAR T cells, DHFR was genetically fused to the carboxyl terminal of a third-generation CAR molecule targeting CD19 (CD19-CAR), constructing the CD19-CAR-DHFR fusion.ResultsThe CD19-CAR-DHFR molecule level was shown to be modulated by TMP. Importantly, the incorporation of DHFR had no impact on the recognition specificity and normal function of the CAR molecule. Little adverse effect on cell proliferation and apoptosis was detected. It was proved that TMP could regulate cytokine secretion and the in vitro cytotoxicity of CD19-CAR-DHFR T cells. Furthermore, the in vivo anti-tumor efficacy was demonstrated to be controllable through the manipulation of TMP administration. The approach to control CD19-CAR also succeeded in 19-BBZ(71), another CD19-targeting CAR with a different structure.ConclusionsThe proposed approach based on DHFR and TMP provides a facile strategy to bring CAR T-cell therapy under conditional user control, and the strategy may have the potential to be transplantable.     

Identification of tetratricopeptide repeat 1 as an adaptor protein that interacts with heterotrimeric G proteins and the small GTPase Ras

The biological functions of heterotrimeric G proteins and small GTPases are modulated by both extracellular stimuli and intracellular regulatory proteins. Using Saccharomyces cerevisiae two-hybrid screening, we identified tetratricopeptide repeat 1 (TPR1), a 292-amino-acid protein with three TPR motifs, as a Galpha16-binding protein. The interaction was confirmed both in vitro and in transfected mammalian cells, where TPR1 also binds to several other Galpha proteins. TPR1 was found to interact with Ha-Ras preferentially in its active form. Overexpression of TPR1 promotes accumulation of active Ras. TPR1 was found to compete with the Ras-binding domain (RBD) of Raf-1 for binding to the active Ras, suggesting that it may also compete with Ras GTPase-activating protein, thus contributing to the accumulation of GTP-bound Ras. Expression of Galpha16 strongly enhances the interaction between TPR1 and Ras. Removal of the TPR1 N-terminal 112 residues abolishes potentiation by Galpha16 while maintaining the interaction with Galpha16 and the ability to discriminate active Ras from wild-type Ras. We have also observed that LGN, a Galphai-interacting protein with seven TPR motifs, binds Ha-Ras. Thus, TPR1 is a novel adaptor protein for Ras and selected Galpha proteins that may be involved in protein-protein interaction relating to G-protein signaling.     

Selection of phage-displayed fab antibodies on the active conformation of ras yields a high affinity conformation-specific antibody preventing the binding of c-Raf kinase to Ras

The Ras proteins cycle in the cell between an inactive state and an active state. In the active state, Ras signals via the switch I region to effectors like c-Raf kinase, leading to cell growth. Since Ras mutations in cancer are often associated with the presence of permanently active Ras, molecules that prevent downstream signaling may be of interest. Here, we show that by selection on the active conformation of Ras, using a recently described large phage antibody repertoire [de Haard et al. (1999) J. Biol. Chem. 274, 18218-18230], a Fab antibody (Fab H2) was identified that exclusively binds to active Ras, and not to inactive Ras. Using surface plasmon resonance (SPR) analysis, the interaction was demonstrated to be of high affinity (7.2 nM). In addition, the interaction with Ras is specific, since binding to the homologous Rap1A protein in BIAcore analysis is at least three orders of magnitude lower, and undetectable in an enzyme-linked immunosorbent assay. The antibody fragment prevents the binding of active Ras to the immobilized Ras-binding domain of c-Raf kinase (Raf-RBD) at an IC(50) value of 135 nM. This value compares well to the K(D) of active Ras-binding to immobilized Raf-RBD using SPR, suggesting identical binding sites. Like the IgG Y13-259, which does not demonstrate preferential binding to either inactive or active Ras, Fab H2 inhibits intrinsic GTPase activity of Ras in vitro. Mapping studies using SPR analysis demonstrate that the binding sites for the antibodies are non-identical. This antibody could be used for dissecting functional differences between Ras effectors. Due to its specificity for active Ras, Fab H2 may well be more selective than previously used anti-Ras antibodies, and thus could be used for gene therapy of cancer with intracellular antibodies.     

Phospholipase C(epsilon): a novel Ras effector

Three classes of mammalian phosphoinositide-specific phospholipase C (PLC) have been characterized, PLCbeta, PLCgamma and PLCdelta, that are differentially regulated by heterotrimeric G-proteins, tyrosine kinases and calcium. Here we describe a fourth class, PLCepsilon, that in addition to conserved PLC domains, contains a GTP exchange factor (GRF CDC25) domain and two C-terminal Ras-binding (RA) domains, RA1 and RA2. The RA2 domain binds H-Ras in a GTP-dependent manner, comparable with the Ras-binding domain of Raf-1; however, the RA1 domain binds H-Ras with a low affinity in a GTP-independent manner. While G(alpha)q, Gbetagamma or, surprisingly, H-Ras do not activate recombinant purified protein in vitro, constitutively active Q61L H-Ras stimulates PLC(epsilon) co-expressed in COS-7 cells in parallel with Ras binding. Deletion of either the RA1 or RA2 domain inhibits this activation. Site-directed mutagenesis of the RA2 domain or Ras demonstrates a conserved Ras-effector interaction and a unique profile of activation by Ras effector domain mutants. These studies identify a novel fourth class of mammalian PLC that is directly regulated by Ras and links two critical signaling pathways.     

Crystallographic analysis reveals a novel second binding site for trimethoprim in active site double mutants of human dihydrofolate reductase

In order to produce a more potent replacement for trimethoprim (TMP) used as a therapy for Pneumocystis pneumonia and targets dihydrofolate reductase from Pneumocystis jirovecii (pjDHFR), it is necessary to understand the determinants of potency and selectivity against DHFR from the mammalian host and fungal pathogen cells. To this end, active site residues in human (h) DHFR were replaced with those from pjDHFR. Structural data are reported for two complexes of TMP with the double mutants Gln35Ser/Asn64Phe (Q35S/N64F) and Gln35Lys/Asn64Phe (Q35K/N64F) of hDHFR that unexpectedly show evidence for the binding of two molecules of TMP: one molecule that binds in the normal folate binding site and the second molecule that binds in a novel subpocket site such that the mutated residue Phe64 is involved in van der Waals contacts to the trimethoxyphenyl ring of the second TMP molecule. Kinetic data for the binding of TMP to hDHFR and pjDHFR reveal an 84-fold selectivity of TMP against pjDHFR (K_i 49 nM) compared to hDHFR (K_i 4093 nM). Two mutants that contain one substitution from pj- and one from the closely related Pneumocystis carinii DHFR (pcDHFR) (Q35K/N64F and Q35S/N64F) show K_i values of 593 and 617 nM, respectively; these K_i values are well above both the K_i for pjDHFR and are similar to pcDHFR (Q35K/N64F and Q35S/N64F) (305 nM). These results suggest that active site residues 35 and 64 play key roles in determining selectivity for pneumocystis DHFR, but that other residues contribute to the unique binding of inhibitors to these enzymes.     

PIRLs: a novel class of plant intracellular leucine-rich repeat proteins

Leucine-rich repeat (LRR) proteins feature tandem leucine-rich motifs that form a protein-protein interaction domain. Plants contain diverse classes of LRR proteins, many of which take part in signal transduction. We have identified a novel family of nine Arabidopsis LRR proteins that, based on predicted intracellular location and LRR motif consensus sequence, are related to Ras-binding LRR proteins found in signaling complexes in animals and yeast. This new class has been named plant intracellular Ras group-related LRR proteins (PIRLs). We have characterized PIRL cDNAs, rigorously defined gene and protein annotations, investigated gene family evolution and surveyed mRNA expression. While LRR regions suggested a relationship to Ras group LRR proteins, outside of their LRR domains PIRLs differed from Ras group proteins, exhibiting N- and C-terminal regions containing low complexity stretches and clusters of charged amino acids. PIRL genes grouped into three subfamilies based on sequence relationships and gene structures. Related gene pairs and dispersed chromosomal locations suggested family expansion by ancestral genomic or segmental duplications. Expression surveys revealed that all PIRL mRNAs are actively transcribed, with three expressed differentially in leaves, roots or flowers. These results define PIRLs as a distinct, plant-specific class of intracellular LRR proteins that probably mediate protein interactions, possibly in the context of signal transduction. T-DNA knock-out mutants have been isolated as a starting point for systematic functional analysis of this intriguing family.     

Chemoattractant-induced Ras activation during Dictyostelium aggregation

Ras proteins are highly conserved molecular switches that regulate cellular response to external stimuli. Dictyostelium discoideum contains an extensive family of Ras proteins that function in regulation of mitosis, cytoskeletal function and motility, and the onset of development. Little is known about the events that lead to the activation of Ras proteins in Dictyostelium, primarily owing to a lack of a biochemical assay to measure the levels of activated Ras. We have adapted an assay, used successfully to measure activated Ras in mammalian cells, to monitor activation of two Dictyostelium Ras proteins, RasC and RasG. We have found that the Ras-binding domain (RBD) of mammalian Raf1 was capable of binding to the activated form of RasG, but not to the activated form of RasC; however, the RBD of Schizosaccharomyces pombe Byr2 was capable of binding preferentially to the activated forms of both RasC and RasG. Using this assay, we discovered that RasC and RasG showed a rapid and transient activation when aggregation-competent cells were stimulated with the chemoattractant cAMP, and this activation did not occur in a number of cAMP signalling mutants. These data provide further evidence of a role for both RasC and RasG in the early development of Dictyostelium.