Mechanism of inhibition of polypeptide chain initiation in calcium-depleted Ehrlich ascites tumor cells.

Protein synthesis in Ehrlich ascites tumor cells is inhibited when cellular calcium is depleted by the addition of EGTA to the growth medium. This inhibition is at the level of polypeptide chain initiation as evidenced by a disaggregation of polyribosomes accompanied by a significant elevation in 80-S monomers. To identify direct effects of calcium on the protein synthesis apparatus we have developed a calcium-dependent, cell-free protein-synthesizing system from the Ehrlich cells by using 1,2-bis(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA), a recently developed chelator with a high (greater than 10(5)) selectivity for calcium (pKa = 6.97) over magnesium (pKa = 1.77). BAPTA inhibits protein synthesis by 70% at 1 mM and 90% at 2 mM. This effect was reversed by calcium but not by other cations tested. The levels of 43-S complexes (i.e., 40-S subunits containing bound methionyl-tRNAf.eIF-2.GTP) were significantly lower in the calcium-deprived incubations, indicating either inhibition of the rate of formation or decreased stability of 43-S complexes. Analysis of 43-S complexes on CsCl gradients showed that in BAPTA-treated lysates, 40-S subunits containing eIF-3, completely disappeared and the residual methionyl-tRNA-containing complexes were bound to 40-S subunits lacking eIF-3. Our results demonstrate a direct involvement of Ca2+ in protein synthesis and we have localized the effect of calcium deprivation to decreased binding of eIF-2 and eIF-3 to 40-S subunits.

A cytosolic protein catalyzes the release of GDP from p21ras.

The rate of release of guanine nucleotides from the ras proteins (Ras) is extremely slow in the presence of Mg2+. It seemed likely, therefore that a factor might exist to accelerate the release of guanosine diphosphate (GDP), and hence the exchange of GDP for guanosine triphosphate (GTP). Such a factor has now been discovered in rat brain cytosol. Brain cytosol was found to catalyze, by orders of magnitude, the release of guanine nucleotides from recombinant v-H-Ras protein bound with [alpha-32P]GDP. This effect occurred even in the presence of a large excess of Mg2+, but was destroyed by heat or by incubation of the cytosol for an hour at 37 degrees C in the absence of phosphatase inhibitors. The effect was observed with either v-H-Ras or c-H-Ras, but not with p25rab3A, a small G protein with about 30% similarity to Ras. The effect could not be mimicked by addition of recombinant Ras-GAP or purified GEF, a guanine nucleotide exchange factor involved in the regulation of eukaryotic protein synthesis. By gel filtration chromatography, the factor appears to possess a molecular size between 100,000 and 160,000 daltons. This protein (Ras-guanine nucleotide-releasing factor, or Ras-GRF) may be involved in the activation of p21ras.

Complexes of Ras.GTP with Raf-1 and mitogen-activated protein kinase kinase.

The guanosine triphosphate (GTP)-binding protein Ras functions in regulating growth and differentiation; however, little is known about the protein interactions that bring about its biological activity. Wild-type Ras or mutant forms of Ras were covalently attached to an insoluble matrix and then used to examine the interaction of signaling proteins with Ras. Forms of Ras activated either by mutation (Gly12Val) or by binding of the GTP analog, guanylyl-imidodiphosphate (GMP-PNP) interacted specifically with Raf-1 whereas an effector domain mutant, Ile36Ala, failed to interact with Raf-1. Mitogen-activated protein kinase (MAP kinase) activity was only associated with activated forms of Ras. The specific interaction of activated Ras with active MAP kinase kinase (MAPKK) was confirmed by direct assays. Thus the forming of complexes containing MAPKK activity and Raf-1 protein are dependent upon the activity of Ras.

Inhibition of the EGF-activated MAP kinase signaling pathway by adenosine 3',5'-monophosphate.

Mitogen-activated protein (MAP) kinases p42mapk and p44mapk are activated in cells stimulated with epidermal growth factor (EGF) and other agents. A principal pathway for MAP kinase (MAPK) activation by EGF consists of sequential activations of the guanine nucleotide exchange factor Sos, the guanosine triphosphate binding protein Ras, and the protein kinases Raf-1, MAPK kinase (MKK), and MAPK. Because adenosine 3',5'-monophosphate (cAMP) does not activate MAPK and has some opposing physiologic effects, the effect of increasing intracellular concentrations of cAMP with forskolin and 3-isobutyl-1-methylxanthine on the EGF-stimulated MAPK pathway was studied. Increased concentrations of cAMP blocked activation of Raf-1, MKK, and MAPK in Rat1hER fibroblasts, accompanied by a threefold increase in Raf-1 phosphorylation on serine 43 in the regulatory domain. Phosphorylation of Raf-1 in vitro and in vivo reduces the apparent affinity with which it binds to Ras and may contribute to the blockade by cAMP.

The 3Rs of life: Ras, Raf and growth regulation.

Cellular Ras proteins are essential elements in normal signal transduction pathways while activated Ras proteins are prevalent in many different forms of human cancers. Here, we discuss the mechanism through which Ras proteins, either cellular or activated, transmit a proliferative signal by activating cytoplasmic serine/threonine kinases.

The mitogen-activated protein kinase cascade is activated by B-Raf in response to nerve growth factor through interaction with p21ras.

Nerve growth factor (NGF) activates the mitogen-activated protein (MAP) kinase cascade through a p21ras-dependent signal transduction pathway in PC12 cells. The linkage between p21ras and MEK1 was investigated to identify those elements which participate in the regulation of MEK1 activity. We have screened for MEK activators using a coupled assay in which the MAP kinase cascade has been reconstituted in vitro. We report that we have detected a single NGF-stimulated MEK-activating activity which has been identified as B-Raf. PC12 cells express both B-Raf and c-Raf1; however, the MEK-activating activity was found only in fractions containing B-Raf. c-Raf1-containing fractions did not exhibit a MEK-activating activity. Gel filtration analysis revealed that the B-Raf eluted with an apparent M(r) of 250,000 to 300,000, indicating that it is present within a stable complex with other unidentified proteins. Immunoprecipitation with B-Raf-specific antisera quantitatively precipitated all MEK activator activity from these fractions. We also demonstrate that B-Raf, as well as c-Raf1, directly interacted with activated p21ras immobilized on silica beads. NGF treatment of the cells had no effect on the ability of B-Raf or c-Raf1 to bind to activated p21ras. These data indicate that this interaction was not dependent upon the activation state of these enzymes; however, MEK kinase activity was found to be associated with p21ras following incubation with NGF-treated samples at levels higher than those obtained from unstimulated cells. These data provide direct evidence that NGF-stimulated B-Raf is responsible for the activation of the MAP kinase cascade in PC12 cells, whereas c-Raf1 activity was not found to function within this pathway.

Association of MEK1 with p21ras.GMPPNP is dependent on B-Raf.

We have previously reported that immobilized p21ras forms a GMPPNP-dependent complex with a MEK activity. Furthermore, the association of the MEK activity was found to be independent of the presence of Raf-1. We have extended those observations to show that MEK1 is the MEK activity previously described to associate with immobilized p21ras.GMPPNP. The association between MEK1 and immobilized p21ras.GMPPNP increased its specific activity towards p42MAPK. We detected the specific association of B-Raf with immobilized p21ras.GMPPNP. In contrast to Raf-1-immunodepleted lysates, preclearance of the cytosolic B-Raf significantly reduced, by 96%, the amount of MEK1 activity associated with immobilized p21ras.GMPPNP. The decrease in MEK1 activity correlated with complete loss in the binding of both B-Raf and MEK1 proteins with immobilized p21ras.GMPPNP. These data suggest that the p21ras.GMPPNP-dependent activation of MEK1 in brain extracts is dependent on the presence of the B-Raf protein kinase.

RAS and RAF-1 form a signalling complex with MEK-1 but not MEK-2.

Recent studies have demonstrated the existence of a physical complex containing p21ras (RAS), p74raf-1 (RAF-1), and MEK-1. Although it is clear that formation of this complex depends on the activation state of RAS, it is not known whether this complex is regulated by the activation state of the cell and whether MEK-2 is also present in the complex. To analyze the regulation and specificity of this complex, we utilized immobilized RAS to probe lysates of cultured NIH 3T3 fibroblasts and analyzed the proteins complexing with RAS following serum starvation or stimulation. Complex formation among RAS, RAF-1, and MEK-1 was dependent only on RAS:GMP-PNP and not on cell stimulation. Incubations of lysates with immobilized RAS depleted all RAF-1 from the lysate but bound only a small fraction of cytosolic MEK-1, and further MEK-1 could bind immobilized RAS only if exogenous RAF-1 was added to the lysate. This indicates that binding of MEK-1 to RAS depends on the presence of RAF-1 or an equivalent protein. In contrast to MEK-1, MEK-2 was not detected in the RAS signalling complex. A proline-rich region of MEK-1 containing a phosphorylation site appears to be essential for signalling complex formation. Consistent with the preferential binding of MEK-1 to RAS:RAF-1, the basal activity of MEK-1 in v-ras-transformed cells was found to be elevated sixfold, whereas MEK-2 was elevated only twofold, suggesting that the RAS signalling pathway favors MEK-1 activation.

Mechanism of inhibition of Raf-1 by protein kinase A.

The cytoplasmic Raf-1 kinase is essential for mitogenic signalling by growth factors, which couple to tyrosine kinases, and by tumor-promoting phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate, which activate protein kinase C (PKC). Signalling by the Raf-1 kinase can be blocked by activation of the cyclic AMP (cAMP)-dependent protein kinase A (PKA). The molecular mechanism of this inhibition is not precisely known but has been suggested to involve attenuation of Raf-1 binding to Ras. Using purified proteins, we show that in addition to weakening the interaction of Raf-1 with Ras, PKA can inhibit Raf-1 function directly via phosphorylation of the Raf-1 kinase domain. Phosphorylation by PKA interferes with the activation of Raf-1 by either PKC alpha or the tyrosine kinase Lck and even can downregulate the kinase activity of Raf-1 previously activated by PKC alpha or amino-terminal truncation. This type of inhibition can be dissociated from the ability of Raf-1 to associate with Ras, since (i) the isolated Raf-1 kinase domain, which lacks the Ras binding domain, is still susceptible to inhibition by PKA, (ii) phosphorylation of Raf-1 by PKC alpha alleviates the PKA-induced reduction of Ras binding but does not prevent the downregulation of Raf-1 kinase activity by PKA and (iii) cAMP agonists antagonize transformation by v-Raf, which is Ras independent.

Ha-ras and N-ras regulate MAPK activity by distinct mechanisms in vivo.

The Ras GTPases function as molecular switches, regulating a multiplicity of biological events. However the contribution, if any, of a specific c-Ras isoform (Ha-, N-, or Ki-ras A or B) in the regulation of a given biological or biochemical process, is unknown. Murine C3H1OT1/2 fibroblasts transformed with activated (G12V)Ha-ras or (Q61K)N-ras proliferate in serum-free media and have constitutive MAPK activity. The growth factor antagonist, suramin, inhibited the serum-independent proliferation of Ha-ras transformed fibroblasts, but not the serum-independent proliferation of N-ras transformed cells. The inhibition of cell proliferation was concomitant with the abrogation of the constitutive MAPK activity in the Ha-ras transformed fibroblasts. Analysis of the Ras-signalling complexes in immunoprecipitates from Ha-ras transformed cells revealed that Raf-1 co-immunoprecipitated with endogenous c-N-ras but not (G12V)Ha-ras. Pretreatment with suramin resulted in the loss of Raf-1 from c-N-ras immunoprecipitates. A c-N-ras antisense oligonucleotide, which down-regulated c-N-ras protein levels, abrogated the constitutive MAPK activity and serum-independent proliferation of (G12V)Ha-ras transformed cells. The data suggest that Raf-1 has a higher affinity for N-ras then Ha-ras in vivo, and c-N-ras function is required for the serum-independent proliferation of Ha-ras transformed cells.

Oncogenic Ras-induced secretion of a novel inhibitor of skeletal myoblast differentiation.

Expression of oncogenic H-Ras in 23A2 myoblasts (A2:H-Ras cells) is sufficient to induce both a transformed phenotype and a differentiation-defective phenotype. Because oncogenic Ras is known to induce the secretion of several different growth factors involved in maintaining the transformed phenotype of both fibroblast and epithelial cells, we explored the possibility that expression of oncogenic Ras in 23A2 myoblasts might lead to the secretion of a factor which inhibits differentiation. The differentiation of 23A2 myoblasts was inhibited (i) by coculture with an equal number of A2:H-Ras cells, (ii) by culture with an equal number of A2:H-Ras cells in the same tissue culture medium on an insert which allowed equilibration of molecules smaller than 1 micron, and (iii) by culture in media previously conditioned by A2:H-Ras cells. Similar results were obtained when 23A2 myoblasts expressing oncogenic N-Ras were substituted for A2:H-Ras cells in each assay. No inhibition of differentiation was observed, however, when differentiation-defective E1A-expressing 23A2 cells or C3H10T1/2 fibroblasts were substituted for A2:H-Ras cells. The differentiation inhibitor(s) in media conditioned by A2:H-Ras cells is heat stable, larger than 3 kD, and sensitive to the non-specific growth factor antagonist, suramin. Western analyses failed to detect either FGF-2 or TGFbeta (the known inhibitors of myoblast differentiation) in media conditioned by A2:H-Ras cells. Furthermore, while FGF-2 is a potent activator of MAP kinase and TGFbeta is a potent inhibitor of mink lung epithelial cell (CCL64) growth, conditioned media from A2:H-Ras cells does not activate MAP kinase and does not inhibit the growth of CCL64 cells. These results indicate that expression of oncogenic Ras induces the secretion of a novel inhibitor of skeletal myoblast differentiation. Furthermore, these results are the first to implicate an autocrine/paracrine mechanism in the inhibition of differentiation by oncogenic Ras.

Differences in the interaction of p21c-Ha-ras-GMP-PNP with full-length neurofibromin and GTPase-activating protein.

Neurofibromin, the product of the neurofibromatosis type 1 gene, was found to form a stable complex with immobilized p21c-Ha-ras-GMP-PNP (a non-hydrolyzable GTP analog). This complex, detectable as early as 30 min after addition of crude brain extract, is extremely stable, with less than 50% dissociating after 5 h at 4 degrees C. We interpret this to suggest that the dissociation of full-length neurofibromin from p21c-Ha-ras-GMP-PNP is tightly linked to the hydrolysis of GTP to GDP. Failure to remove a significant proportion of the bound neurofibromin in the presence of EDTA and GDP implies that the binding of neurofibromin to p21c-Ha-ras-GMP-PNP results in the ras protein becoming resistant to guanine nucleotide exchange. Under conditions in which neurofibromin quantitatively binds to p21c-Ha-ras-GMP-PNP, we were unable to detect a complex between p21c-Ha-ras and GAP (GTPase-activating protein). The failure to detect GAP binding to immobilized p21c-Ha-ras-GMP-PNP cannot be explained by the known differences in affinities of the GAP-related domain of neurofibromin and GAP for p21c-Ha-ras-GTP. GAP is, however, able to interact biochemically with immobilized p21c-Ha-ras, suggesting a difference in the interaction between GAP and neurofibromin with p21c-Ha-ras-GMP-PNP.

Catalytic properties, tissue and intracellular distribution of neurofibromin.

The neurofibromatosis type 1 (NF1) gene encodes a protein, neurofibromin, that shows homology with members of the GTPase-activating protein (GAP) family. To study neurofibromin, rabbit polyclonal antisera were raised against two synthetic peptides. These antisera immunoprecipitated a specific protein of about 240 kDa in lysates of adult murine and rat tissues both in the soluble (S100) and to a lesser degree in the particulate (P100) fractions. The neurofibromin immunoprecipitated from the lysates of several murine organs stimulated the intrinsic GTPase activity of p21 c-Ha-ras protein. Based on immunoblotting, immunoprecipitation and GTPase assays, neurofibromin appears to be at least 10-fold more abundant in the brain than in the other murine organs. The GTPase-stimulatory activity of full-length neurofibromin, like the catalytic GAP-related domain, is inhibited by arachidonic acid and the detergent dodecyl maltoside, while phosphatidic acid, containing arachidonic and stearic acid, is non-inhibitory. Immunofluorescence analysis with anti-neurofibromin sera in NIH3T3 cells suggests that at least some of the cellular protein associates with cytoplasmic structures that are distinct from actin or tubulin filaments.

Distinct signaling pathways regulate transformation and inhibition of skeletal muscle differentiation by oncogenic Ras.

Expression of oncogenic Ras in 23A2 skeletal myoblasts is sufficient to induce both a transformed phenotype and a differentiation-defective phenotype, but the signaling pathways activated by oncogenic Ras in these cells and their respective contribution to each phenotype have not been explored. In this study, we investigated MAP kinase activity in control 23A2 myoblasts and in 23A2 myoblasts rendered differentiation-defective by the stable expression of an oncogenic (G12V)Ha-ras gene (Ras9 cells). The MAP kinase immunoprecipitated from Ras9 cells was 30-40% more active than that from control 23A2 cells. To establish if this elevated MAP kinase activity is essential to the maintenance of the oncogenic Ras-induced phenotype, we utilized the selective MAP kinase kinase 1 (MEK1) inhibitor, PD 098059. PD 098059 decreased the MAP kinase activity in Ras9 cells to the level found in 23A2 cells. PD 098059 did not affect the ability of 23A2 myoblasts to differentiate. PD 098059 reverted the transformed morphology of Ras9 cells but did not restore the ability of these cells to express the muscle-specific myosin heavy chain gene or to form muscle fibers. Treatment with PD 098059 also did not affect the ability of oncogenic Ha-Ras to establish a non-myogenic phenotype in C3H10T1/2 cells co-expressing MyoD. These results demonstrate that the activation of MAP kinase is necessary for the transformed morphology of Ras9 cells but is not required by oncogenic Ras to establish or to maintain a differentiation-defective phenotype. While these data do not rule out the possibility that constitutive signaling by MEK1 or MAP kinase could inhibit myoblast differentiation, they clearly demonstrate that other pathways activated by oncogenic Ras are sufficient to inhibit differentiation.

Cloning of TACC1, an embryonically expressed, potentially transforming coiled coil containing gene, from the 8p11 breast cancer amplicon.

Amplification of several chromosomal regions have been observed in human breast carcinomas. One such region, 8p11, is amplified in 10-15% of tumor samples. Although the FGFR1 gene is located close to this region, and is often included within the amplicon, the observation that tumors exhibiting 8p11 amplification do not always overexpress FGFR1 suggests that another gene located close to FGFR1 is involved in the tumorigenic process. We now report the precise location of four expressed sequence tags (ESTs) within this region and the cloning of a novel gene, designated TACC1 (transforming acidic coiled coil gene 1), which encodes an 8 kb transcript and which is expressed at high levels during early embryogenesis. Constitutive expression of this gene under the control of the cytomegalovirus (CMV) promoter in mouse fibroblasts, results in cellular transformation and anchorage independent growth, suggesting that inappropriate expression can impart a proliferative advantage. This observation raises the possibility that amplification of TACC1 could promote malignant growth, thereby making TACC1 an attractive candidate for the gene promoting tumorigenicity as a result of the 8p11 amplification in human breast cancers.

Different structural requirements within the switch II region of the Ras protein for interactions with specific downstream targets.

Ras proteins function through the formation of specific complexes with Raf-1, B-raf, PI-3 kinase and RalGDS. These interactions all require Ras-GTP with an intact effector binding domain (Switch I region). We have examined the requirements of the Switch II region (amino acids 60-72) for the production of stable interactions between Ras and its downstream effectors. A point mutation at position 65 or 64 combined with additional mutations at either position 65 or 71 rendered nucleotide-free Ras protein unable to stably interact with Ras specific guanine nucleotide exchange factors. Ha-Ras containing point mutations at positions 65 and 71 possessed a twofold higher affinity for B-raf and consequently MEK1. The point mutation at 64, in combination with additional point mutations at either position 65 or 71, resulted in a protein which failed to interact with either PI-3 kinase or neurofibromin, though these Ras mutants effectively bound both Raf-1 and B-raf. An activated form of Ras, Q61L-Ras, associated with all effector proteins independent of the bound guanine nucleotide. Q61L-Ras-GDP was almost as effective as wild type Ras-GMPPNP in the in vitro activation of MEK1 and MAP kinase. Competitive studies with the catalytic domain if neurofibromin, NF1-GRD, demonstrated that its interaction with Ras-GMPPNP is mutually exclusive with both Raf-1 and B-raf. These data suggest that rasGAP and neurofibromin are unable to downregulate Ras-GTP complexed to Raf-1 or B-raf.

Regulation of p53 expression by the RAS-MAP kinase pathway.

Activation of MAP kinase leads to the activation of p53-dependent pathways, and vice-versa. Although the amount of p53 protein increases in response to MAP kinase-dependent signaling, the basis of this increase is not yet fully understood. We have isolated the mutant cell line AP14, defective in p53 expression, from human HT1080 fibrosarcoma cells, which have an activated ras allele. The expression of p53 mRNA and protein is approximately 10-fold lower in AP14 cells than in the parental cells. The high constitutive phosphorylation and activities of the MAP kinases ERK1 and ERK2 in HT1080 cells are greatly reduced in AP14 cells, although the levels of these proteins are unchanged, suggesting that the defect in the mutant cells affects the steady-state phosphorylation of ERK1 and ERK2. Overexpression of ERK2 in AP14 cells restored both MAP kinase activity and p53 expression, and incubation of the mutant cells with the phosphatase inhibitor orthovanadate resulted in strong coordinate elevation of MAP kinase activity and p53 expression. The levels of expression of the p53-regulated gene p21 parallel those of p53 throughout, showing that basal p21 expression depends on p53. The levels of p53 mRNA increased by 5-8-fold when activated ras was introduced into wild-type cells, and the levels of the p53 and p21 proteins decreased substantially in wild-type cells treated with the MEK inhibitor U0216. We conclude that MAP kinase-dependent pathways help to regulate p53 levels by regulating the expression of p53 mRNA.

Ras isoform-specific signaling: location, location, location.

The proteins that compose the Ras family of small guanosine triphosphatases share a remarkably high degree of sequence similarity, yet recent evidence indicates that they may have unique biological properties. How is it that similar proteins carry out different jobs in the cell? Wolfman addresses this question by surveying recent reports that indicate that different biological roles may be born out of distinct subcellular localizations of the Ras proteins. It appears that the small differences in their amino acid sequences and their different posttranslational modifications may be all that is necessary to direct various Ras proteins to different sites.

Signal transduction and the ras gene family: molecular switches of unknown function.

The ras family consists of 20 or more genes that encode small GTP/GDP-binding proteins, of 20-26 kDa, the functions of which are unknown. This article discusses possible roles of the ras proteins in signal transduction and the interaction of p21(ras) and other members of the ras family with GTPase-accelerating proteins (GAPS) that may be regulatory elements of the signaling machinery.

Mitogen-activated protein kinase kinase (MEK) activity is required for inhibition of skeletal muscle differentiation by insulin-like growth factor 1 or fibroblast growth factor 2.

Both insulin-like growth factor 1 (IGF-1) and fibroblast growth factor 2 (FGF-2) are key modulators of skeletal myoblast differentiation. The critical signaling pathways used by either IGF-1 or FGF-2 to inhibit differentiation have not been determined. In this study, we show that both IGF-1 and FGF-2 inhibit the differentiation of 23A2 myoblasts and that both stimulate signaling through mitogen-activated protein kinase (MAPK) kinase (MEK) to MAPK roughly 8-fold in 23A2 myoblasts. We used the selective chemical inhibitor of MEK, PD 098059, to determine if signaling by MEK is required by IGF-1 or FGF-2 to inhibit differentiation. PD 098059 did not affect the ability of 23A2 myoblasts to differentiate. Addition of PD 098059 to the culture medium 10 min before the addition of IGF-1 or FGF-2 completely blocked the signal from MEK to MAPK and restored the ability of the 23A2 myoblasts to differentiate in the presence of either IGF-1 or FGF-2. The peak of signaling through MEK to MAPK in response to either IGF-1 or FGF-2 occurred within the first hour with maximal activation observed after 10 min. This signal remained elevated (at roughly 70% above basal) for at least 48 h. PD 098059 was added to the culture 60 min after IGF-1 or FGF-2 to test whether this initial peak of signaling was sufficient for the inhibition of differentiation. The restoration of myogenic potential seen when cells were preincubated with PD 098059 was essentially identical to that seen when PD 098059 was added to cultures after the initial peak of signaling from MEK to MAPK, suggesting that persistent signaling through MEK is required for the inhibition of differentiation by either IGF-1 or FGF-2.