Two SH2 domains of p120 Ras GTPase-activating protein bind synergistically to tyrosine phosphorylated p190 Rho GTPase-activating protein

p120 GTPase-activating protein (GAP) is a negative regulator of Ras that functions at a key relay point in signal transduction pathways that control cell proliferation. Among other proteins, p120 GAP associates with p190, a GAP for the Ras-related protein, Rho. To characterize the p120.p190 interaction further, we used bacterially expressed glutathione S-transferase fusion polypeptides to map the regions of p120 necessary for its interactions with p190. Our results show that both the N-terminal and the C-terminal SH2 domains of p120 are individually capable of binding p190 expressed in a baculovirus/insect cell system. Moreover, the two SH2 domains together on one polypeptide bind synergistically to p190, and this interaction is dependent on tyrosine phosphorylation of p190. In addition, mutation of the highly conserved Arg residues in the critical FLVR sequences of both SH2 domains of full-length p120 reduces binding to tyrosine-phosphorylated p190. The dependence on p190 phosphorylation for complex formation with p120 SH2 domains observed in vitro is consistent with analysis of the native p120.p190 complexes formed in vivo. These findings suggest that SH2-phosphotyrosine interaction is one mechanism by which the cell regulates p120.p190 association and thus may be a means for coordinating the Ras- and Rho-mediated signaling pathways.     

Structural determinants required for the interaction between Rho GTPase and the GTPase-activating domain of p190

The Rho family small GTP-binding proteins are subjected to regulation by Rho GTPase-activating proteins (GAPs) in the course of transmitting diverse intracellular signals. To understand the mechanism of GAP-catalyzed GTP hydrolysis of Rho GTPases, we have studied the interaction between RhoA and p190, the RasGAP binding phosphoprotein which has been implicated as a Rho-specific GAP, by delineating the structural determinants of RhoA and p190 GAP domain (p190GD) that are involved in their functional coupling. Besides the conserved residues Tyr34, Thr37, and Phe39 in the switch I region of RhoA which are required for p190GD interaction, chimeras made between RhoA and Cdc42, a close relative of RhoA with which p190GD interacts 50-fold less efficiently, revealed that residues outside the switch I and neighboring regions of RhoA, residues 85-122 in particular, contain the major p190GD-specifying determinant(s). Mutation of the unique Asp90 of RhoA in this region mostly abolished p190GD stimulation, whereas the corresponding reverse mutation of Cdc42 (S88D) was able to respond to p190GD-catalysis similarly as RhoA. Further kinetic analysis of these mutants provided evidence that Asp90 of RhoA contributes primarily to the specific binding interaction with p190GD. On the other hand, two charged residues of p190GD, Arg1283 and Lys1321, which are located in the putative G-protein binding helix pocket of GAP domain, were found to be involved in different aspects of interaction with RhoA. The R1283L mutant of p190GD lost GAP activity but retained the ability to bind to RhoA, while K1321A failed to stimulate and to bind to RhoA. These results indicate that residue Asp90 constitutes the second GAP-interactive site in RhoA which is mostly responsible for conferring p190GD-specificity, and suggest that the role of p190GD in the GTPase reaction of RhoA is in part to supply active site residue Arg1283 for efficient catalysis.     

Regulation of RhoA GTP hydrolysis by the GTPase-activating proteins p190, p50RhoGAP, Bcr, and 3BP-1

The small GTP-binding protein RhoA becomes inactivated by hydrolyzing bound GTP to GDP through its intrinsic GTPase activity which is further stimulated by a family of Rho GTPase-activating proteins (GAPs). Here we have compared the kinetics of interaction between recombinant RhoA and the RhoGAP domains of p190, p50RhoGAP, Bcr, and 3BP-1. The intrinsic rate of GTP hydrolysis by RhoA is relatively slow when compared to other Rho-family GTPases such as Cdc42 or Rac1 with a rate constant of 0.022 min-1, which can be further stimulated at least 4000-fold by p190 or p50RhoGAP. The RhoGAP domains of Bcr and 3BP-1, which were thought to be inactive toward RhoA, are also found capable of stimulating the GTPase activity of RhoA in a dose-dependent manner. The supreme catalytic activities of p190 and p50RhoGAP toward RhoA reside mostly in their lower Km values (1.79 and 2.83 microM, respectively) which correlate well with their binding affinity for GMP-PNP-bound RhoA (2.18 and 2. 47 microM, respectively), in contrast with Bcr and 3BP-1 which interact with the activated RhoA with much higher Km (89 microM). However, the mechanisms of catalysis by p190 and p50RhoGAP are distinct in at least three aspects: (1) p50RhoGAP displays an effect of product inhibition by binding to the GDP-bound form of RhoA with a Kd of 6 microM in comparison with the Kd for p190 of 33 microM; (2) the Km of p190 increases drastically upon the increase of salt and Mg2+ concentrations, conditions under which only modest changes of Km for p50RhoGAP are observed; and (3) p50RhoGAP remains partially active toward the effector domain mutants of RhoA, Y34K, and T37A, whereas p190 is completely inactive toward Y34K and T37A. These results suggest that there exists a unique mechanism of functional interaction between RhoA and individual RhoGAP which involves distinct structural determinants of the small G-protein to cause the apparent differences in kinetic properties.     

The tumor suppression function of p21Waf is contained in its N-terminal half ('half-WAF')

The Waf-1 encoded protein, p21, mediates p53 suppression of tumor cell growth. Overexpression of p21 in the H1299 tumor cell line suppresses colony formation similar to that resulted from p53 overexpression. In an effort to localize the tumor suppression function within the structure of p21 we utilized vectors constructed with systematic truncations of p21 and tested their efficiency in suppressing tumor cell growth. We demonstrate that the N-terminal half of the p21 molecule (residues 1-80 and 1-89) shows better tumor cell growth suppression than the entire p21 molecule whereas the C-terminal half of p21 does not show this effect. These results may have implications for gene therapy of cancer.     

The N-terminal globular domain of Eph receptors is sufficient for ligand binding and receptor signaling

The Eph family of receptor protein-tyrosine kinases (RTKs) have recently been implicated in patterning and wiring events in the developing nervous system. Eph receptors are unique among other RTKs in that they fall into two large subclasses that show distinct ligand specificities and for the fact that they themselves might function as 'ligands', thereby activating bidirectional signaling. To gain insight into the mechanisms of ligand-receptor interaction, we have mapped the ligand binding domain in Eph receptors. By using a series of deletion and domain substitution mutants, we now report that an N-terminal globular domain of the Nuk/Cek5 receptor is the ligand binding domain of the transmembrane ligand Lerk2. Using focus formation assays, we show that the Cek5 globular domain is sufficient to confer Lerk2-dependent transforming activity on the Cek9 orphan receptor. Extending our binding studies to other members of both subclasses of receptors, it became apparent that the same domain is used for binding of both transmembrane and glycosylphosphatidyl-anchored ligands. Our studies have determined the first structural elements involved in ligand-receptor interaction and will allow more fine-tuned genetic experiments to elucidate the mechanism of action of these important guidance molecules.     

Ribosome binding to mitochondria is regulated by GTP and the transit peptide

The association between ribosomes and the pore proteins at the endoplasmic reticulum membrane is important to co-translational translocation. To determine if a similar association occurs between the ribosome and mitochondrial membrane protein(s) during protein import in higher eukaryotes, we examined ribosome-mitochondria binding. By using spectral measurements, analysis of mitochondrial associated RNA, and electron microscopy, we demonstrated that ribosomes stably bind to purified rat liver mitochondria in vitro. Binding of ribosomes to mitochondria was markedly reduced by GTP and nearly abolished by the non-hydrolyzable GTP analogue, guanosine-5'-[thio]-triphosphate (GTPgammaS), but was only modestly reduced by GDP or ATP and unaffected by CTP. The initial rate of GTP hydrolysis by mitochondria was increased by ribosomes, whereas the rate of ATP hydrolysis by mitochondria was not affected. Ribosomes programmed with mRNA for 92 amino acids of the N terminus of mitochondrial malate dehydrogenase bound to mitochondria, but unlike unprogrammed rat liver ribosomes, neither GTP nor GDP disrupted binding; however, GTPgammaS did. These data show that receptors specific for ribosomes are present on the mitochondrial membrane, and a GTP-dependent process mediates this binding. The presence of a nascent chain alters these binding characteristics. These findings support the hypothesis that a co-translational translocation pathway exists for import of proteins into mitochondria.     

The essential yeast RNA binding protein Np13p is methylated

Arginine methylation is a prevalent modification found in many RNA binding proteins, yet little is known about its functional consequences. Using a monoclonal antibody, 1E4, we have shown that the yeast NPL3 gene product Np13p, an essential RNA binding protein with repeated RGG motifs, is arginine-methylated in vivo. The 1E4 epitope can be generated by incubating recombinant Np13p with partially purified bovine arginine methyltransferase block this reaction. Np13p methylation requires S-adenosyl-L-methionine and also occurs in yeast extracts. An Np13p deletion mutant lacking the RGG domain is not a substrate for methylation, suggesting that the methylation sites lie within the RGG motifs. The discovery of arginine methylation in a genetically tractable organism provides a powerful entrée to understanding the function of this modification, particularly in view of the many roles postulated for Np13p in RNA processing and transport. The recent discovery of phosphorylated serine residues within the RGG domain suggests a hypothesis in which a molecular switch governed by methylation and phosphorylation regulates the biochemical properties of the Np13p RGG domain.     

Molecular characterization of the GTPase-activating domain of ADP-ribosylation factor domain protein 1 (ARD1)

ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins recognized as critical components in intracellular vesicular transport and phospholipase D activation. Both guanine nucleotide-exchange proteins and GTPase-activating proteins (GAPs) for ARFs have been cloned recently. A zinc finger motif near the amino terminus of the ARF1 GAP was required for stimulation of GTP hydrolysis. ARD1 is an ARF family member that differs from other ARFs by the presence of a 46-kDa amino-terminal extension. We had reported that the ARF domain of ARD1 binds specifically GDP and GTP and that the amino-terminal extension acts as a GAP for the ARF domain of ARD1 but not for ARF proteins. The GAP domain of ARD1, synthesized in Escherichia coli, stimulated hydrolysis of GTP bound to the ARF domain of ARD1. Using ARD1 truncations, it appears that amino acids 101-190 are critical for GAP activity, whereas residues 190-333 are involved in physical interaction between the two domains of ARD1 and are required for GTP hydrolysis. The GAP function of the amino-terminal extension of ARD1 required two arginines, an intact zinc finger motif, and a group of residues which resembles a sequence present in Rho/Rac GAPs. Interaction between the two domains of ARD1 required two negatively charged residues (Asp427 and Glu428) located in the effector region of the ARF domain and two basic amino acids (Arg249 and Lys250) found in the amino-terminal extension. The GAP domain of ARD1 thus is similar to ARF GAPs but differs from other GAPs in its covalent association with the GTP-binding domain.     

The charged region of Hsp90 modulates the function of the N-terminal domain

Hsp90, an abundant heat shock protein that is highly expressed even under physiological conditions, is involved in the folding of key molecules of the cellular signal transduction system such as kinases and steroid receptors. It seems to contain two chaperone sites differing in substrate specificity. Binding of ATP or the antitumor drug geldanamycin alters the substrate affinity of the N-terminal chaperone site, whereas both substances show no influence on the C-terminal one. In wild-type Hsp90 the fragments containing the chaperone sites are connected by a highly charged linker of various lengths in different organisms. As this linker region represents the most striking difference between bacterial and eukaryotic Hsp90s, it may be involved in a gain of function of eukaryotic Hsp90s. Here, we have analyzed a fragment of yeast Hsp90 consisting of the N-terminal domain and the charged region (N272) in comparison with the isolated N-terminal domain (N210). We show that the charged region causes an increase in the affinity of the N-terminal domain for nonnative protein and establishes a crosstalk between peptide and ATP binding. Thus, the binding of peptide to N272 decreases its affinity for ATP and geldanamycin, whereas the ATP-binding properties of the monomeric N-terminal domain N210 are not influenced by peptide binding. We propose that the charged region connecting the two chaperone domains plays an important role in regulating chaperone function of Hsp90.     

DNA bending is essential for the site-specific recognition of DNA response elements by the DNA binding domain of the tumor suppressor protein p53

We have used circular permutation assays to determine the extent and location of the DNA bend induced by the DNA binding domain of human wild type p53 (p53DBD) upon binding to several naturally occurring DNA response elements. We have found that p53DBD binding induces axial bending in all of the response elements investigated. In particular, response elements having a d(CATG) sequence at the junction of two consensus pentamers in each half-site favor highly bent complexes (bending angle is approximately 50 degrees ), whereas response elements having d(CTTG) bases at this position are less bent (bending angles from approximately 37 to approximately 25 degrees ). Quantitative electrophoretic mobility shift assays of different complexes show a direct correlation between the DNA bending angle and the binding affinity of the p53DBD with the response elements, i.e. the greater the stability of the complex, the more the DNA is bent by p53DBD binding. The study provides evidence that the energetics of DNA bending, as determined by the presence or absence of flexible sites in the response elements, may contribute significantly to the overall binding affinity of the p53DBD for different sequences. The results therefore suggest that both the structure and the stability of the p53-DNA complex may vary with different response elements. This variability may be correlated with variability in p53 function.     

Toxin-induced activation of the G protein p21 Rho by deamidation of glutamine

Pathogenic Escherichia coli are responsible for a variety of diseases, including diarrhoea, haemolytic uraemic syndrome, kidney infection, septicaemia, pneumonia and meningitis. Toxins called cytotoxic necrotizing factors (CNFs) are among the virulence factors produced by uropathogenic (CNF1) or enteropathogenic (CNF2) E. coli strains that cause diseases in humans and animals, respectively. CNFs induce an increase in the content of actin stress fibres and focal contacts in cultured cells. Effects of CNFs on the actin cytoskeleton correlated with a decrease in the electrophoretic mobility of the GTP-binding protein Rho and indirect evidence indicates that CNF1 might constitutively activate Rho. Here we show that CNF1 catalyses the deamidation of a glutamine residue at position 63 of Rho, turning it into glutamic acid, which inhibits both intrinsic GTP hydrolysis and that stimulated by its GTPase-activating protein (GAP). Thus, this deamidation of glutamine 63 by CNF1 leads to the constitutive activation of Rho, and induces the reorganization of actin stress fibres. To our knowledge, CNF1 is the first example of a bacterial toxin acting by deamidation of a specific target protein.     

The GTPase and Rho GAP domains of p190, a tumor suppressor protein that binds the M(r) 120,000 Ras GAP, independently function as anti-Ras tumor suppressors

p190 is a Tyr-phosphorylatable G protein of M(r) 190,000 that binds NH2-terminal SH2 domains of GAP1, a Ras GAP of M(r) 120,000. p190 contains at least two functional domains: a GTPase domain at the NH2 terminus and a GAP domain at the COOH terminus that can attenuate signal-transducing activity of three distinct G proteins (Rac, Rho, and CDC42). Here, we demonstrate that overexpression of either an antisense p190 RNA or a dominant negative mutant (Asn36) of p190 GTPase domain (residues 1-251) but not the wild-type p190 GTPase domain is able to transform normal NIH/3T3 fibroblasts. Furthermore, overexpression of either the wild-type p190 GTPase domain or the COOH-terminal GAP domain can suppress v-Ha-Ras-induced malignant transformation. These results indicate that p190 contains at least two distinct anti-Ras tumor suppressor domains, the GTPase and GAP domains, and suggest that one of the mechanisms underlying the suppression of Ras-transformation by p190 is the attenuation by p190 GAP domain of Rac/Rho/CDC42 signalings, which are essential for Ras-transformation. In fact, the p190 GAP domain alone suppresses the expression of the c-Fos gene, which is mediated by Rac/Rho/CDC42 and is required for oncogenicity of Ras.     

Calcium binding by the N-terminal cellulose-binding domain from Cellulomonas fimi beta-1,4-glucanase CenC

The interaction of the N-terminal cellulose-binding domain, CBDN1, from Cellulomonas fimi beta-1,4-glucanase CenC with calcium was investigated using NMR spectroscopy and calorimetry. CBDN1 binds a single calcium ion with an equilibrium association constant of approximately 10(5) M-1 at 35 degreesC and pH 6.0. Binding is exothermic (-42 +/- 2 kJ mol-1) under these conditions and is accompanied by a small negative change in heat capacity (DeltaCp = -0.41 +/- 0.16 kJ mol-1 K-1). From an NMR line shape analysis, the rate constants for calcium association and dissociation were found to be (5 +/- 2) x 10(7) s-1 M-1 and (4.5 +/- 0.6) x 10(2) s-1, respectively. The rapid association kinetics indicate that the calcium-binding site on CBDN1 is accessible and, to the first approximation, preformed. Based on patterns of chemical shift perturbations, and structural comparisons with the Bacillus sp. 1, 3-1,4-beta-glucanases, the backbone carbonyl oxygens of Thr8, Gly30, and Asp142 and a side chain carboxyl oxygen of Asp142 are postulated to form the calcium-binding site of CBDN1. Consistent with the calcium-independent affinity of CBDN1 for cellopentaose, this exposed site is located on the face of CBDN1 opposite to that forming the oligosaccharide-binding cleft. The midpoint denaturation temperature of CBDN1 is increased by approximately 8 degreesC at pH 6.0 in the presence of saturating amounts of calcium, confirming that metal ion binding is thermodynamically linked to native-state stability.     

The N-terminal domain of human topoisomerase IIalpha is a DNA-dependent ATPase

We have constructed clones encoding N-terminal fragments of human DNA topoisomerase IIalpha. We show that the N-terminal domain (approximately 50 kDa) has an intrinsic ATPase activity that can be stimulated by DNA. The enzyme obeys Michaelis-Menten kinetics showing a approximately 6-fold increase in kcat in the presence of DNA. Cross-linking studies indicate that the N-terminal domain is a dimer in the absence and presence of nucleotides. Using site-directed mutagenesis, we have identified the catalytic residue for ATP hydrolysis as Glu86. Phosphorylation of the N-terminal domain with protein kinase C does not affect the ATPase activity. The ATPase domain of human topoisomerase IIalpha shows significant differences from its counterpart in DNA gyrase and we discuss the mechanistic implications of these data.     

Regulation of SNARE complex assembly by an N-terminal domain of the t-SNARE Sso1p

The fusion of intracellular transport vesicles with their target membranes requires the assembly of SNARE proteins anchored in the apposed membranes. Here we use recombinant cytoplasmic domains of the yeast SNAREs involved in Golgi to plasma membrane trafficking to examine this assembly process in vitro. Binary complexes form between the target membrane SNAREs Sso1p and Sec9p; these binary complexes can subsequently bind to the vesicle SNARE Snc2p to form ternary complexes. Binary and ternary complex assembly are accompanied by large increases in alpha-helical structure, indicating that folding and complex formation are linked. Surprisingly, we find that binary complex formation is extremely slow, with a second-order rate constant of approximately 3 M(-1) s(-1). An N-terminal regulatory domain of Sso1p accounts for slow assembly, since in its absence complexes assemble 2,000-fold more rapidly. Once binary complexes form, ternary complex formation is rapid and is not affected by the presence of the regulatory domain. Our results imply that proteins that accelerate SNARE assembly in vivo act by relieving inhibition by this regulatory domain.     

TAR-RNA binding by HIV-1 Tat protein is selectively inhibited by its L-enantiomer

An oligoribonucleotide, corresponding to the Tat-interactive top half of the HIV-1 TAR RNA stem-loop, was synthesized in both the natural D- and the enantiomeric L-configurations. The affinity of Tat for the two RNAs, assessed by competition binding experiments, was found to be identical and is reduced 10-fold for both, upon replacement of the critical bulge residue U23 with cytidine. It is suggested that this interaction of the flexible Tat protein depends strongly upon the tertiary structure of a binding pocket within TAR, but not upon its handedness, and may be described by a 'hand-in-mitten' model.     

The specificity of the N-terminal SH2 domain of SHP-2 is modified by a single point mutation

SH2 domains are small protein domains of approximately 100 amino acids that bind to phosphotyrosine (pY) in the context of a specific sequence surrounding the target pY. In general, the residues C-terminal to the pY of the binding target are considered most important for defining the binding specificity, and in particular the pY + 1 and pY + 3 residues (i.e., the first and third amino acids C-terminal to the pY). However, our previous studies with the SH2 domains of the protein tyrosine phosphatase SHP-2 [Huyer, G., Li, Z. M., Adam, M., Huckle, W. R., and Ramachandran, C. (1995) Biochemistry 34, 1040-1049] indicated important interactions with the pY - 2 residue as well. In the SH2 domains of SHP-2, the highly conserved alphaA2 Arg is replaced by Gly. A comparison of the published crystal structures of the Src SH2 domain and the N-terminal SH2 domain of SHP-2 complexed with high-affinity peptides suggested that the alphaA2 Gly of SHP-2 creates a gap which is filled by the side chain of the pY - 2 residue of the bound peptide. It was predicted that replacing this Gly with Arg would alter or eliminate the involvement of the pY - 2 residue in binding. The alphaA2 Gly --> Arg mutant was constructed, and indeed, this mutant no longer required residues N-terminal to the target pY for high-affinity binding, making its specificity more like that of other SH2 domains. The alphaA2 Gly is clearly involved in directing the unusual requirement for the pY - 2 residue in the binding sequence of this SH2 domain, which has important implications for its in vivo targeting and specificity.     

The N-terminal region of the luteovirus readthrough domain determines virus binding to Buchnera GroEL and is essential for virus persistence in the aphid

Luteoviruses and the luteovirus-like pea enation mosaic virus (PEMV; genus Enamovirus) are transmitted by aphids in a circulative, nonreplicative manner. Acquired virus particles persist for several weeks in the aphid hemolymph, in which a GroEL homolog, produced by the primary endosymbiont of the aphid, is abundantly present. Six subgroup II luteoviruses and PEMV displayed a specific but differential affinity for Escherichia coli GroEL and GroEL homologs isolated from the endosymbiotic bacteria of both vector and nonvector aphid species. These observations suggest that the basic virus-binding capacity resides in a conserved region of the GroEL molecule, although other GroEL domains may influence the efficiency of binding. Purified luteovirus and enamovirus particles contain a major 22-kDa coat protein (CP) and lesser amounts of an approximately 54-kDa readthrough protein, expressed by translational readthrough of the CP into the adjacent open reading frame. Beet western yellows luteovirus (BWYV) mutants devoid of the readthrough domain (RTD) did not bind to Buchnera GroEL, demonstrating that the RTD (and not the highly conserved CP) contains the determinants for GroEL binding. In vivo studies showed that virions of these BWYV mutants were significantly less persistent in the aphid hemolymph than were virions containing the readthrough protein. These data suggest that the Buchnera GroEL-RTD interaction protects the virus from rapid degradation in the aphid. Sequence comparison analysis of the RTDs of different luteoviruses and PEMV identified conserved residues potentially important in the interaction with Buchnera GroEL.     

Crystal structure of the GTPase-activating domain of human p120GAP and implications for the interaction with Ras

Ras-related GTP-binding proteins function as molecular switches which cycle between GTP-bound 'on'- and GDP-bound 'off'-states. GTP hydrolysis is the common timing mechanism that mediates the return from the 'on' to the 'off'-state. It is usually slow but can be accelerated by orders of magnitude upon interaction with GTPase-activating proteins (GAPs). In the case of Ras, a major regulator of cellular growth, point mutations are found in approximately 30% of human tumours which render the protein unable to hydrolyse GTP, even in the presence of Ras-GAPs. The first structure determination of a GTPase-activating protein reveals the catalytically active fragment of the Ras-specific p120GAP (ref. 2), GAP-334, as an elongated, exclusively helical protein which appears to represent a novel protein fold. The molecule consists of two domains, one of which contains all the residues conserved among different GAPs for Ras. From the location of conserved residues around a shallow groove in the central domain we can identify the site of interaction with Ras x GTP. This leads to a model for the interaction between Ras and GAP that satisfies numerous biochemical and genetic data on this important regulatory process.