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 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.     

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.     

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.     

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.     

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.     

Cross-talk between cellular signalling pathways suggested by phorbol-ester-induced adenylate cyclase phosphorylation

Receptor-mediated activation of both adenylate cyclase and phosphatidylinositide hydrolysis systems occurs through guanine nucleotide regulatory proteins and ultimately leads to specific activation of either cyclic AMP-dependent protein kinase A or Ca2+/phospholipid-dependent protein kinase C. Given the remarkable diversity of agents that influence cellular metabolism through these pathways and the similarities of their components, interactions between the two signalling systems could occur. In fact, stimulation of cells with 12-O-tetradecanoyl phorbol-13-acetate (TPA), a phorbol ester that activates protein kinase C, influences hormone-sensitive adenylate cyclase. In some cells TPA induces desensitization of receptor-mediated stimulation of adenylate cyclase, whereas in others, such as frog erythrocytes, phorbol ester treatment results in increased agonist-stimulated as well as basal, guanine nucleotide- and fluoride ion-stimulated adenylate cyclase activities. We show here that TPA produces phosphorylation of the catalytic unit of adenylate cyclase in frog erythrocytes. Moreover, purified protein kinase C can directly phosphorylate in vitro the catalytic unit of adenylate cyclase purified from bovine brain. These results suggest that phosphorylation of the catalytic unit of adenylate cyclase by protein kinase C may be involved in the phorbol ester-induced enhancement of adenylate cyclase activity. In addition to providing the first direct demonstration of a covalent modification of the catalytic unit of adenylate cyclase, these results provide a potential biochemical mechanism for a regulatory link between the two major transmembrane signalling systems.     

A nucleotide regulatory site for somatostatin inhibition of adenylate cyclase in S49 lymphoma cells

The cyc- variants of S49 lymphoma cells have served as powerful tools for studying the components and mechanisms of hormone-induced adenylate cyclase stimulation, as these cells are deficient in the guanine nucleotide regulatory site (Ns) mediating hormone, guanine nucleotide, cholera toxin and fluoride-induced stimulations of the enzyme. Because of this deficiency, membranes of these cells have been used for reconstitution of the system by inserting the coupling component derived from other cell types. The hormone-sensitive adenylate cyclase is not only stimulated by hormones but can also be inhibited by a wide variety of hormones and neurotransmitters, and there is some evidence that hormonal inhibition may be mediated by a distinct guanine nucleotide regulatory site. Studies in cyc- cells lacking a functional Ns may therefore answer this unresolved, important question. We have recently observed that stable GTP analogues can inhibit cyc- adenylate cyclase stimulated by purified, preactivated Ns or forskolin, which can activate adenylate cyclase even in the absence of a functional Ns (ref. 10). The data indicated that these Ns-deficient cells contain an inhibitory guanine nucleotide site, Ni. To strengthen this concept, we investigated whether the cyc- adenylate cyclase can be inhibited by a hormone. We report here that somatostatin decreases cyclic AMP levels in cyc- cells, inhibits the forskolin-stimulated adenylate cyclase and causes a concomitant increase in a high affinity GTPase activity in cyc- membranes. The data strongly suggest that both the hormone- and guanine nucleotide-induced adenylate cyclase inhibitions in cyc- cells are mediated by Ni and that the mechanisms of activation and inactivation of Ni are similar to those established for Ns.     

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.     

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.     

Differential regulation of rasGAP and neurofibromatosis gene product activities

The ras-encoded p21ras proteins bind GTP very tightly, but catalyse hydrolysis to GDP very slowly. In humans, two genes encode proteins that stimulate this GTPase activity (GAP, or GTPase-activating proteins), one of relative molecular mass 120,000, referred to as p120-GAP, and another NF1-GAP, which is encoded by the neurofibromatosis type-1 gene. Both GAPs are widely expressed in mammalian tissues. Here we show that although they will both bind oncogenic mutants of p21ras, neither will stimulate their GTPase activity. NF1-GAP binds to the p21ras proteins up to 300 times more efficiently than p120-GAP. The two GAPs are inhibited to different extents by certain lipids: micromolar concentrations of arachidonate, phosphatidate and phosphatidylinositol-4,5-bisphosphate affect only NF1-GAP. This inhibition does not compete with p21ras, and lipid-inactivated NF1-GAP can still bind p21ras. We used the detergent dodecyl maltoside, which inhibits only NF1-GAP, to distinguish between the two activities in cell extracts and found both types present together in several mammalian cell lines. In contrast, GAP activity in extracts of Xenopus oocytes was not affected by dodecyl maltoside. By these criteria, the mammalian cells contain both GAP activities and the oocytes have only p120-like GAP activity. These results indicate that more than one GAP regulates p21ras in the same cell.     

Normal p21N-ras couples bombesin and other growth factor receptors to inositol phosphate production

Many receptors, in response to ligand activation, trigger inositol phospholipid breakdown, which leads to rapid intracellular responses. The sustained activation of this pathway is believed to be at least one of the factors involved in the stimulation of cell growth and there has been much speculation that certain oncogenes use this pathway to effect uncontrolled cellular proliferation. It has been suggested, by analogy with the receptor-mediated control of adenylate cyclase, that the receptor stimulation of inositol phospholipid metabolism is mediated through a guanine nucleotide regulatory protein (G-protein) called Gp (or Np). Although such a species has not been identified, there is now strong experimental evidence that this process is mediated by a G-protein distinct from the stimulatory and inhibitory G-proteins (Gs and Gi, respectively). The ras genes code for a plasma membrane protein, p21, whose only known biochemical property is a high-affinity GTPase activity. We show here that the expression of normal p21N-ras in NIH 3T3 fibroblasts leads to the coupling of certain growth factor receptors to stimulated inositol phosphate production. We propose that the N-ras proto-oncogene encodes a protein which couples the receptors for certain growth factors to the stimulation of phospholipase C. Thus, N-ras p21 may be the putative Gp or a functionally related protein.     

Requirement for ras proto-oncogene function during serum-stimulated growth of NIH 3T3 cells

Human tumours often contain DNA sequences not found in normal tissues which are able to transform cultured NIH 3T3 cells. In some tumours the gene responsible for this transformation belongs to the cellular ras gene family. A specific type of mutation is responsible for converting the cellular proto-oncogene into a ras oncogene capable of inducing transformation. In a study of the function of a cellular ras gene, its protein product (produced in a bacterial cell) was microinjected into NIH 3T3 cells; the recipient cells became morphologically transformed and were induced to initiate DNA synthesis in the absence of added serum, but only when cellular ras protein was injected at much higher concentrations than required with protein of the transforming ras gene. To further analyse the function of the cellular ras gene, we have now injected monoclonal antibodies against ras proteins into NIH 3T3 cells. We report here that NIH 3T3 cells induced to divide by adding serum to the culture medium are unable to enter the S phase of the cell cycle after microinjection of anti-ras antibody, showing that the protein product of the ras proto-oncogene is required for initiation of the S-phase in NIH 3T3 cells.     

A product of the prune locus of Drosophila is similar to mammalian GTPase-activating protein.

The X-linked prune (pn) eye-colour mutation of Drosophila melanogaster has a highly specific, complementary lethal interaction with the conditional dominant Killer of prune (awdK-pn) mutation. Although awdK-pn flies have no apparent phenotype on their own, pn awdK-pn double mutants die as second or third larval instars. The awd locus encodes a nucleoside diphosphate kinase, an enzyme that catalyses the transfer of high-energy phosphate bonds between nucleoside diphosphates and nucleoside triphosphates, which is essential for the normal development of Drosophila. Analysis of the pn locus has suggested that the complementary DNA, TcD37, encodes a putative pn+ product. Here we report the nucleotide sequence of TcD37 and the similarity of its deduced protein product to the catalytic domain of mammalian GTPase-activating proteins (GAPs); GAPs stimulate the GTPase activity of Ras (ref. 6), which are plasma membrane-bound proteins involved in the regulation of cell proliferation and differentiation. These results suggest that the Drosophila TcD37 protein participates in a biochemical pathway similar to that of Ras and GAPs in mammals and yeast. We propose that the interaction between pn and awd is due to a neomorphic mutation that enhances the ability of AwdK-pn nucleoside diphosphate kinase to induce a regulatory GTPase into a GTP-bound 'on' state, whereas Pn modulates the activity of this GTPase either by switching it to a GDP-bound 'off' state or by interfering with its effector function.     

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.     

盘基网柄菌细胞发育中gp150分子与凋亡蛋白作用分析

用Percoll密度梯度技术分离和收集盘基网柄菌前柄和前孢子细胞,Western blot分析gp150分子和胱天蛋白酶在前孢子细胞和前柄细胞两种类型细胞中的表达情况.结果显示：只能在前柄细胞中检测到gp150蛋白条带,并随细胞发育蛋白的量逐渐增加,提示gp150蛋白的表达量与发育时间,前柄细胞分化有密切关系;在前柄细胞中能检测到31.5 kD和37.5 kD分子量大小的凋亡蛋白,且蛋白量也是随发育时间有所增加,在两种类型细胞中都可检测38.2 kD的凋亡蛋白.这些数据表明盘基网柄菌细胞凋亡过程中有类似Caspase-3的蛋白表达,它们的存在与细胞凋亡存在密切关系; gp150分子的表达与胱天蛋白酶的激活可能存在一定关系.     

Altered Gs and adenylate cyclase activity in human GH-secreting pituitary adenomas

Gs and Gi are guanine nucleotide-binding, heterotrimer proteins that regulate the activity of adenylate cyclase, and are responsible for transferring stimulatory and inhibitory hormonal signals, respectively, from cell surface receptors to the enzyme catalytic unit. These proteins can be directly activated by agents such as GTP and analogues, fluoride and magnesium. Decreased amounts of Gs and Gi, and even the absence of Gs, have been described, whereas an altered Gs has been reported in a cultured cell line (UNC variant of S49 lymphoma cells), but has never been observed in human disease states. We have found a profoundly altered Gs protein in a group of human growth hormone-secreting pituitary adenomas, characterized by high secretory activity and intracellular cyclic AMP levels. In the membranes from these tumours no stimulation of adenylate cyclase activity by growth hormone-releasing hormone, by GTP or by fluoride was observed. Indeed, the last two agents caused an inhibition, probably mediated by Gi. In contrast, adenylate cyclase stimulation by Mg2+ was enormously increased. This altered pattern of adenylate cyclase regulation was reproduced when a cholate extract of the tumour membranes (which contains G proteins) was reconstituted with Gs-free, cyc- S49 cell membranes. Inasmuch as secretion from somatotrophic cells is known to be a cAMP-dependent function, the alteration of Gs could be the direct cause of the high secretory activity of the tumours in which it occurs.     

Monoclonal antibodies define differential ras gene expression in malignant and benign colonic diseases

DNAS of some human tumours can transform NIH 3T3 fibroblast cells, thus demonstrating the transforming potential of human ras genes (Hu-rasHa, Hu-rasKi, and Hu-rasN, respectively Harvey, Kirsten and neuroblastoma ras genes). Only a small percentage of a given type of human carcinoma, however, scores positive in this assay system. Activation of ras and subsequent transformation of NIH 3T3 cells are either by a point mutation in the ras gene or enhanced expression of the normal, or proto-onc, ras gene. If the transformation of a given human tumour involves the enhanced expression of the normal or cellular ras gene and the resulting gene product, the tumour DNA would probably score negative in the NIH 3T3 transfection assay. In human colon carcinoma, for example, lesions at position 12 of Hu-rasKi have been found. None of nine colon carcinomas obtained at biopsy, however, contain the ras lesion at this position, using a Hu-rasHa probe; one other colon carcinoma does appear to contain amplified proto-onc ras, and other colon carcinomas do have increased levels of ras RNA. There are at least three explanations for these observations. Either very few colon carcinomas contain point-mutated ras, the lesion in the majority of colon carcinomas is at a position other than 12 or ras activation in many colon carcinomas involves the enhanced expression of either the point-mutated or proto-onc form of a ras gene. We have now used monoclonal antibodies directed against a synthetic peptide reflecting sequences of the human T24 ras gene product to define ras p21 protein expression in a spectrum of colonic disease states. Immunohistochemical analyses of individual cells within tissue sections reveal differences in ras p21 expression in colon carcinomas compared with normal colonic epithelium, benign colon tumours and inflammatory or dysplastic colon lesions. Our data suggest that ras p21 expression is correlated with depth of carcinoma within the bowel wall, and is probably a relatively late event in colon carcinogenesis.     

Abolition of the expression of inhibitory guanine nucleotide regulatory protein Gi activity in diabetes

Many cell-surface receptors for hormones appear to exert their effects on target cells by interacting with specific guanine nucleotide binding regulatory proteins (G-proteins) which couple receptors to their second-messenger signal generation systems. A common intracellular second messenger, which is used by many hormones, is cyclic AMP. This is produced by adenylate cyclase, whose activity is controlled by two G-proteins, Gs which mediates stimulatory effects and Gi inhibitory effects on adenylate cyclase activity. In liver, the hormone glucagon increases intracellular cAMP concentrations by activating adenylate cyclase by a Gs-mediated process. This effect of glucagon is antagonised by the hormone insulin, although the molecular mechanism by which insulin elicits its actions is obscure. However, insulin receptors exhibit a tyrosyl kinase activity and appear to interact with G-proteins, perhaps by causing phosphorylation of them. In type I diabetes, circulating insulin levels are abnormally low, giving rise to gross perturbations of metabolism as well as to a variety of complications such as ionic disturbances, neuropathies of the nervous system, respiratory and cardiovascular aberrations and predisposition to infection. We show here that experimentally-induced type I diabetes leads to the loss of expression of Gi in rat liver. As it has been suggested that Gi may couple receptors to K+-channels as well as mediating the inhibition of adenylate cyclase, aberrations in the control of expression of this key regulatory protein in type I diabetes may be expected to lead to pleiotropic effects.     

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.