Death effector domain protein PEA-15 potentiates Ras activation of extracellular signal receptor-activated kinase by an adhesion-independent mechanism

PEA-15 is a small, death effector-domain (DED)-containing protein that was recently demonstrated to inhibit tumor necrosis factor-alpha-induced apoptosis and to reverse the inhibition of integrin activation due to H-Ras. This led us to investigate the involvement of PEA-15 in Ras signaling. Surprisingly, PEA-15 activates the extracellular signal receptor-activated kinase (ERK) mitogen-activated protein kinase pathway in a Ras-dependent manner. PEA-15 expression in Chinese hamster ovary cells resulted in an increased mitogen-activated protein kinase kinase and ERK activity. Furthermore, PEA-15 expression leads to an increase in Ras guanosine 5'-triphosphate loading. PEA-15 bypasses the anchorage dependence of ERK activation. Finally, the effects of PEA-15 on integrin signaling are separate from those on ERK activation. Heretofore, all known DEDs functioned in the regulation of apoptosis. In contrast, the DED of PEA-15 is essential for its capacity to activate ERK. The ability of PEA-15 to simultaneously inhibit apoptosis and potentiate Ras-to-Erk signaling may be of importance for oncogenic processes.     

The dominant negative Ras mutant, N17Ras, can inhibit signaling independently of blocking Ras activation

Ras plays an important role in a variety of cellular functions, including growth, differentiation, and oncogenic transformation. For instance, Ras participates in the activation of Raf, which phosphorylates and activates mitogen-activated protein kinase kinase (MEK), which then phosphorylates and activates extracellular signal-regulated kinase (ERK), a mitogen-activated protein (MAP) kinase. Activation of MAP kinase appears to be essential for propagating a wide variety of extracellular signals from the plasma membrane to the nucleus. N17Ras, a GDP-bound dominant negative mutant, is used widely as an interfering mutant to assess Ras function in vivo. Surprisingly, we observed that expression of N17Ras inhibited the activity and phosphorylation of Elk-1, a physiological substrate of MAP kinases, in response to phorbol myristate acetate. The activity and phosphorylation of the MAP kinase hemagglutinin epitope (HA)-ERK1 were not affected by N17Ras in response to the same stimulus. Additionally, expression of N17Ras, but not L61S186Ras, a GTP-bound interfering mutant, inhibited MEK-induced Elk-1 phosphorylation, suggesting that inhibition of Elk-1 may be unique to GDP-bound Ras mutants. Finally, we observed that V12Ras-induced focus formation in NIH3T3 cells is inhibited by coexpression of GDP-bound Ras mutants, such as N17, A15, and N17N69. Therefore, N17Ras and V12 Ras may be codominant with respect to Elk-1 activation and cellular transformation. These results indicate that N17Ras appears to have at least two distinguishable functions: interference with endogenous Ras activation and inhibition of Elk-1 and transfomation. Furthermore, our data imply the possibility that GDP-bound Ras, like N17Ras, may have a direct role in signal transduction.     

The methionine-rich domain of the 54 kd protein subunit of the signal recognition particle contains an RNA binding site and can be crosslinked to a signal sequence.

The 54 kd protein subunit of the signal recognition particle (SRP54) has been shown to bind signal sequences by UV crosslinking. Primary structure analysis and phylogenetic comparisons have suggested that SRP54 is composed of two domains: an amino-terminal domain that contains a putative GTP-binding site (G-domain) and a carboxy-terminal domain that contains a high abundance of methionine residues (M-domain). Partial proteolysis of SRP revealed that the two proposed domains of SRP54 indeed represent structurally discrete entities. Upon proteolysis the intact G-domain was released from SRP, whereas the M-domain remained attached to the core of the particle. Reconstitution experiments demonstrated that the isolated M-domain associates with 7SL RNA in the presence of SRP19. In addition, we observed a specific binding of the M-domain directly to 4.5S RNA of Escherichia coli, which contains a structural motif also present in 7SL RNA. This shows that the M-domain contains an RNA binding site, and suggests that SRP54 may be linked to the rest of SRP through this domain by a direct interaction with 7SL RNA. Using UV crosslinking, we found that in an in vitro translation system the preprolactin signal sequence contacts SRP through the M-domain of SRP54. These results imply that the M-domain contains the signal sequence binding site of SRP54, although we cannot exclude that the G-domain may also be in proximity to bound signal sequences.(ABSTRACT TRUNCATED AT 250 WORDS)     

The human estrogen receptor hormone binding domain dimerizes independently of ligand activation

High level expression of biochemically active human estrogen receptor hormone binding domain (hER-HBD) was achieved using a Saccharomyces cerevisae expression system. Using dissociation kinetic analysis, density gradient centrifugation and cross-linking studies, a spontaneous dimerization activity of hER-HBD independent of the presence of the DNA binding domain, ligand, and of elevated temperature is demonstrated.     

Coupling of agonist binding to effector domain activation in metabotropic glutamate-like receptors

Many membrane receptors are made of a ligand binding domain and an effector domain mediating intracellular signaling. This is the case for the metabotropic glutamate-like G-protein-coupled receptors. How ligand binding leads to the active conformation of the effector domain in such receptors is largely unknown. Here, we used an evolutionary trace analysis and mutagenesis to identify critical residues involved in the allosteric coupling between the Venus flytrap ligand binding domain (VFT) and the heptahelical G-protein activating domain of the metabotropic glutamate-like receptors. We have shown that a conserved interdomain disulfide bridge is required for this allosteric interaction. Taking into account that these receptors are homodimers, this finding provides important new information explaining how the different conformations of the dimer of VFT lead to different signaling of such dimeric receptors.     

Critical binding and regulatory interactions between Ras and Raf occur through a small, stable N-terminal domain of Raf and specific Ras effector residues.

Genetic and biochemical evidence suggests that the Ras protooncogene product regulates the activation of the Raf kinase pathway, leading to the proposal that Raf is a direct mitogenic effector of activated Ras. Here we report the use of a novel competition assay to measure in vitro the relative affinity of the c-Raf-1 regulatory region for Ras-GTP, Ras-GDP, and 10 oncogenic and effector mutant Ras proteins. c-Raf-1 associates with normal Ras and the oncogenic V12 and L61 forms of Ras with equal affinity. The moderately transforming mutant Ras[E30K31] also bound to the c-Raf-1 regulatory region with normal affinity. Transformation-defective Ras effector mutants Ras[N33], Ras[S35], and Ras[N38] bound poorly. In contrast, the transformation defective Ras[G26I27] and Ras[E45] mutants bound to the c-Raf-1 regulatory region with nearly wild-type affinity. A stable, high-affinity Ras-binding region of c-Raf-1 was mapped to a 99-amino-acid subfragment of the first 257 residues. The smallest Ras-binding region identified consisted of N-terminal residues 51 to 131, although stable expression of the domain and high-affinity binding were improved by the presence of residues 132 to 149. Deletion of the Raf zinc finger region did not reduce Ras-binding affinity, while removal of the first 50 amino acids greatly increased affinity. Phosphorylation of Raf[1-149] by protein kinase A on serine 43 resulted in significant inhibiton of Ras binding. demonstrating that the mechanism of cyclic AMP downregulation results through structural changes occurring exclusively in this small Ras-binding domain.     

The Ras-GTPase-activating protein SH3 domain is required for Cdc2 activation and mos induction by oncogenic Ras in Xenopus oocytes independently of mitogen-activated protein kinase activation.

The Ras-GTPase-activating protein (RasGAP) is an important modulator of p21ras - dependent signal transduction in Xenopus oocytes and in mammalian cells. We investigated the role of the RasGAP SH3 domain in signal transduction with a monoclonal antibody against the SH3 domain of RasGaP. This antibody prevented the activation of the maturation-promoting factor complex (cyclin B-p34cdc2) by oncogenic Ras. The antibody appears to be specific because as little as 5 ng injected per oocyte reduced the level of Cdc2 activation by 50% whereas 100 ng of nonspecific immunoglobulin G did not affect Cdc2 activation. The antibody blocked the Cdc2 activation induced by oncogenic Ras but not that induced by progesterone, which acts independently of Ras. A peptide corresponding to positions 317 to 326 of a sequence in the SH3 domain of human RasGAP blocked Cdc2 activation, whereas a peptide corresponding to positions 273 to 305 of a sequence in the N-terminal moiety of the SH3 domain of RasGAP had no effect. The antibody did not block the mitogen-activated protein (MAP) kinase cascade (activation of MAPK/ERK kinase [MEK], MAP kinase, and S6 kinase p90rsk). Surprisingly, injection of the negative MAP kinase mutant protein ERK2 K52R (containing a K-to-R mutation at position 52) blocked the Cdc2 activation induced by oncogenic Ras as well as blocking the activation of MAP kinase. Thus, MAP kinase is also implicated in the regulation of Cdc2 activity. In this study, we further investigated the regulation of the synthesis of the c-mos oncogene product, which is necessary for the activation of Cdc2. We report that the synthesis of the c-mos oncogene product, which is necessary for the activation antibody to the SH3 domain of RasGAP and by injecting the negative MAP kinase mutant protein ERK2 K52R. These results suggest that oncogenic Ras activates two signaling mechanisms: the MAP kinase cascade and a signaling pathway implicating the SH3 domain of RasGAP. These mechanisms might control Mos protein expression implicated in Cdc2 activation.     

Crystal structure and functional analysis of Ras binding to its effector phosphoinositide 3-kinase gamma

Ras activation of phosphoinositide 3-kinase (PI3K) is important for survival of transformed cells. We find that PI3Kgamma is strongly and directly activated by H-Ras G12V in vivo or by GTPgammaS-loaded H-Ras in vitro. We have determined a crystal structure of a PI3Kgamma/Ras.GMPPNP complex. A critical loop in the Ras binding domain positions Ras so that it uses its switch I and switch II regions to bind PI3Kgamma. Mutagenesis shows that interactions with both regions are essential for binding PI3Kgamma. Ras also forms a direct contact with the PI3Kgamma catalytic domain. These unique Ras/PI3Kgamma interactions are likely to be shared by PI3Kalpha. The complex with Ras shows a change in the PI3K conformation that may represent an allosteric component of Ras activation.     

Extracellular signal-regulated activation of Rap1 fails to interfere in Ras effector signalling.

The small GTPase Rap1 has been implicated in both negative and positive control of Ras-mediated signalling events. We have investigated which extracellular signals can activate Rap1 and whether this activation leads to a modulation of Ras effector signalling, i.e. the activation of ERK and the small GTPase Ral. We found that Rap1 is rapidly activated following stimulation of a large variety of growth factor receptors. These receptors include receptor tyrosine kinases for platelet-derived growth factor (PDGF) and epithelial growth factor (EGF), and G protein-coupled receptors for lysophosphatidic acid (LPA), thrombin and endothelin. At least three distinct pathways may transduce a signal towards Rap1 activation: increase in intracellular calcium, release of diacylglycerol and cAMP synthesis. Surprisingly, activation of endogenous Rap1 fails to affect Ras-dependent ERK activation. In addition, we found that although overexpression of active Rap1 is able to activate the Ral pathway, activation of endogenous Rap1 in fibroblasts does not result in Ral activation. Rap1 also does not negatively influence Ras-mediated Ral activation. We conclude that activation of Rap1 is a common event upon growth factor treatment and that the physiological function of Rap1 is likely to be different from modulation of Ras effector signalling.     

High-pressure NMR study of the complex of a GTPase Rap1A with its effector RalGDS. A conformational switch in RalGDS revealed from non-linear pressure shifts

Unusually large non-linear 1H and 15N nuclear magnetic resonance chemical shifts against pressure have been detected for individual amide groups of the Ras-binding domain of Ral guanine dissociation stimulator (GDS). The non-linear response is largest in the region of the protein remote from the Rap1A-binding site, which increases by about two-fold by the complex formation with its effector protein Rap1A. The unusual non-linearity is explained by the increasing population of another conformer (N'), lying energetically above the basic native conformer (N), at higher pressure. It is considered likely that the conformational change from N to N' in the Ras-binding domain of RalGDS works as a switch to transmit the effector signal further to molecules of different RalGDS-dependent signaling pathways.     

The Bcr kinase downregulates Ras signaling by phosphorylating AF-6 and binding to its PDZ domain

The protein kinase Bcr is a negative regulator of cell proliferation and oncogenic transformation. We identified Bcr as a ligand for the PDZ domain of the cell junction and Ras-interacting protein AF-6. The Bcr kinase phosphorylates AF-6, which subsequently allows efficient binding of Bcr to AF-6, showing that the Bcr kinase is a regulator of the PDZ domain-ligand interaction. Bcr and AF-6 colocalize in epithelial cells at the plasma membrane. In addition, Bcr, AF-6, and Ras form a trimeric complex. Bcr increases the affinity of AF-6 to Ras, and a mutant of AF-6 that lacks a specific phosphorylation site for Bcr shows a reduced binding to Ras. Wild-type Bcr, but not Bcr mutants defective in binding to AF-6, interferes with the Ras-dependent stimulation of the Raf/MEK/ERK pathway. Since AF-6 binds to Bcr via its PDZ domain and to Ras via its Ras-binding domain, we propose that AF-6 functions as a scaffold-like protein that links Bcr and Ras to cellular junctions. We suggest that this trimeric complex is involved in downregulation of Ras-mediated signaling at sites of cell-cell contact to maintain cells in a nonproliferating state.     

The physiological role of RNase T can be explained by its unusual substrate specificity

Escherichia coli RNase T, the enzyme responsible for the end-turnover of tRNA and for the 3' maturation of 5 S and 23 S rRNAs and many other small, stable RNAs, was examined in detail with respect to its substrate specificity. The enzyme was found to be a single-strand-specific exoribonuclease that acts in the 3' to 5' direction in a non-processive manner. However, although other Escherichia coli exoribonucleases stop several nucleotides downstream of an RNA duplex, RNase T can digest RNA up to the first base pair. The presence of a free 3'-hydroxyl group is required for the enzyme to initiate digestion. Studies with RNA homopolymers and a variety of oligoribonucleotides revealed that RNase T displays an unusual base specificity, discriminating against pyrimidine and, particularly, C residues. Although RNase T appears to bind up to 10 nucleotides in its active site, its specificity is defined largely by the last 4 residues. A single 3'-terminal C residue can reduce RNase T action by >100-fold, and 2-terminal C residues essentially stop the enzyme. In vivo, the substrates of RNase T are similar in that they all contain a double-stranded stem followed by a single-stranded 3' overhang; yet, the action of RNase T on these substrates differs. The substrate specificity described here helps to explain why the different substrates yield different products, and why certain RNA molecules are not substrates at all.     

A consensus motif in the RFX DNA binding domain and binding domain mutants with altered specificity.

The RFX DNA binding domain is a novel motif that has been conserved in a growing number of dimeric DNA-binding proteins, having diverse regulatory functions, in eukaryotic organisms ranging from yeasts to humans. To characterize this novel motif, we have performed a detailed dissection of the site-specific DNA binding activity of RFX1, a prototypical member of the RFX family. First, we have performed a site selection procedure to define the consensus binding site of RFX1. Second, we have developed a new mutagenesis-selection procedure to derive a precise consensus motif, and to test the accuracy of a secondary structure prediction, for the RFX domain. Third, a modification of this procedure has allowed us to isolate altered-specificity RFX1 mutants. These results should facilitate the identification both of additional candidate genes controlled by RFX1 and of new members of the RFX family. Moreover, the altered-specificity RFX1 mutants represent valuable tools that will permit the function of RFX1 to be analyzed in vivo without interference from the ubiquitously expressed endogenous protein. Finally, the simplicity, efficiency, and versatility of the selection procedure we have developed make it of general value for the determination of consensus motifs, and for the isolation of mutants exhibiting altered functional properties, for large protein domains involved in protein-DNA as well as protein-protein interactions.     

Analysis of Ras and Rap activation in living cells using fluorescent Ras binding domains

Ras GTPases regulate cellular growth and differentiation and are modulated by myriad stimuli including growth factors, cytokines, antigens, and UV irradiation. Ras GTPases are molecular switches that are active when GTP-bound and inactive when GDP-bound. The ability of these GTPases to signal requires that the GTP-bound form engage downstream effectors, interactions that occur only on the cytosolic surface of cellular membranes. Ras family proteins include H-Ras, N-Ras, K-Ras, and Rap1. Insight into the regulation and signaling properties of these molecules has come largely from in vitro studies relying on cellular extracts prepared following cellular stimulation. Since Ras GTPases are expressed on multiple cellular compartments that include the plasma membrane, vesicles derived from the plasma membrane, and other internal membranes such as the ER and Golgi complex, analysis of how their spatial distribution modulates signaling has remained unknown. We have developed fluorescent, GFP-based probes capable of selectively binding GTP-bound Ras or Rap1 in living cells. We have used these reporters to examine sites of cellular activation of Ras and Rap1 during growth factor stimulation. These studies have revealed new insights into the platforms from which these GTPases signal and have led to the hypothesis that GTPase signaling is modulated in a compartmentalized fashion. Here, we describe the design and implementation of fluorescent probes for Ras and Rap1.     

The Hantaan virus M-segment glycoproteins G1 and G2 can be expressed independently.

The two glycoproteins of Hantaan virus (HTV), G1 and G2, are encoded as a continuous single open reading frame in the M segment of the virion RNA. They are believed to be synthesized contemporaneously via a polypeptide precursor which is then processed to yield two glycoproteins, both of which appear in the Golgi complex of the cell. To study the properties of G1 and G2 as separate entities, we have constructed vaccinia virus recombinants which contain the sequences for each glycoprotein individually. Both glycoproteins made from these recombinants appear normal on sodium dodecyl sulfate-polyacrylamide gels compared with HTV products made in virus-infected cells. Interestingly, in the independently expressed G2 recombinant, a stretch of hydrophobic amino acids preceding the mature G2 N terminus appears to contain the signals necessary for translocation across membranes and proper glycosylation; partial deletion of this hydrophobic sequence results in production of an nonglycosylated form of G2. Thus, both G1 and G2 appear able to be expressed in an authentic fashion quite independently of each other, using their own signal sequences. In addition, it appears that the G1 from vaccinia virus recombinants contains the motif(s) necessary for cellular targeting of the HTV glycoproteins, while G2 from vaccinia virus recombinants remains strongly associated with the endoplasmic reticulum. In contrast, cells doubly infected with G1-vaccinia virus and G2-vaccinia virus recombinants show the G2 in a predominantly perinuclear (Golgi-like) distribution, presumably targeted there through association with G1. A carboxy-terminal deletion of G1 (2-43-Vac), which lacks 82 amino acids proximal to the start of the mature G2, retains a Golgi-like distribution.     

A distinct class of dominant negative Ras mutants: cytosolic GTP-bound Ras effector domain mutants that inhibit Ras signaling and transformation and enhance cell adhesion.

Cytosolic GTP-bound Ras has been shown to act as a dominant negative (DN) inhibitor of Ras by sequestering Raf in non-productive cytosolic complexes. Nevertheless, this distinct class of DN mutants has been neither well characterized nor extensively used to analyze Ras signaling. In contrast, DN Ras17N, which functions by blocking Ras guanine nucleotide exchange factors, has been well characterized and is widely used. Cytosolic GTP-bound Ras mutants could be used to inhibit particular Ras effectors by introducing additional mutations (T35S, E37G or Y40C) that permit them to associate selectively with and inhibit Raf, RalGDS, or phosphoinositide 3-kinase, respectively. When the wild-type Ras effector binding region is used, cytosolic Ras should associate with all Ras effectors, even those that are not yet identified, making these DN Ras mutants effective inhibitors of multiple Ras functions. We generated cytosolic GTP-bound H-, N-, and K-Ras, and we assessed their ability to inhibit Ras-induced phenotypes. In fibroblasts, cytosolic H-, N-, and K-Ras inhibited Ras-induced Elk-1 activation and focus formation, induced a flattened cell morphology, and increased adhesion to fibronectin through modulation of a beta(1)-subunit-containing integrin, thereby demonstrating that DN activity is not limited to a subset of Ras isoforms. We also generated cytosolic GTP-bound Ras effector domain mutants (EDMs), each of which reduced the ability of cytosolic GTP-bound Ras proteins to inhibit Elk-1 activation and to induce cell flattening, implicating multiple pathways in these phenotypes. In contrast, Ras-induced focus formation, platelet-derived growth factor (PDGF)-, or Ras-induced phospho-Akt levels and cell adhesion to fibronectin were affected by T35S and Y40C EDMs, whereas PDGF- or Ras-induced phospho-Erk levels were affected only by the T35S EDM, implying that a more limited set of Ras-mediated pathways participate in these phenotypes. These data constitute the first extensive characterization of this functionally distinct class of DN Ras inhibitor proteins.     

The mechanism of group I self-splicing: an internal guide sequence can be provided in trans.

We have reconstituted a group I self-splicing reaction between two RNA molecules with different functional RNA parts: a substrate molecule containing the 5' splice site and a functional internal guide sequence (IGS), and a ribozyme molecule with core structure elements and splice sites but a mutated IGS. The 5' exon of the substrate molecule is ligated in trans to the 3' exon of the ribozyme molecule, suggesting that the deficient IGS in the ribozyme can be replaced by an externally added IGS present on the substrate molecule. This result is different from catalysis mediated by proteins where it is not possible to dissect the specificity of an enzyme from its catalytic activity.