Aberrant regulation of ras proteins in malignant tumour cells from type 1 neurofibromatosis patients.

Defects in the NF1 gene have been implicated in the inherited disorder neurofibromatosis type 1, which is characterized by several developmental abnormalities including an increased frequency of benign and malignant tumours of neural crest origin (neurofibromas and neurofibrosarcomas respectively). The NF1 gene encodes a ubiquitous protein homologous to p120GAP, the GTPase-activating protein (GAP) for the products of the ras protooncogenes. When expressed in non-mammalian systems, the region of the NF1 gene homologous to p120GAP produces a protein with GAP-like activity. Here we present evidence that the ras proteins in malignant tumour cell lines from patients with type 1 neurofibromatosis are in a constitutively activated state, as judged by the guanine nucleotide bound to them, and are necessary for cellular proliferation. These cells contain p21ras and p120GAP that are both functionally wild type, but barely any functional NF1 protein. Our results show that the NF1 protein is normally essential for correct negative regulation of ras proteins in the cell, even in the presence of normal p120GAP, and they support the hypothesis that NF1 is a tumour-suppressor gene whose product acts upstream of ras.     

Bcr encodes a GTPase-activating protein for p21rac

More than thirty small guanine nucleotide-binding proteins related to the ras-encoded oncoprotein, termed Ras or p21ras, are known. They regulate many fundamental processes in all eukaryotic cells, such as growth, vesicle traffic and cytoskeletal organization. GTPase-activating proteins (GAPs) accelerate the intrinsic rate of GTP hydrolysis of Ras-related proteins, leading to down-regulation of the active GTP-bound form. For p21ras, two GAP proteins are known, rasGAP and the neurofibromatosis (NF1) gene product. There is evidence that rasGAP may also be a target protein for regulation by Ras and be involved in downstream signalling. We have purified a GAP protein for p21rho, which is involved in the regulation of the actin cytoskeleton. Partial sequencing of rhoGAP reveals significant homology with the product of the bcr (breakpoint cluster region) gene, the translocation breakpoint in Philadelphia chromosome-positive chronic myeloid leukaemias. We show here that the carboxy-terminal domains of the bcr-encoded protein (Bcr) and of a Bcr-related protein, n-chimaerin, are both GAP proteins for the Ras-related GTP-binding protein, p21rac. This result suggest that Bcr could be a target for regulation by Rac and has important new implications for the role of bcr translocations in leukaemia.     

Cloning of bovine GAP and its interaction with oncogenic ras p21

The plasma membrane-bound mammalian ras proteins of relative molecular mass 21,000 (ras p21) share biochemical and structural properties with other guanine nucleotide-binding regulatory proteins (G-proteins). Oncogenic ras p21 variants result from amino acid substitutions at specific positions that cause p21 to occur predominantly complexed to GTP in vivo. Recently, a GTPase activating protein (GAP) with cytosolic activity has been discovered that stimulates the GTPase activity of normal but not of oncogenic ras p21. GAP might be either a negative regulatory agent which acts further upstream in the regulatory pathway or the downstream target of ras p21. We have identified a protein from bovine brain with apparent relative molecular mass 125,000 that has GAP activity. Here, using pure GAP in a kinetic competition assay, we show that GAP interacts preferentially with the active GTP complexes of both normal and oncogenic Harvey (Ha) ras p21 compared with the inactive GDP complexes. We also report the cloning and sequencing of the complementary DNA for bovine GAP. Regions of GAP share amino acid similarity with the noncatalytic domain of adenylate cyclase from the yeast Saccharomyces cerevisiae and with regions conserved between phospholipase C-148, the crk oncogene product and the nonreceptor tyrosine kinases.     

The cytoplasmic protein GAP is implicated as the target for regulation by the ras gene product

About 30% of human tumours contain a mutation in one of the three ras genes leading to the production of p21ras oncoproteins that are thought to make a major contribution to the transformed phenotype of the tumour. The biochemical mode of action of the ras proteins is unknown but as they bind GTP and GDP and have an intrinsic GTPase activity, they may function like regulatory G proteins and control cell proliferation by regulating signal transduction pathways at the plasma membrane. It is assumed that an external signal is detected by a membrane molecule (or detector) that stimulates the conversion of p21.GDP to p21.GTP which then interacts with a target molecule (or effector) to generate an internal signal. Recently a cytoplasmic protein, GAP, has been identified that interacts with the ras proteins, dramatically increasing the GTPase activity of normal p21 but not of the oncoproteins. We report here that GAP appears to interact with p21ras at a site previously identified as the 'effector' site, strongly implicating GAP as the biological target for regulation by p21.     

Yeast and mammalian ras proteins have conserved biochemical properties

Mammalian ras oncogenes encode polypeptides of relative molecular mass (Mr) 21,000 (p21) which bind GTP and GDP. Oncogenic ras-encoded proteins differ from their normal homologues by an amino acid substitution for Gly 12, Ala 59 or Gln 61. Recently, we and others have observed that normal p21, encoded by the Ha-ras gene, has a GTP hydrolytic activity that is reduced by the oncogenic substitutions Val 12 or Thr 59. The yeast Saccharomyces cerevisiae contains two ras-related genes, RASsc1 and RASsc2, the expression of either of which is sufficient for viability. RASsc1 and RASsc2 encode proteins of 309 (SC1) and 322 (SC2) residues which are 62% homologous to mammalian p21 in their 172-amino acid N-terminal sequences. We report here that the N-terminal domain of SC1 binds GTP and GDP and has a GTP hydrolytic activity that is reduced in the variants SC1[Thr 66] and SC1[Leu 68] which are analogous to oncogenic Ha[Thr 59] and Ha[Leu 61], respectively. These results suggest that yeast and mammalian ras proteins have similar biochemical and possibly biological functions.     

Association between GTPase activators for Rho and Ras families.

The ras-related low-molecular-mass GTPases participate in signal transduction involving a variety of cellular functions, including cell-cycle progression, cellular differentiation, cytoskeletal organization, protein transport and secretion. The cycling of these proteins between GTP-bound and GDP-bound states is partially controlled by GTPase activating proteins (GAPs) which stimulate the intrinsic GTP-hydrolysing activity of specific GTPases. The ras GTPase-activating protein (Ras-GAP) forms a complex with a second protein, p190 (M(r) 190,000), in growth-factor stimulated and tyrosine-kinase transformed cells. At its carboxy-terminal end, p190 contains a region that is conserved in the breakpoint cluster region, n-chimaerin, and Rho-GAP. Each of these three proteins exhibits GAP activity for at least one member of the rho family of small GTPases. We have tested recombinant p190 protein for GAP activity on GTPases of the ras, rho and rab families, and show here that p190 can function as a GAP specifically for members of the rho family. Consequently, the formation of a complex between Ras-GAP and p190 in growth-factor stimulated cells may allow the coupling of signalling pathways that involve ras and rho GTPases.     

Suppression of c-ras transformation by GTPase-activating protein

The ras genes are required for normal cell growth and mediate transformation by oncogenes encoding protein tyrosine kinases. Normal ras can transform cells in vitro and in vivo, but mutationally activated ras does so much more efficiently, and highly transforming mutant versions of ras have been isolated from a variety of human and animal tumours. The ras genes encode membrane-associated, guanine nucleotide-binding proteins that are active when GTP is bound and inactive when GDP is bound. The slow intrinsic GTPase activity of normal mammalian Ras proteins can be greatly accelerated by the GTPase-activating protein (GAP), which is predominantly cytoplasmic. This activity of GAP, which can increase with cell density in contact-inhibited cells, suggests that it functions as a negative, upstream regulator of ras. Other studies, however, show that GAP interacts with a region of ras-encoded protein implicated in ras effector function, which raises the possibility that GAP might also be a downstream target of ras. Mutationally activated ras-encoded proteins also interact with GAP, although they are resistant to its catalytic activity. In an attempt to define the role of GAP in ras-mediated transformation, we examined the effects on transformation of normal or mutant ras when cells overexpress GAP. We found that GAP suppresses transformation of NIH 3T3 cells by normal Ha-ras (c-ras) but does not inhibit transformation by activated Ha-ras (v-ras). These results support the hypothesis that GAP functions as a negative regulator of normal ras and make it unlikely that GAP alone is the ras target.     

The GTPase-activating protein Rap1GAP uses a catalytic asparagine

Rap1 is a Ras-like guanine-nucleotide-binding protein (GNBP) that is involved in a variety of signal-transduction processes. It regulates integrin-mediated cell adhesion and might activate extracellular signal-regulated kinase. Like other Ras-like GNBPs, Rap1 is regulated by guanine-nucleotide-exchange factors (GEFs) and GTPase-activating proteins (GAPs). These GAPs increase the slow intrinsic GTPase reaction of Ras-like GNBPs by many orders of magnitude and allow tight regulation of signalling. The activation mechanism involves stabilization of the catalytic glutamine of the GNBP and, in most cases, the insertion of a catalytic arginine of GAP into the active site. Rap1 is a close homologue of Ras but does not possess the catalytic glutamine essential for GTP hydrolysis in all other Ras-like and Galpha proteins. Furthermore, RapGAPs are not related to other GAPs and apparently do not use a catalytic arginine residue. Here we present the crystal structure of the catalytic domain of the Rap1-specific Rap1GAP at 2.9 A. By mutational analysis, fluorescence titration and stopped-flow kinetic assay, we demonstrate that Rap1GAP provides a catalytic asparagine to stimulate GTP hydrolysis. Implications for the disease tuberous sclerosis are discussed.     

PDGF induction of tyrosine phosphorylation of GTPase activating protein

The cascade of biochemical events triggered by growth factors and their receptors is central to understanding normal cell-growth regulation and its subversion in cancer. Ras proteins (p21ras) have been implicated in signal transduction pathways used by several growth factors, including platelet-derived growth factor (PDGF). These guanine nucleotide-binding Ras proteins specifically interact with a cellular GTPase-activating protein (GAP). Here we report that in intact quiescent fibroblasts, both AA and BB homodimers of PDGF rapidly induce tyrosine phosphorylation of GAP under conditions in which insulin and basic fibroblast growth factor (bFGF) are ineffective. Although GAP is located predominantly in the cytosol, most tyrosine-phosphorylated GAP is associated with the cell membrane, the site of p21ras biological activity. These results provide a direct biochemical link between activated PDGF-receptor tyrosine kinases and the p21ras-GAP mitogenic signalling system.     

Phosphorylation of GAP and GAP-associated proteins by transforming and mitogenic tyrosine kinases.

The critical pathways through which protein-tyrosine kinases induce cellular proliferation and malignant transformation are not well defined. As microinjection of antibodies against p21ras can block the biological effects of both normal and oncogenic tyrosine kinases, it is likely that they require functional p21ras to transmit their mitogenic signals. No biochemical link has been established, however, between tyrosine kinases and p21ras. We have identified a non-catalytic domain of cytoplasmic tyrosine kinases, SH2, that regulates the activity and specificity of the kinase domain. The presence of two adjacent SH2 domains in the p21ras GTPase-activating protein (GAP) indicates that GAP might interact directly with tyrosine kinases. Here we show that GAP, and two co-precipitating proteins of relative molecular masses 62,000 and 190,000 (p62 and p190) are phosphorylated on tyrosine in cells that have been transformed by cytoplasmic and receptor-like tyrosine kinases. The phosphorylation of these polypeptides correlates with transformation in cells expressing inducible forms of the v-src or v-fps encoded tyrosine kinases. Furthermore, GAP, p62 and p190 are also rapidly phosphorylated on tyrosine in fibroblasts stimulated with epidermal growth factor. Our results suggest a mechanism by which tyrosine kinases might modify p21ras function, and implicate GAP and its associated proteins as targets of both oncoproteins and normal growth factor receptors with tyrosine kinase activity. These data support the idea that SH2 sequences direct the interactions of cytoplasmic proteins involved in signal transduction.     

Co-capping of ras proteins with surface immunoglobulins in B lymphocytes

Cellular ras genes encode a family of membrane-associated proteins (p21ras) that bind guanine nucleotide and possess a low intrinsic GTPase activity. The p21ras proteins are ubiquitously expressed in mammalian cells and are thought to be involved in a growth-promoting signal transduction pathway; their mode of action, however, remains unknown. The ligand-induced movement of cell-surface receptors seems to be a primary event in the transduction of several extracellular signals that control cell growth and differentiation. In B lymphocytes, surface immunoglobulin receptors crosslinked by antibody or other multivalent ligands form aggregates called patches, which then collect into a single assembly, a cap, at one pole of the cell. This process constitutes the initial signal for the activation of a B cell. Here we show by immunofluorescence microscopy that p21ras co-caps with surface immunoglobulin molecules in mouse splenic B lymphocytes. In contrast, no apparent change in the distribution of p21ras occurs during the capping of concanavalin A receptors. The redistribution of p21ras is apparent at the early stages (patching) of immunoglobulin capping and is inhibited by metabolic inhibitors and the cytoskeleton-disrupting agents colchicine and cytochalasin D. The distribution of another membrane-associated guanine nucleotide-binding regulatory protein, the Gi alpha subunit, is not affected by surface immunoglobulin capping. These findings demonstrate that p21ras can migrate in a directed manner along the plasma membrane and suggest that p21ras may be a component of the signalling pathway initiated by the capping of surface immunoglobulin in B lymphocytes.     

Epidermal growth factor stimulates guanine nucleotide binding activity and phosphorylation of ras oncogene proteins

Several human tumour cell lines contain genes that can transform NIH 3T3 cells into malignant cells. Certain genes have been classified as members of the ras oncogene family, namely, Ha-ras, Ki-ras or N-ras. The proteins encoded by the ras family are generally small (Ha-ras, for example, encodes a protein of molecular weight 21,000 named p21), and are associated with the inner surface of the plasma membrane. The only known biochemical property common to all forms of the ras proteins is the ability to bind guanine nucleotides, a property which may be closely related to the transforming ability of ras proteins. A GTP-dependent, apparent autophosphorylation (on threonine 59) activity has been identified only in the case of the v-Ha-ras protein. Although the role of these biochemical activities in the transformation process remains unclear, we have initiated studies to determine the possible biochemical interactions of ras proteins with other membrane components. We report here the evidence that epidermal growth factor enhances the guanine nucleotide binding activity of activated c-Ha-ras or v-Ha-ras p21, and phosphorylation of v-Ha-ras p21, suggesting that some mitogenic growth factors may regulate those activities.     

Stimulation of p21ras upon T-cell activation

External signals that control the activity of proteins encoded by the ras proto-oncogenes have not previously been characterized. It is now shown that stimulation of the antigen receptor of T lymphocytes causes a rapid activation of p21ras. The mechanism seems to involve a decrease in the activity of GAP, the GTPase-activating protein, on stimulation of protein kinase C. In lymphocytes, p21ras may therefore be an important mediator of the action of protein kinase C.     

TBC-domain GAPs for Rab GTPases accelerate GTP hydrolysis by a dual-finger mechanism

Rab GTPases regulate membrane trafficking by cycling between inactive (GDP-bound) and active (GTP-bound) conformations. The duration of the active state is limited by GTPase-activating proteins (GAPs), which accelerate the slow intrinsic rate of GTP hydrolysis. Proteins containing TBC (Tre-2, Bub2 and Cdc16) domains are broadly conserved in eukaryotic organisms and function as GAPs for Rab GTPases as well as GTPases that control cytokinesis. An exposed arginine residue is a critical determinant of GAP activity in vitro and in vivo. It has been expected that the catalytic mechanism of TBC domains would parallel that of Ras and Rho family GAPs. Here we report crystallographic, mutational and functional analyses of complexes between Rab GTPases and the TBC domain of Gyp1p. In the crystal structure of a TBC-domain-Rab-GTPase-aluminium fluoride complex, which approximates the transition-state intermediate for GTP hydrolysis, the TBC domain supplies two catalytic residues in trans, an arginine finger analogous to Ras/Rho family GAPs and a glutamine finger that substitutes for the glutamine in the DxxGQ motif of the GTPase. The glutamine from the Rab GTPase does not stabilize the transition state as expected but instead interacts with the TBC domain. Strong conservation of both catalytic fingers indicates that most TBC-domain GAPs may accelerate GTP hydrolysis by a similar dual-finger mechanism.     

The ras oncogene product p21 is not a regulatory component of adenylate cyclase

Harvey (Ha-MSV) and Kirsten (Ki-MSV) murine sarcoma viruses induce tumours in animals and transform various cells in culture because of the expression of the ras oncogene product, p21 (ref. 1). Proto-oncogenes homologous with these genes are highly conserved evolutionarily and activated ras oncogenes have been detected in many human cancers. Whether c-ras oncogenes are directly responsible for human carcinogenesis is uncertain; however, it is clear that p21 mediates virus-induced transformation, although by an unknown mechanism. Epithelial and fibroblast cell lines transformed with Ha-MSV and Ki-MSV express p21 (ref. 8) and exhibit reduced adenylate cyclase activity. Like the guanine nucleotide regulatory proteins, Ns and Ni, which mediate stimulation and inhibition, respectively, of adenylate cyclase, p21 is a membrane-associated GTP binding protein, which exhibits GTPase activity. These similarities suggest that p21 and the adenylate cyclase regulatory proteins are related in cellular function, and that p21 depresses adenylate cyclase by inhibiting the activity of Ns or acting as Ni. We have therefore now examined the structural and functional similarities between p21 and Ns and Ni and find no evidence that p21 regulates adenylate cyclase activity by acting as one of these regulatory proteins.     

RanGAP mediates GTP hydrolysis without an arginine finger

GTPase-activating proteins (GAPs) increase the rate of GTP hydrolysis on guanine nucleotide-binding proteins by many orders of magnitude. Studies with Ras and Rho have elucidated the mechanism of GAP action by showing that their catalytic machinery is both stabilized by GAP binding and complemented by the insertion of a so-called 'arginine finger' into the phosphate-binding pocket. This has been proposed as a universal mechanism for GAP-mediated GTP hydrolysis. Ran is a nuclear Ras-related protein that regulates both transport between the nucleus and cytoplasm during interphase, and formation of the mitotic spindle and/or nuclear envelope in dividing cells. Ran-GTP is hydrolysed by the combined action of Ran-binding proteins (RanBPs) and RanGAP. Here we present the three-dimensional structure of a Ran-RanBP1-RanGAP ternary complex in the ground state and in a transition-state mimic. The structure and biochemical experiments show that RanGAP does not act through an arginine finger, that the basic machinery for fast GTP hydrolysis is provided exclusively by Ran and that correct positioning of the catalytic glutamine is essential for catalysis.     

GTPase inhibiting mutations activate the alpha chain of Gs and stimulate adenylyl cyclase in human pituitary tumours

A subset of growth hormone-secreting human pituitary tumours carries somatic mutations that inhibit GTPase activity of a G protein alpha chain, alpha(s). The resulting activation of adenylyl cyclase bypasses the cells' normal requirement for trophic hormone. Amino acids substituted in the putative gsp oncogene identify a domain of G protein alpha-chains required for intrinsic ability to hydrolyse GTP. This domain may serve as a built-in counter-part of the separate GTPase-activating proteins required for GTP hydrolysis by small GTP-binding proteins such as p21ras.     

Structural differences between a ras oncogene protein and the normal protein

One of the most commonly found transforming ras oncogenes in human tumours has a valine codon replacing the glycine codon at position 12 of the normal c-Ha-ras gene. To understand the structural reasons behind cell transformation arising from this single amino acid substitution, we have determined the crystal structure of the GDP-bound form of the mutant protein, p21(Val-12), encoded by this oncogene. We report here the overall structure of p21(Val-12) at 2.2 A resolution and compare it with the structure of the normal c-Ha-ras protein. One of the major differences is that the loop of the transforming ras protein that binds the beta-phosphate of the guanine nucleotide is enlarged. Such a change in the 'catalytic site' conformation could explain the reduced GTPase activity of the mutant, which keeps the protein in the GTP bound 'signal on' state for a prolonged period time, ultimately causing cell transformation.     

An artefact explains the apparent association of the transferrin receptor with a ras gene product

There is substantial evidence implicating ras genes in a number of human neoplasms. The ras genes of several human tumours display mutational changes which are likely to be responsible for their transforming activity. Normal cells also express ras genes, over-expression of which can induce cellular transformation. ras genes encode proteins of approximately 21,000 molecular weight (MW) (p21) that are localized to the inner surface of the plasma membrane. Much effort is being focused on the elucidation of the physiological function of ras-encoded proteins in normal and transformed cells, concentrating on interactions between p21 and other cellular elements. Recently, Finkel and Cooper reported that p21 in extracts of human bladder carcinoma cells is involved in a molecular complex with the transferrin receptor of these cells. This report aroused considerable interest, particularly as expression of the transferrin receptor has been linked to cell proliferation. I present here evidence that the apparent association of p21 and the transferrin receptor is an artefact of the immunoprecipitation technique.     

Transient reversion of ras oncogene-induced cell transformation by antibodies specific for amino acid 12 of ras protein

The proteins encoded by the ras oncogene are thought to trigger expression of the transformed phenotype in some types of cancer cells. In human cells, the ras protein family consists of several members including normal (proto-oncogene) and mutant (oncogene) forms. In general, the proto-oncogene forms are thought to be involved in the normal growth control of cells, while the mutant forms (which apparently result from somatic mutation of the normal ras genes) appear to be responsible, in part, for the loss of normal growth control. On microinjection into living normal cells, the purified ras oncogene protein (p21) induces a characteristic loss of growth control in cells within several hours. The mutant forms of the different ras proteins typically contain a single amino-acid change, usually at position 12 or less frequently at position 61. Here we report that microinjection of antibodies specific for amino acid 12 of the oncogenic v-Ki-ras protein into cells transformed by this protein causes a transient reversion of the cells to a normal phenotype. The fact that this antibody inhibits binding of GTP to the v-Ki-ras protein supports the notion that GTP binding is essential to the transforming function of this oncogene product.