ABSTRACT
RAP1 proteins belong to the RAS family of small GTPases that operate as molecular switches by cycling between GDP-bound inactive and GTP-bound active states. The C-terminal anchors of RAP1 proteins are known to direct membrane localization, but how these anchors organize RAP1 on the plasma membrane (PM) has not been investigated. Using high-resolution imaging, we show that RAP1A and RAP1B form spatially segregated nanoclusters on the inner leaflet of the PM, with further lateral segregation between GDP-bound and GTP-bound proteins. The C-terminal polybasic anchors of RAP1A and RAP1B differ in their amino acid sequences and exhibit different lipid binding specificities, which can be modified by single-point mutations in the respective polybasic domains (PBD). Molecular dynamics simulations reveal that single PBD mutations substantially reduce the interactions of the membrane anchors with the PM lipid phosphatidylserine. In summary, we show that aggregate lipid binding specificity encoded within the C-terminal anchor determines PM association and nanoclustering of RAP1A and RAP1B. Taken together with previous observations on RAC1 and KRAS, the study reveals that the PBD sequences of small GTPase membrane anchors can encode distinct lipid binding specificities that govern PM interactions.
Subject(s)
Amino Acid Sequence , Cell Membrane , Molecular Dynamics Simulation , rap GTP-Binding Proteins , rap GTP-Binding Proteins/metabolism , rap GTP-Binding Proteins/chemistry , rap GTP-Binding Proteins/genetics , Cell Membrane/metabolism , Cell Membrane/chemistry , Humans , rap1 GTP-Binding Proteins/metabolism , rap1 GTP-Binding Proteins/chemistry , rap1 GTP-Binding Proteins/genetics , Protein Binding , Phosphatidylserines/chemistry , Phosphatidylserines/metabolism , Binding SitesABSTRACT
Geranylgeranyltransferase type-I (GGTase-I) represents an important drug target since it contributes to the function of many proteins that are involved in tumor development and metastasis. This led to the development of GGTase-I inhibitors as anti-cancer drugs blocking the protein function and membrane association of e.g., Rap subfamilies that are involved in cell differentiation and cell growth. In the present study, we developed a new NanoBiT assay to monitor the interaction of human GGTase-I and its substrate Rap1B. Different Rap1B prenylation-deficient mutants (C181G, C181S, and ΔCQLL) were designed and investigated for their interaction with GGTase-I. While the Rap1B mutants C181G and C181S still exhibited interaction with human GGTase-I, mutant ΔCQLL, lacking the entire CAAX motif (defined by a cysteine residue, two aliphatic residues, and the C-terminal residue), showed reduced interaction. Moreover, a specific, peptidomimetic and competitive CAAX inhibitor was able to block the interaction of Rap1B with GGTase-I. Furthermore, activation of both Gαs-coupled human adenosine receptors, A2A (A2AAR) and A2B (A2BAR), increased the interaction between GGTase-I and Rap1B, probably representing a way to modulate prenylation and function of Rap1B. Thus, A2AAR and A2BAR antagonists might be promising candidates for therapeutic intervention for different types of cancer that overexpress Rap1B. Finally, the NanoBiT assay provides a tool to investigate the pharmacology of GGTase-I inhibitors.
Subject(s)
Alkyl and Aryl Transferases/metabolism , Enzyme Inhibitors/pharmacology , Peptide Fragments/pharmacology , Protein Interaction Domains and Motifs/drug effects , rap GTP-Binding Proteins/metabolism , Adenosine A2 Receptor Antagonists/pharmacology , Alkyl and Aryl Transferases/chemistry , Alkyl and Aryl Transferases/genetics , Humans , Protein Prenylation , Substrate Specificity , Xanthines/pharmacology , rap GTP-Binding Proteins/chemistry , rap GTP-Binding Proteins/geneticsABSTRACT
During development, neurons form synapses with their fate-determined targets. While we begin to elucidate the mechanisms by which extracellular ligand-receptor interactions enhance synapse specificity by inhibiting synaptogenesis, our knowledge about their intracellular mechanisms remains limited. Here we show that Rap2 GTPase (rap-2) and its effector, TNIK (mig-15), act genetically downstream of Plexin (plx-1) to restrict presynaptic assembly and to form tiled synaptic innervation in C. elegans. Both constitutively GTP- and GDP-forms of rap-2 mutants exhibit synaptic tiling defects as plx-1 mutants, suggesting that cycling of the RAP-2 nucleotide state is critical for synapse inhibition. Consistently, PLX-1 suppresses local RAP-2 activity. Excessive ectopic synapse formation in mig-15 mutants causes a severe synaptic tiling defect. Conversely, overexpression of mig-15 strongly inhibited synapse formation, suggesting that mig-15 is a negative regulator of synapse formation. These results reveal that subcellular regulation of small GTPase activity by Plexin shapes proper synapse patterning in vivo.
Subject(s)
Caenorhabditis elegans Proteins/chemistry , Nerve Tissue Proteins/chemistry , Protein Serine-Threonine Kinases/chemistry , Receptors, Cell Surface/chemistry , rap GTP-Binding Proteins/chemistry , Animals , Caenorhabditis elegans/chemistry , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/genetics , Guanosine Diphosphate/chemistry , Guanosine Triphosphate/chemistry , Mutation , Nerve Tissue Proteins/genetics , Neurogenesis/genetics , Neurons/chemistry , Protein Serine-Threonine Kinases/genetics , Receptors, Cell Surface/genetics , Signal Transduction/genetics , Synapses/chemistry , Synapses/genetics , Synapses/pathology , rap GTP-Binding Proteins/geneticsABSTRACT
Rap1 proteins are members of the Ras subfamily of small GTPases involved in many biological responses, including adhesion, cell proliferation, and differentiation. Like all small GTPases, they work as molecular allosteric units that are active in signaling only when associated with the proper membrane compartment. Prenylation, occurring in the cytosol, is an enzymatic posttranslational event that anchors small GTPases at the membrane, and prenyl-binding proteins are needed to mask the cytoplasm-exposed lipid during transit to the target membrane. However, several of these proteins still await discovery. In this study, we report that cyclase-associated protein 1 (CAP1) binds Rap1. We found that this binding is GTP-independent, does not involve Rap1's effector domain, and is fully contained in its C-terminal hypervariable region (HVR). Furthermore, Rap1 prenylation was required for high-affinity interactions with CAP1 in a geranylgeranyl-specific manner. The prenyl binding specifically involved CAP1's C-terminal hydrophobic ß-sheet domain. We present a combination of experimental and computational approaches, yielding a model whereby the high-affinity binding between Rap1 and CAP1 involves electrostatic and nonpolar side-chain interactions between Rap1's HVR residues, lipid, and CAP1 ß-sheet domain. The binding was stabilized by the lipid insertion into the ß-solenoid whose interior was occupied by nonpolar side chains. This model was reminiscent of the recently solved structure of the PDEδ-K-Ras complex; accordingly, disruptors of this complex, e.g. deltarasin, blocked the Rap1-CAP1 interaction. These findings indicate that CAP1 is a geranylgeranyl-binding partner of Rap1.
Subject(s)
Cell Cycle Proteins/metabolism , Cytoskeletal Proteins/metabolism , Diterpenes/metabolism , Protein Prenylation , Thyroid Epithelial Cells/metabolism , rap GTP-Binding Proteins/metabolism , Animals , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Cells, Cultured , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/genetics , Diterpenes/chemistry , Humans , Models, Molecular , Molecular Dynamics Simulation , Protein Conformation , Rats , rap GTP-Binding Proteins/chemistry , rap GTP-Binding Proteins/geneticsABSTRACT
Activation of transmembrane receptor integrin by talin is essential for inducing cell adhesion. However, the pathway that recruits talin to the membrane, which critically controls talin's action, remains elusive. Membrane-anchored mammalian small GTPase Rap1 is known to bind talin-F0 domain but the binding was shown to be weak and thus hardly studied. Here we show structurally that talin-F0 binds to human Rap1b like canonical Rap1 effectors despite little sequence homology, and disruption of the binding strongly impairs integrin activation, cell adhesion, and cell spreading. Furthermore, while being weak in conventional binary binding conditions, the Rap1b/talin interaction becomes strong upon attachment of activated Rap1b to vesicular membranes that mimic the agonist-induced microenvironment. These data identify a crucial Rap1-mediated membrane-targeting mechanism for talin to activate integrin. They further broadly caution the analyses of weak protein-protein interactions that may be pivotal for function but neglected in the absence of specific cellular microenvironments.
Subject(s)
Integrins/metabolism , Talin/chemistry , Talin/metabolism , rap GTP-Binding Proteins/chemistry , rap GTP-Binding Proteins/metabolism , Amino Acid Sequence , Animals , Cell Adhesion/physiology , Cell Line , Cell Membrane/metabolism , Guanosine Triphosphate/metabolism , Humans , Mice , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Talin/geneticsABSTRACT
Macropinocytosis involves the uptake of large volumes of fluid, which is regulated by various small GTPases. The Dictyostelium discoideum protein GflB is a guanine nucleotide exchange factor (GEF) of Rap1, and is involved in chemotaxis. Here, we studied the role of GflB in macropinocytosis, phagocytosis and cytokinesis. In plate culture of vegetative cells, compared with the parental strain AX2, gflB-knockout (KO) cells were flatter and more polarized, whereas GflB-overproducing cells were rounder. The gflB-KO cells exhibited impaired crown formation and retraction, particularly retraction, resulting in more crowns (macropinocytic cups) per cell and longer crown lifetimes. Accordingly, gflB-KO cells showed defects in macropinocytosis and also in phagocytosis and cytokinesis. F-actin levels were elevated in gflB-KO cells. GflB localized to the actin cortex most prominently at crowns and phagocytic cups. The villin headpiece domain (VHP)-like N-terminal domain of GflB directly interacted with F-actin in vitro Furthermore, a domain enriched in basic amino acids interacted with specific membrane cortex structures such as the cleavage furrow. In conclusion, GflB acts as a key local regulator of actin-driven membrane protrusion possibly by modulating Rap1 signaling pathways.
Subject(s)
Dictyostelium/cytology , Dictyostelium/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Pinocytosis , Protozoan Proteins/metabolism , rap GTP-Binding Proteins/metabolism , Actin Cytoskeleton/metabolism , Actins/metabolism , Amino Acid Sequence , Cell Movement , Cell Shape , Cell Surface Extensions/metabolism , Cytokinesis , Gene Knockout Techniques , Green Fluorescent Proteins/metabolism , Guanine Nucleotide Exchange Factors/chemistry , Morphogenesis , Phagocytosis , Protein Binding , Protein Domains , Protozoan Proteins/chemistry , Subcellular Fractions/metabolism , rap GTP-Binding Proteins/chemistryABSTRACT
Two isoforms of the small GTPase Rap1, Rap1A and Rap1B, participate in cell adhesion; Rap1A promotes steady state adhesion, while Rap1B regulates dynamic changes in cell adhesion. These events depend on the prenylation of Rap1, which promotes its membrane localization. Here, we identify previously unsuspected differences in the regulation of prenylation of Rap1A versus Rap1B, due in part to their different phosphorylation-dependent interactions with the chaperone protein SmgGDS-607. Previous studies indicate that the activation of Gαs protein-coupled receptors (GPCRs) phosphorylates S-179 and S-180 in the polybasic region (PBR) of Rap1B, which inhibits Rap1B binding to SmgGDS-607 and diminishes Rap1B prenylation and membrane localization. In this study, we investigate how phosphorylation in the PBR of multiple small GTPases, including K-Ras4B, RhoA, Rap1A, and Rap1B, affects their binding to SmgGDS, with emphasis on differences between Rap1A and Rap1B. We identify the amino acids in SmgGDS-607 necessary for binding of Rap1A and Rap1B, and present homology models examining the binding between Rap1A or Rap1B and SmgGDS-607. Unlike Rap1B, phosphorylation in the PBR of Rap1A does not detectably inhibit its prenylation or its binding to SmgGDS-607. Activation of GPCRs suppresses Rap1A prenylation, but unlike this effect on Rap1B, the GPCR-mediated suppression of Rap1A prenylation can occur independently of Rap1A phosphorylation and does not detectably diminish Rap1A membrane localization. These data demonstrate unexpected evolutionarily conserved differences in the ability of GPCRs to regulate the prenylation of Rap1B compared to Rap1A.
Subject(s)
Guanine Nucleotide Exchange Factors/metabolism , Prenylation , Protein Processing, Post-Translational , rap GTP-Binding Proteins/metabolism , rap1 GTP-Binding Proteins/metabolism , Amino Acid Sequence , Cell Line , Humans , Models, Molecular , Molecular Sequence Data , Phosphorylation , Protein Binding , Protein Conformation , Protein Interaction Mapping , Sequence Alignment , rap GTP-Binding Proteins/chemistry , rap1 GTP-Binding Proteins/chemistryABSTRACT
Rap2B GTPase, a member of Ras-related protein superfamily, was first discovered from a platelet cDNA library in the early 1990s. Since then, it has been reported to play an important role in regulating cellular processes including cytoskeletal organization, cell growth, and proliferation. It can be stimulated and suppressed by a wide range of external and internal inducers, circulating between GTP-bound active state and GDP-bound inactive state. Increasing focus on Ras signaling pathway reveals critical effects of Rap2B on tumorigenesis. In particular, Rap2B behaves in a p53-dependent manner in regulation of apoptosis and migration. Apart from being an oncogenic activator, Rap2B has been found to participate in many other physiological events via diverse downstream effectors. In this review, we present recent studies on the structure, regulation, and multiple biological functions of Rap2B, shedding light on its potential status in treatment of cancer as well as other diseases.
Subject(s)
Gene Expression Regulation, Neoplastic , Neoplasms/pathology , rap GTP-Binding Proteins/chemistry , rap GTP-Binding Proteins/metabolism , Animals , Humans , Neoplasms/genetics , Neoplasms/metabolism , Signal Transduction , rap GTP-Binding Proteins/geneticsABSTRACT
Rap1B is a small GTPase involved in the regulation of numerous cellular processes including synaptic plasticity, one of the bases of memory. Like other members of the Ras family, the active GTP-bound form of Rap1B can bind to a large number of effector proteins and so transmit signals to downstream components of the signaling pathways. The structure of Rap1B bound only to a nucleotide has yet to be solved, but might help reveal an inactive conformation that can be stabilized by a small molecule drug. Unlike other Ras family proteins such as H-Ras and Rap2A, Rap1B crystallizes in an intermediate state when bound to a non-hydrolyzable GTP analog. Comparison with H-Ras and Rap2A reveals conservative mutations relative to Rap1B, distant from the bound nucleotide, which control how readily the protein may adopt the fully activated form in the presence of GTP. High resolution crystallographic structures of mutant proteins show how these changes may influence the hydrogen bonding patterns of the key switch residues.
Subject(s)
Mutation , Protein Structure, Tertiary , rap GTP-Binding Proteins/chemistry , rap GTP-Binding Proteins/genetics , Amino Acid Sequence , Animals , Binding Sites/genetics , Crystallography, X-Ray , Guanosine Diphosphate/chemistry , Guanosine Diphosphate/metabolism , Models, Molecular , Molecular Sequence Data , Protein Binding , Rats , Sequence Homology, Amino Acid , rap GTP-Binding Proteins/metabolismABSTRACT
Plexins are cell surface receptors that bind semaphorins and transduce signals for regulating neuronal axon guidance and other processes. Plexin signaling depends on their cytoplasmic GTPase activating protein (GAP) domain, which specifically inactivates the Ras homolog Rap through an ill-defined non-canonical catalytic mechanism. The plexin GAP is activated by semaphorin-induced dimerization, the structural basis for which remained unknown. Here we present the crystal structures of the active dimer of zebrafish PlexinC1 cytoplasmic region in the apo state and in complex with Rap. The structures show that the dimerization induces a large-scale conformational change in plexin, which opens the GAP active site to allow Rap binding. Plexin stabilizes the switch II region of Rap in an unprecedented conformation, bringing Gln63 in Rap into the active site for catalyzing GTP hydrolysis. The structures also explain the unique Rap-specificity of plexins. Mutational analyses support that these mechanisms underlie plexin activation and signaling. DOI:http://dx.doi.org/10.7554/eLife.01279.001.
Subject(s)
Cell Adhesion Molecules/chemistry , Guanosine Triphosphate/chemistry , Nerve Tissue Proteins/chemistry , Zebrafish Proteins/chemistry , Zebrafish/genetics , rap GTP-Binding Proteins/chemistry , Amino Acid Sequence , Animals , Catalytic Domain , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/metabolism , Crystallography, X-Ray , Gene Expression Regulation , Guanosine Triphosphate/metabolism , Humans , Mice , Models, Molecular , Molecular Sequence Data , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Protein Binding , Protein Multimerization , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Alignment , Sequence Homology, Amino Acid , Signal Transduction , Zebrafish/metabolism , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism , rap GTP-Binding Proteins/genetics , rap GTP-Binding Proteins/metabolismABSTRACT
Continuum electrostatics methods are commonly used to calculate electrostatic potentials in proteins and at protein-protein interfaces to aid many types of biophysical studies. Despite their ubiquity throughout the biophysical literature, these calculations are difficult to test against experimental data to determine their accuracy and validity. To address this, we have calculated the Boltzmann-weighted electrostatic field at the midpoint of a nitrile bond placed at a variety of locations on the surface of the protein RalGDS, both in its monomeric form as well as when docked to four different constructs of the protein Rap, and compared the computation results to vibrational absorption energy measurements of the nitrile oscillator. This was done by generating a statistical ensemble of protein structures using enhanced molecular dynamics sampling with the Amber03 force field, followed by solving the linear Poisson-Boltzmann equation for each structure using the Applied Poisson-Boltzmann Solver (APBS) software package. Using a two-stage focusing strategy, we examined numerous second stage box dimensions, grid point densities, box locations, and compared the numerical result to the result obtained from the sum of the numeric reaction field and the analytic Coulomb field. It was found that the reaction field method yielded higher correlation with experiment for the absolute calculation of fields, while the numeric solutions yielded higher correlation with experiment for the relative field calculations. Finer grid spacing typically improved the calculation, although this effect was less pronounced in the reaction field method. These sorts of calculations were also very sensitive to the box location, particularly for the numeric calculations of absolute fields using a 10(3) Å(3) box.
Subject(s)
Models, Molecular , Static Electricity , ral Guanine Nucleotide Exchange Factor/chemistry , rap GTP-Binding Proteins/chemistry , Algorithms , Linear Models , Molecular Docking Simulation , Molecular Dynamics Simulation , Nitriles/chemistry , Probability , Protein Conformation , Software , Thiocyanates/chemistry , VibrationABSTRACT
The principle virulence factors in Clostridium difficile pathogenesis are TcdA and TcdB, homologous glucosyltransferases capable of inactivating small GTPases within the host cell. We present crystal structures of the TcdA glucosyltransferase domain in the presence and absence of the co-substrate UDP-glucose. Although the enzymatic core is similar to that of TcdB, the proposed GTPase-binding surface differs significantly. We show that TcdA is comparable with TcdB in its modification of Rho family substrates and that, unlike TcdB, TcdA is also capable of modifying Rap family GTPases both in vitro and in cells. The glucosyltransferase activities of both toxins are reduced in the context of the holotoxin but can be restored with autoproteolytic activation and glucosyltransferase domain release. These studies highlight the importance of cellular activation in determining the array of substrates available to the toxins once delivered into the cell.
Subject(s)
Bacterial Toxins/chemistry , Clostridioides difficile/enzymology , Enterotoxins/chemistry , Glucosyltransferases/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Bacterial Toxins/metabolism , Enterotoxins/metabolism , Enzyme Activation/physiology , Glucosyltransferases/metabolism , Protein Structure, Tertiary , Uridine Diphosphate Glucose/chemistry , Uridine Diphosphate Glucose/metabolism , rap GTP-Binding Proteins/chemistry , rap GTP-Binding Proteins/metabolismABSTRACT
Signaling by the small G-protein Rap is under tight regulation by its GEFs and GAPs. These are multi-domain proteins that are themselves controlled by distinct upstream pathways, and thus couple different extra- and intracellular cues to Rap. The individual RapGEFs and RapGAPs are, in addition, targeted to specific cellular locations by numerous anchoring mechanisms and, consequently, may control different pools of Rap. Here, we review the various activating signals and targeting mechanisms of these proteins and discuss their contribution to the spatiotemporal regulation and biological functions of the Rap proteins.
Subject(s)
Cyclic AMP/metabolism , GTPase-Activating Proteins/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Models, Molecular , Monomeric GTP-Binding Proteins/metabolism , rap GTP-Binding Proteins/metabolism , Cyclic AMP/chemistry , GTPase-Activating Proteins/chemistry , Guanine Nucleotide Exchange Factors/chemistry , Monomeric GTP-Binding Proteins/chemistry , Signal Transduction , rap GTP-Binding Proteins/chemistry , rap1 GTP-Binding Proteins/chemistry , rap1 GTP-Binding Proteins/metabolismABSTRACT
RUN domain is present in several proteins related to the functions of Rap and Rab family GTPases. Accumulating evidence supports the hypothesis that RUN domain-containing proteins act as a component of vesicle traffic and might be responsible for an interaction with a filamentous network linked to actin cytoskeleton or microtubules. That is to say, on one hand, RUN domains associate with Rab or Rap family proteins, on the other hand, they also might interact with motor proteins such as kinesin or myosin via intervention molecules. In this review, we summarize the background and current status of RUN domain research with an emphasis on the interaction between RUN domain and motor proteins with respect to the vesicle traffic on filamentous network.
Subject(s)
Molecular Motor Proteins/metabolism , rab GTP-Binding Proteins/physiology , rap GTP-Binding Proteins/physiology , Biological Transport , Protein Structure, Tertiary , rab GTP-Binding Proteins/chemistry , rap GTP-Binding Proteins/chemistryABSTRACT
We have recently shown that ADP-induced activation of protein kinase C (PKC) requires the co-stimulation of both P2Y1 and P2Y12 receptors. In this work, we show that inhibition of ADP-mediated phosphorylation of pleckstrin, the main PKC substrate, caused by antagonists of the P2Y12 receptor can be reversed by stimulation of the α2-adrenergic receptor by epinephrine. However, we also observed that addition of epinephrine alone caused a marked phosphorylation of pleckstrin. This effect occurred in the absence of Gq stimulation, as it was not associated to intracellular Ca2+ release. Epinephrine-induced pleckstrin phosphorylation was time- and dose-dependent, and was inhibited by the α2-adrenergic antagonist yohimbin. Phosphorylation of pleckstrin did not occur when platelet stimulation with epinephrine was performed in the presence of the ADP scavenger apyrase, and was suppressed by antagonists of both P2Y1 and P2Y12 ADP receptors. Importantly, no release of dense granules was measured in epinephrine-treated platelets. Addition of epinephrine to platelets was also able to stimulate Rap1b activation. Similarly to pleckstrin phosphorylation, however, this effect was prevented in the presence of apyrase or upon pharmacologic blockade of either P2Y1 or P2Y12 receptors. These results indicate that sub-threshold amounts of ADP in the medium are essential to allow epinephrine stimulation of α2-adrenergic receptor to elicit platelet responses, and reveal a novel synergism among strong stimulation of Gz and sub-threshold stimulation of both Gq and Gi, able to dissociate PKC activation from intracellular Ca2+ mobilisation.
Subject(s)
Epinephrine/chemistry , Receptors, Purinergic P2Y12/metabolism , Receptors, Purinergic P2Y1/metabolism , Blood Proteins/chemistry , Calcium/chemistry , Cytosol/metabolism , Dose-Response Relationship, Drug , GTP-Binding Protein alpha Subunits/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/chemistry , GTP-Binding Protein alpha Subunits, Gq-G11/chemistry , Humans , Phosphoproteins/chemistry , Phosphorylation , Signal Transduction , Yohimbine/pharmacology , rap GTP-Binding Proteins/chemistryABSTRACT
The molecular mechanism by which dual-specificity RasGAPs of the Gap1 subfamily activate the GTP hydrolysis of both Rap and Ras is an unresolved phenomenon. RasGAPs and RapGAPs use different strategies to stimulate the GTPase reaction of their cognate G-proteins. RasGAPs contribute an arginine finger to orient through the Gln61 of Ras the nucleophilic water molecule. RapGAP contributes an asparagine (Asn thumb) into the active site to substitute for the missing Gln61. Here, by using steady-state kinetic assays and time-resolved Fourier-transform infrared spectroscopy (FTIR) experiments with wild type and mutant proteins, we unravel the remarkable mechanism for the specificity switch. The plasticity of GAP1(IP4BP) and RASAL is mediated by the extra GTPase-activating protein (GAP) domains, which promote a different orientation of Ras and Rap's switch-II and catalytic residues in the active site. Thereby, Gln63 in Rap adopts the catalytic role normally taken by Gln61 of Ras. This re-orientation requires specific interactions between switch-II of Rap and helix-alpha6 of GAPs. This supports the notion that the specificities of fl proteins versus GAP domains are potentially different.
Subject(s)
rap GTP-Binding Proteins/metabolism , ras GTPase-Activating Proteins/metabolism , Amino Acid Sequence , GTPase-Activating Proteins/metabolism , Guanosine Triphosphate/metabolism , Humans , Models, Molecular , Molecular Sequence Data , Mutation , Protein Binding , Protein Structure, Tertiary , Receptors, Cytoplasmic and Nuclear/metabolism , Spectroscopy, Fourier Transform Infrared , rap GTP-Binding Proteins/chemistry , rap GTP-Binding Proteins/genetics , ras GTPase-Activating Proteins/chemistry , ras GTPase-Activating Proteins/genetics , ras Proteins/metabolismABSTRACT
RhoA and RhoC are highly related Rho GTPases, but differentially control cellular behaviour. We combined molecular, cellular, and biochemical experiments to characterise differences between these highly similar GTPases. Our findings demonstrate that enhanced expression of RhoC results in a striking increase in the migration and invasion of pancreatic carcinoma cells, whereas forced expression of RhoA decreases these actions. These isoform-specific functions correlate with differences in the cellular activity of RhoA and RhoC in human cells, with RhoC being more active than RhoA in activity assays and serum-response factor-dependent gene transcription. Subcellular localisation studies revealed that RhoC is predominantly localised in the membrane-containing fraction, whereas RhoA is mainly localised in the cytoplasmic fraction. These differences are not mediated by a different interaction with RhoGDIs. In vitro GTP/GDP binding analyses demonstrate different affinity of RhoC for GTP[S] and faster intrinsic and guanine nucleotide exchange factor (GEF)-stimulated GDP/GTP exchange rates compared to RhoA. Moreover, the catalytic domains of SopE and Dbs are efficacious GEFs for RhoC. mRNA expression of RhoC is markedly enhanced in advanced pancreatic cancer stages, and thus the differences discovered between RhoA and RhoC might provide explanations for their different influences on cell migration and tumour invasion.
Subject(s)
Cell Movement , Neoplasm Invasiveness , Pancreatic Neoplasms/pathology , rho GTP-Binding Proteins/metabolism , rhoA GTP-Binding Protein/metabolism , Cell Line, Tumor , Deep Brain Stimulation , Humans , Pancreatic Neoplasms/metabolism , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , rap GTP-Binding Proteins/chemistry , rap GTP-Binding Proteins/genetics , rap GTP-Binding Proteins/metabolism , rho GTP-Binding Proteins/chemistry , rho GTP-Binding Proteins/genetics , rhoA GTP-Binding Protein/chemistry , rhoA GTP-Binding Protein/genetics , rhoC GTP-Binding ProteinABSTRACT
Rap1b has been implicated in the transduction of the cAMP mitogenic response. Agonists that increase intracellular cAMP rapidly activate (i.e. GTP binding) and phosphorylate Rap1b on Ser(179) at its C terminus. cAMP-dependent protein kinase (PKA)-mediated phosphorylation of Rap1b is required for cAMP-dependent mitogenesis, tumorigenesis, and inhibition of AKT activity. However, the role of phosphorylation still remains unknown. In this study, we utilized amide hydrogen/deuterium exchange mass spectroscopy (DXMS) to assess potential conformational changes and/or mobility induced by phosphorylation. We report here DXMS data comparing exchange rates for PKA-phosphorylated (Rap1-P) and S179D phosphomimetic (Rap1-D) Rap1b proteins. Rap1-P and Rap1-D behaved exactly the same, revealing an increased exchange rate in discrete regions along the protein; these regions include a domain around the phosphorylation site and unexpectedly the two switch loops. Thus, local effects induced by Ser(179) phosphorylation communicate allosterically with distal domains involved in effector interaction. These results provide a mechanistic explanation for the differential effects of Rap1 phosphorylation by PKA on effector protein interaction.
Subject(s)
rap GTP-Binding Proteins/chemistry , rap GTP-Binding Proteins/metabolism , Allosteric Regulation , Amino Acid Sequence , Cell Line , Deuterium Exchange Measurement , Humans , Mass Spectrometry , Models, Molecular , Phosphorylation , Protein Conformation , Protein Structure, TertiaryABSTRACT
Epac proteins are activated by binding of the second messenger cAMP and then act as guanine nucleotide exchange factors for Rap proteins. The Epac proteins are involved in the regulation of cell adhesion and insulin secretion. Here we have determined the structure of Epac2 in complex with a cAMP analogue (Sp-cAMPS) and RAP1B by X-ray crystallography and single particle electron microscopy. The structure represents the cAMP activated state of the Epac2 protein with the RAP1B protein trapped in the course of the exchange reaction. Comparison with the inactive conformation reveals that cAMP binding causes conformational changes that allow the cyclic nucleotide binding domain to swing from a position blocking the Rap binding site towards a docking site at the Ras exchange motif domain.
Subject(s)
Carrier Proteins/chemistry , Carrier Proteins/metabolism , Cyclic AMP/analogs & derivatives , Guanine Nucleotide Exchange Factors/chemistry , Guanine Nucleotide Exchange Factors/metabolism , Thionucleotides/chemistry , Thionucleotides/metabolism , rap GTP-Binding Proteins/metabolism , Amino Acid Motifs , Animals , Binding Sites , Carrier Proteins/ultrastructure , Crystallography, X-Ray , Cyclic AMP/chemistry , Cyclic AMP/metabolism , Enzyme Activation , Guanine Nucleotide Exchange Factors/ultrastructure , Humans , Mice , Microscopy, Electron , Models, Molecular , Protein Binding , Protein Conformation , rap GTP-Binding Proteins/chemistry , rap GTP-Binding Proteins/ultrastructureABSTRACT
Rap1b and Rap2b are the only members of the Rap family of GTPases expressed in circulating human platelets. Rap1b is involved in the inside-out activation of integrins, while the role of Rap2b is still poorly understood. In this work, we investigated the localization of Rap proteins to specific microdomains of plasma membrane called lipid rafts, implicated in signal transduction. We found that Rap1b was not associated to lipid rafts in resting platelets, and did not translocate to these microdomains in stimulated cells. By contrast, about 20% of Rap2b constitutively associated to lipid rafts, and this percentage did not increase upon platelet stimulation. Rap2b interaction with lipid rafts also occurred in transfected HEK293T cell. Upon metabolic labelling with [(3)H]palmitate, incorporation of the label into Rap2b was observed. Palmitoylation of Rap2b did not occur when Cys176 or Cys177 were mutated to serine, or when the C-terminal CAAX motif was deleted. Contrary to CAAX deletion, Cys176 and Cys177 substitution did not alter the membrane localization of Rap2b, however, relocation of the mutants within lipid rafts was completely prevented. In intact platelets, disruption of Rap2b interaction with lipid rafts obtained by cholesterol depletion caused a significant inhibition of aggregation. Importantly, agonist-induced activation of Rap2b was concomitantly severely impaired. These results demonstrate that Rap2b, but not the more abundant Rap1b, is associated to lipid rafts in human platelets. This interaction is supported by palmitoylation of Rap2b, and is important for a complete agonist-induced activation of this GTPase.