Identification of a c-Jun N-terminal kinase-2-dependent signal amplification cascade that regulates c-Myc levels in ras transformation.

Ras is one of the most frequently activated oncogenes in cancer. Two mitogen-activated protein kinases (MAPKs) are important for ras transformation: extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase 2 (JNK2). Here we present a downstream signal amplification cascade that is critical for ras transformation in murine embryonic fibroblasts. This cascade is coordinated by ERK and JNK2 MAPKs, whose Ras-mediated activation leads to the enhanced levels of three oncogenic transcription factors, namely, c-Myc, activating transcription factor 2 (ATF2) and ATF3, all of which are essential for ras transformation. Previous studies show that ERK-mediated serine 62 phosphorylation protects c-Myc from proteasomal degradation. ERK is, however, not alone sufficient to stabilize c-Myc but requires the cooperation of cancerous inhibitor of protein phosphatase 2A (CIP2A), an oncogene that counteracts protein phosphatase 2A-mediated dephosphorylation of c-Myc. Here we show that JNK2 regulates Cip2a transcription via ATF2. ATF2 and c-Myc cooperate to activate the transcription of ATF3. Remarkably, not only ectopic JNK2, but also ectopic ATF2, CIP2A, c-Myc and ATF3 are sufficient to rescue the defective ras transformation of JNK2-deficient cells. Thus, these data identify the key signal converging point of JNK2 and ERK pathways and underline the central role of CIP2A in ras transformation.

Sensitivity of wild type and mutant ras alleles to Ras specific exchange factors: Identification of factor specific requirements.

We have investigated the productive interaction between the four mammalian Ras proteins (H-, N-, KA- and KB-Ras) and their activators, the mammalian exchange factors mSos1, GRF1 and GRP, by using a modified Saccharomyces cerevisiae whose growth is dependent on activation of a mammalian Ras protein by its activator. All four mammalian Ras proteins were activated with similar efficiencies by the individual exchange factors. The H-Ras mutant V103E, which is competent for membrane localization, nucleotide binding, intrinsic and stimulated GTPase activity as well as intrinsic exchange, was defective for activation by all factors tested, suggesting that the integrity of this residue is necessary for catalyzed exchange. However, when other H-Ras mutants were studied, some distinct sensitivities to the exchange factors were observed. GRP-mediated, but not mSos1-mediated, exchange was blocked in additional mutants, suggesting different structural requirements for GRP. Analysis of Ras-mediated gene activation in murine fibroblasts confirmed these results.

Oncogenic Ras induces p19ARF and growth arrest in mouse embryo fibroblasts lacking p21Cip1 and p27Kip1 without activating cyclin D-dependent kinases.

Oncogenic Ras induces two products of the INK4a/ARF tumor suppressor locus (p16(INK4a) and p19(ARF)) in primary human and rodent fibroblasts, ultimately leading to a permanent state of cell cycle arrest resembling replicative senescence. Whereas p16(INK4a) antagonizes the activities of cyclin D-dependent kinases, p19(ARF) activates the p53 transcription factor. Immortalized rodent fibroblast cell lines that lack INK4a/ARF function, ARF alone, or p53 are resistant to the growth inhibitory effects of oncogenic Ras and instead continue to proliferate and undergo morphological transformation. Primary mouse embryo fibroblasts lacking Cip1 and Kip1 genes encoding inhibitors of cyclin-dependent kinase-2 were used to further explore the effects of oncogenic Ras on arrest of the cell division cycle. Although early passage primary fibroblast strains that lack both p21(Cip1) and p27(Kip1) fail to assemble cyclin D-dependent kinases, oncogenic Ras retained its ability to induce p19(ARF), but not p16(INK4a), protecting Cip/Kip-null cells from proliferating and undergoing transformation. Under these conditions, Ras did not induce G(1) phase arrest but instead triggered DNA synthesis, abnormal nuclear divisions, failure of cytokinesis, and emergence of polyploid cells. Therefore, in the absence of p16(INK4a), p21(Cip1), and p27(Kip1), oncogenic Ras affects the functions of genes required for completion of the cell cycle.

Rac and Cdc42 are potent stimulators of E2F-dependent transcription capable of promoting retinoblastoma susceptibility gene product hyperphosphorylation.

The Rho family of GTPases plays an important and diverse role in reorganization of the actin cytoskeleton, transcriptional regulation, and multiple aspects of cell growth. Our study has examined their potential links to the cell cycle machinery. We find that constitutively active mutants of Rac and Cdc42, but not Rho, are potent inducers of E2F transcriptional activity in NIH 3T3 fibroblasts. Furthermore, activated Rac and Cdc42, but again not Rho, are capable of inducing cyclin D1 accumulation and pRB hyperphosphorylation in serum-deprived cells, outlining one route leading to enhanced E2F-mediated transcription. The inhibitory effect of the cyclin-dependent kinase inhibitors, p16(ink4), p21(cip1), and p27(cip) on Rac/Cdc42-mediated E2F transcription corroborates a role for pRB family members and their functional inactivation by cyclin-dependent kinases in generating E2F activity. While the up-regulation of E2F transcriptional activity by Rac or Cdc42, not Rho, suffices for entry into S phase and DNA synthesis in Rat-1 R12 cells, this is clearly not the case in NIH 3T3, where additional requirements must exist.

The Ras-specific exchange factors mouse Sos1 (mSos1) and mSos2 are regulated differently: mSos2 contains ubiquitination signals absent in mSos1.

We have compared aspects of the mouse sos1 (msos1) and msos2 genes, which encode widely expressed, closely related Ras-specific exchange factors. Although an msos1 plasmid did not induce phenotypic changes in NIH 3T3 cells, addition of a 15-codon myristoylation signal to its 5' end enabled the resulting plasmid, myr-sos1, to induce approximately one-half as many foci of transformed cells as a v-H-ras control. By contrast, an isogenic myr-sos2 plasmid, which was made by fusing the first 102 codons from myr-sos1 at homologous sequences to an intact msos2 cDNA, did not induce focal transformation directly, although it could form foci in cooperation with c-H-ras. Pulse-chase experiments indicated that the half-life of Sos1 in NIH 3T3 cells was greater than 18 h, while that of Sos2 was less than 3 h. While in vitro-translated Sos1 was stable in a rabbit reticulocyte lysate, Sos2 was degraded in the lysate, as were each of two reciprocal chimeric Sos1-Sos2 proteins, albeit at a slower rate. In the lysate, Sos2 and the two chimeric proteins could be stabilized by ATPgammaS. Unlike Sos1, Sos2 was specifically immunoprecipitated by antiubiquitin antibodies. In a myristoylated version, the chimeric gene encoding Sos2 at its C terminus made a stable protein in NIH 3T3 cells and induced focal transformation almost as efficiently as myr-msos1, while the myristoylated protein encoded by the other chimera was unstable and defective in the transformation assay. We conclude that mSos2 is much less stable than mSos1 and is degraded by a ubiquitin-dependent process. A second mSos2 degradation signal, mapped to the C terminus in the reticulocyte lysate, does not seem to function under the growth conditions of the NIH 3T3 cells.

Rasp21 sequences opposite the nucleotide binding pocket are required for GRF-mediated nucleotide release.

The substrate requirements for the catalytic activity of the mouse Cdc25 homolog Guanine nucleotide Release Factor, GRF, were determined using the catalytic domain of GRF expressed in insect cells and E. coli expressed H-Ras mutants. We found a requirement for the loop 7 residues in Ras (amino acids 102 to 105) for stimulation of guanine nucleotide release. The dependence on the switch I and II regions of Rasp21 (encompassing the residues that shift position in the GTP- versus GDP-bound protein), which had been seen with Sdc25-mediated exchange, was also found for GRF. In addition, the sensitivity of H-Ras to GRF was abolished when residues 130-139 were replaced by proline-aspartic acid-glutamine, whereas substitution of the entire loop 8 (residues 123-130 replaced by leucine-isoleucine-arginine) had no effect on the stimulation of guanine nucleotide release by GRF. Substrate activity of Ras proteins were independent of their post-translational processing, GDP release was stimulated threefold more effectively by GRF than was GTP release, and no major differences were found between the mammalian N-, H- and K-Ras proteins. Examining the responsiveness of the Ras protein from S. pombe and the human Ras like proteins RhoA, Rap1A, Rac1 and G25K revealed a strict Ras specificity; of these only S. pombe Ras was GRF sensitive.

Novel determinants of H-Ras plasma membrane localization and transformation.

Although it is well-established that modification of Ras by farnesol is a critical step for its membrane association and transforming activity, the contribution of other C-terminal sequences and palmitate modification to Ras localization and function remains unclear. We have characterized H-Ras mutant proteins with alterations in the palmitoylated cysteines or in sequences flanking these residues. We found that non-palmitoylated proteins were impaired not only in membrane association but also in transforming activity. Mutations which drastically altered residues adjacent to the palmitoylated cysteine did not abolish palmitoylation. However, despite continued lipid modification the mutant proteins failed to bind to plasma membranes and instead accumulated on internal membranes and, importantly, were not transforming. Addition of an N-terminal myristoylation signal to these defective mutants, or to proteins entirely lacking the C-terminal 25 residues restored both plasma membrane association and transforming activity. Thus, H-Ras does not absolutely require prenylation or palmitoylation nor indeed its hypervariable domain in order to interact with effectors that ultimately cause transformation. However, in this native state, the C-terminus appears to provide a combination of lipids and a previously unrecognized signal for specific plasma membrane targeting that are essential for the correct localization and biological function of H-Ras.

Kinetics of expression of inducible beta-galactosidase in murine fibroblasts: high initial rate compared to steady-state expression.

We have constructed a murine fibroblast cell line in which synthesis of beta-galactosidase can be induced by incubation with isopropyl-beta-D-thiogalactopyranoside (IPTG). This was obtained by transfection by both a plasmid expressing lacI and a second plasmid expressing lacZ from a modified simian virus 40 (SV40) promoter containing a lac operator. We have measured the induction kinetics as well as the basal and induced differential rate of synthesis of beta-galactosidase. The steady-state rate of synthesis is tenfold higher in the presence than in the absence of inducer; we calculate an average of 1200 lacZ polypeptides are synthesized per minute per cell in the induced cultures. However, immediately after induction, the rate of accumulation of beta-galactosidase is up to 50-fold higher than the basal level. Based on our measurements of stability of beta-galactosidase, we suggest that induction may result in a subsequent down-modulation of the transcriptional activity from the induced gene. We hypothesize this inhibition may result from structural changes in DNA components, such as nucleosomes.

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.

Two distinct regions of Ras participate in functional interaction with GDP-GTP exchangers.

We have previously implemented a combined genetic/biochemical approach, for analysis of insertion-deletion mutants, to identify sites of Harvey-Ras participating in the interaction with guanine nucleotide exchangers, using the yeast Cdc25 as a model exchanger. We showed that positions 101-106 may be required for catalyzed exchange. We here present a further improved strategy to define more precisely the residues on Ras participating in this interaction. Non-conservative replacements at positions 103 or 105 abolished response to Cdc25 while substitutions at positions 102 or 104 were partially affected. The same substitutions had no effect on coupling to adenylyl cyclase. Since the strategy enables us to assess Ras functional interaction with both the exchanger and effector simultaneously, we have also examined the effect of substitutions in the distal part of the switch II region (amino acids 69-78). In contrast to other reports, substitutions at positions 69 or 73 prevented Cdc25 response while mutations at position 74 did not prevent this interaction. However, all these substitutions partly affected cyclase activation. These findings establish the crucial role of the 102-105 region in the catalyzed exchange reaction and suggest that the 69-74 area would be required for the functional interaction with both exchangers and effector molecules.

Residues crucial for Ras interaction with GDP-GTP exchangers.

Cdc25 is essential for Ras-mediated activation of adenylyl cyclase in the yeast Saccharomyces cerevisiae. This protein acts by catalyzing GDP-GTP exchange on yeast Ras. Harvey (Ha) ras expressed in S. cerevisiae is also recognized by both Cdc25 and Sdc25, a yeast homolog of Cdc25. Thus it is feasible to examine molecular aspects of mammalian Ras modulation by Cdc25 using the RAS/cAMP pathway in yeast as a model system. Here, we describe mutational analysis of Ha-ras for the identification of residues critical for the ability of Ras to interact with Cdc25 and related guanine nucleotide-release proteins. Mutations within codons 97-108 impaired Ras-mediated activation of adenylyl cyclase in the presence but not in the absence of mammalian GTPase-activating protein. Such mutations, therefore, affected the ability of Ras to undergo GDP-GTP exchange catalyzed by the guanine nucleotide exchanger without preventing Ras activation of the effector. Similar mutations were previously shown to impair the ability of c-ras to transform mammalian cells while having a less drastic effect on v-ras.

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.

Cell transformation by ras and regulation of its protein product.

We are studying the biological activity and regulation of mammalian Ras protein in tumours and in physiological signalling. We have shown that GAP (the GTPase-activating protein) is a potent negative regulator of normal Ras in cells. Reduction or loss of the NF1 gene product neurofibromin, in association with genetic abnormalities of the NF1 locus, has been identified in schwannoma cell lines from patients with neurofibromatosis and in melanoma and neuroblastoma lines from patients without neurofibromatosis. Although loss of neurofibromin in the schwannoma lines was associated with a high proportion of normal Ras protein in the active GTP-bound state, Ras-GTP appeared to be appropriately regulated in the melanoma and neuroblastoma lines, which contain normal levels of GAP. Therefore the GTPase-activating activity of neurofibromin is not essential for negative regulation of Ras in some cell types and the putative tumour suppressor function of neurofibromin in such cell types is independent of its GTPase-activating activity. Mitogen activation of Ras in fibroblasts is mediated primarily by exchange factors, which probably interact with a region on the Ras protein distinct from the region required for interaction with GAP. Multiple full-length cDNAs have identified a mouse gene whose products are related to yeast CDC25 guanine nucleotide exchange factor.

The bovine papillomavirus E5 oncogene can cooperate with ras: identification of p21 amino acids critical for transformation by c-rasH but not v-rasH.

We have previously used a series of insertion-deletion mutants of the mutationally activated v-rasH gene to identify several regions of the encoded protein that are dispensable for cellular transformation (B. M. Willumsen, A. G. Papageorge, H.-F. Kung, E. Bekesi, T. Robins, M. Johnsen, W. C. Vass, and D. R. Lowy, Mol. Cell. Biol. 6:2646-2654, 1986). To determine if some of these amino acids are more important for the biological activity of c-rasH, we have now tested many of the same insertion-deletion mutants in the c-rasH form for their ability to transform NIH 3T3 cells. Since the transforming activity of c-rasH is low, we have used cotransfection with the bovine papillomavirus (BPV) genome to develop a more sensitive transformation assay for c-rasH mutants. The increased sensitivity of the assay, which is seen both in focal transformation and in anchorage-independent growth, is mediated by cooperation between the BPV E5 gene and ras. E5-dependent cooperation was seen for v-rasH as well as for c-rasH, which suggests that the major effect of E5 was to increase the susceptibility of the cell to transformation to a given level of ras activity. The cooperation assay was used to test the potential importance, in c-rasH, of codons 93 to 108, 123 to 130, and 166 to 183, which were nonessential for v-rasH transformation. Relative to the respective transforming activity of wild-type c-rasH and v-rasH, mutants with lesions in codons 102 and 103 were significantly less active in their c-rasH forms than in their v-rasH forms. We conclude that a region including amino acids 102 and 103 encodes a function that is more critical to c-rasH than to v-rasH. Guanine nucleotide exchange is one function that is compatible with such a phenotype.

Regulation of p21ras activity.

The ras genes encode GTP/GDP-binding proteins that participate in mediating mitogenic signals from membrane tyrosine kinases to downstream targets. The activity of p21ras is determined by the concentration of GTP-p21ras, which is tightly regulated by a complex array of positive and negative control mechanisms. GAP and NF1 can negatively regulate p21ras activity by stimulating hydrolysis of GTP bound to p21ras. Other cellular factors can positively regulate p21ras by stimulating GDP/GTP exchange.

Inhibition of cell growth by lovastatin is independent of ras function.

We have investigated the inhibition of cell growth by lovastatin (previously known as mevinolin), an antagonist of hydroxymethylglutaryl coenzyme A reductase which blocks the processing and membrane localization of ras proteins via inhibition of polyisoprenylation. A series of NIH 3T3 cells transformed by oncogenes with activities that are dependent or independent of isoprenylated ras were studied, including cells transformed by myristylated ras protein that is isoprenylation independent. Treatment with lovastatin at concentrations ranging from 5 to 15 microM for up to 96 h resulted in a time- and dose-dependent inhibition of cell growth in all lines tested. The inhibition ranged from 25 to 50% when cells were treated with 5 microM lovastatin for 48 h, to 72-90% for cells treated with 15 microM lovastatin for 96 h. Cells transformed by c-ras, v-ras, v-src, v-raf, and the myristylated ras genes displayed similar sensitivities; the parental NIH 3T3 line was the most resistant of the lines tested. Metabolic labeling of control and lovastatin-treated cells with [35S]methionine or tritiated lipids revealed that 15 microM lovastatin blocked the processing of both endogenous ras and v-ras proteins yet had no effect on the lipidation of myristylated ras proteins. Addition of 300 microM mevalonic acid overcame the inhibition induced by 15 microM lovastatin. Thus the inhibition of cell growth in vitro by lovastatin did not show specificity for cells the transformation of which is dependent upon isoprenylated ras protein. It is therefore likely that the inhibition of other pathways affected by lovastatin, such as cholesterol biosynthesis or the processing of other cellular proteins, are responsible for the growth inhibition by lovastatin.