Proline Reverses the Abnormal Phenotypes of Colletotrichum trifolii Associated with Expression of Endogenous Constitutively Active Ras

Colletotrichum trifolii is the causative organism of alfalfa anthracnose. We previously cloned and characterized the small prototypical G protein, Ras, of C. trifolii, which is involved in the signaling pathways that mediate interaction between the pathogen and its host. Transformants expressing constitutively active forms of Ras have growth medium-dependent phenotypes. In nutrient-rich media (e.g., yeast extract and peptone), the phenotype of the transformants was indistinguishable from that of the wild type. However, during nutrient starvation, the transformants lose polarity, have distended hyphae, and fail to sporulate and produce appressoria. Since peptone caused the phenotype to revert, amino acids were tested singly and in combination to identify the responsible amino acid(s). We found that 1.6 mM proline in the medium reverses the constitutively active Ras phenotype.     

Cdc42 is required for proper growth and development in the fungal pathogen Colletotrichum trifolii

Cdc42 is a highly conserved small GTP-binding protein that is involved in regulating morphogenesis in eukaryotes. In this study, we isolated and characterized a highly conserved Cdc42 gene from Colletotrichum trifolii (CtCdc42), a fungal pathogen of alfalfa. CtCdc42 is, at least in part, functionally equivalent to Saccharomyces cerevisiae Cdc42p, since it restores the temperature-sensitive phenotype of a yeast Cdc42p mutant. Inhibition of CtCdc42 by expression of an antisense CtCdc42 or a dominant negative form of CtCdc42 (DN Cdc42) resulted in appressorium differentiation under noninductive conditions, suggesting that CtCdc42 negatively regulates pathogenic development in this fungus. We also examined the possible linkage between CtCdc42 and Ras signaling. Expression of a dominant active Cdc42 (DA Cdc42) in C. trifolii leads to aberrant hyphal growth under nutrient-limiting conditions. This phenotype was similar to that of our previously reported dominant active Ras (DA Ras) mutant. Also consistent with our observations of the DA Ras mutant, high levels of reactive oxygen species (ROS) were observed in the DA Cdc42 mutant, and proline restored the wild-type phenotype. Moreover, overexpression of DN Cdc42 resulted in a significant decrease in spore germination, virtually no hyphal branching, and earlier sporulation, again similar to what we observed in a dominant negative Ras (DN Ras) mutant strain. Interestingly, coexpression of DA Cdc42 with DN Ras resulted in germination rates close to wild-type levels, while coexpression of DN Cdc42 with the DA Ras mutant restored the wild-type phenotype. These data suggest that CtCdc42 is positioned as a downstream effector of CtRas to regulate spore germination and pathogenic development.     

H-Ras activation promotes cytoplasmic accumulation and phosphoinositide 3-OH kinase association of beta-catenin in epidermal keratinocytes.

The mechanisms underlying downregulation of the cadherin/catenin complexes and beta-catenin signaling during tumor progression are not fully understood. We have analyzed the effect of oncogenic H-Ras on E-cadherin/catenin complex formation/stabilization and beta-catenin distribution in epidermal keratinocytes. Microinjection or stable expression of V12Ras into keratinocytes promotes the loss of E-cadherin and alpha-catenin and relocalization of beta-catenin to the cytoplasm and nucleus. Moreover, these effects are dependent on PI3K (phosphoinositide 3-OH kinase) activity. Interestingly, a strong association of p85alpha and p110alpha subunits of PI3K with beta-catenin is induced in V12Ras-expressing keratinocytes, and in vitro binding assays show a direct interaction between beta-catenin and p85alpha. Overexpression of either V12Ras or constitutively active p110alpha induces metabolic stabilization of beta-catenin and promotes its accumulation in cytoplasmic and nuclear pools. In addition, the interaction of beta-catenin with the adenomatous polyposis coli protein is blocked in V12Ras and p110alpha transformants though no changes in glycogen synthase kinase 3 beta activity could be detected. Nevertheless, in V12Ras transformants the in vivo phosphorylation of beta-catenin in Ser residues is strongly decreased. These results indicate that H-Ras activation induces the relocalization and cytoplasmic stabilization of beta-catenin by a mechanism involving its interaction with PI3K.     

Role of phosphoinositide 3-kinase in the aggressive tumor growth of HT1080 human fibrosarcoma cells

We have developed a model system of human fibrosarcoma cell lines that do or do not possess and express an oncogenic mutant allele of N-ras. HT1080 cells contain an endogenous mutant allele of N-ras, whereas the derivative MCH603 cell line contains only wild-type N-ras. In an earlier study (S. Gupta et al., Mol. Cell. Biol. 20:9294-9306, 2000), we had shown that HT1080 cells produce rapidly growing, aggressive tumors in athymic nude mice, whereas MCH603 cells produced more slowly growing tumors and was termed weakly tumorigenic. An extensive analysis of the Ras signaling pathways (Raf, Rac1, and RhoA) provided evidence for a potential novel pathway that was critical for the aggressive tumorigenic phenotype and could be activated by elevated levels of constitutively active MEK. In this study we examined the role of phosphoinositide 3-kinase (PI 3-kinase) in the regulation of the transformed and aggressive tumorigenic phenotypes expressed in HT1080 cells. Both HT1080 (mutant N-ras) and MCH603 (wild-type N-ras) have similar levels of constitutively active Akt, a downstream target of activated PI 3-kinase. We find that both cell lines constitutively express platelet-derived growth factor (PDGF) and PDGF receptors. Transfection with tumor suppressor PTEN cDNA into HT1080 and constitutively active PI 3-kinase-CAAX cDNA into MCH603 cells, respectively, resulted in several interesting and novel observations. Activation of the PI 3-kinase/Akt pathway, including NF-kappaB, is not required for the aggressive tumorigenic phenotype in HT1080 cells. Activation of NF-kappaB is complex: in MCH603 cells it is mediated by Akt, whereas in HT1080 cells activation also involves other pathway(s) that are activated by mutant Ras. A threshold level of activation of PI 3-kinase is required in MCH603 cells before stimulatory cross talk to the RhoA, Rac1, and Raf pathways occurs, without a corresponding activation of Ras. The increased levels of activation seen were similar to those observed in HT1080 cells, except for Raf and MEK, which were more active than HT1080 levels. This cross talk results in conversion to the aggressive tumorigenic phenotype. This latter observation is consistent with our previous observation that overstimulation of the activity of endogenous members of Ras signaling pathways, activated MEK in particular, is a prerequisite for aggressive tumorigenic growth.     

A Rho-like small GTPase of Entamoeba histolytica contains an unusual amino acid residue in a conserved GDP-stabilization region and is not a substrate for C3 exoenzyme

The Entamoeba histolytica small GTP-binding protein EhRho1 has an unusual amino acid residue at a conserved site found in all known Ras superfamily proteins. EhRho1 has an isoleucine at position 45, which corresponds to position 28 of human Ras and Rac and position 30 of human Rho and Cdc42. All other known small GTPases have an aromatic residue (typically phenylalanine) at this position, and mutation to a leucine renders other Ras proteins constitutively active by reason of diminished affinity for GDP. It was determined that the EhRho1 protein has a half-time of GDP dissociation similar to that of a human Rho protein, HsRhoA, and therefore an isoleucine at this site in EhRho1 is not likely to render EhRho1 constitutively active. It was also found that EhRho1 is not a substrate for the Rho-specific C3 exoenzyme. Thus EhRho1 appears to be an unusual member of the Ras family.     

Signal pathways which promote invasion and metastasis: critical and distinct contributions of extracellular signal-regulated kinase and Ral-specific guanine exchange factor pathways

Approximately 50% of metastatic tumors contain Ras mutations. Ras proteins can activate at least three downstream signaling cascades mediated by the Raf-MEK-extracellular signal-regulated kinase family, phosphatidylinositol-3 (PI3) kinase, and Ral-specific guanine nucleotide exchange factors (RalGEFs). Here we investigated the contribution of RalGEF and ERK activation to the development of experimental metastasis in vivo and associated invasive properties in vitro. Each pathway contributes distinct properties to the metastatic phenotype. Following lateral tail vein injection, 3T3 cells transformed by constitutively active Raf or MEK produced lung metastasis that displayed circumscribed, noninfiltrating borders. In contrast, 3T3 cells transformed by Ras(12V,37G), a Ras effector mutant that activates RalGEF but not Raf or P13 kinase, formed aggressive, infiltrative metastasis. Dominant negative RalB inhibited Ras(12V,37G)-activated invasion and metastasis, demonstrating the necessity of the RalGEF pathway for a fully transformed phenotype. Moreover, 3T3 cells constitutively expressing a membrane-associated form of RalGEF (RalGDS-CAAX) formed invasive tumors as well, demonstrating that activation of a RalGEF pathway is sufficient to initiate the invasive phenotype. Despite the fact that Ras(12V,37G) expression does not elevate ERK activity, inhibition of this kinase by a conditionally expressed ERK phosphatase demonstrated that ERK activity was necessary for Ras(12V,37G)-transformed cells to express matrix-degrading activity in vitro and tissue invasiveness in vivo. Therefore, these experiments have revealed a hitherto-unknown but essential interaction of the RalGEF and ERK pathways to produce a malignant phenotype. The generality of the role of the RalGEF pathway in metastasis is supported by the finding that Ras(12V,37G) increased the invasiveness of epithelial cells as well as fibroblasts.     

Study of the constitutively active form of the alpha subunit of rice heterotrimeric G proteins

We used site-directed mutagenesis to engineer two constitutively active forms of the alpha subunit of a rice heterotrimeric G protein. The recombinant proteins produced from these novel cDNAs had GTP-binding activity but no GTPase activity. A chimeric gene for a constitutively active form of the alpha subunit was introduced into the rice mutant d1, which is defective for the alpha-subunit gene. All the transformants essentially showed a wild-type phenotype compared with normal cultivars, although seed sizes were substantially increased and internode lengths also showed some increase.     

Mechanism of Activation of the Caenorhabditis Elegans Ras Homologue Let-60 by a Novel, Temperature-Sensitive, Gain-of-Function Mutation

The Caenorhabditis elegans let-60 gene encodes a Ras protein that mediates induction of the hermaphrodite vulva. To better understand how mutations constitutively activate Ras and cause unregulated cell division, we have characterized ga89, a temperature-sensitive, gain-of-function mutation in let-60 ras. At 25°, ga89 increases let-60 activity resulting in a multivulva phenotype. At 15°, ga89 decreases let-60 activity resulting in a vulvaless phenotype in let-60(ga89)/Df animals. The ga89 mutation causes a leucine (L) to phenylalanine (F) substitution at amino acid 19, a residue conserved in all Ras proteins. We introduced the L19F change into human H-Ras protein and found that the in vitro GTPase activity of H-Ras became temperature-dependent. Genetic experiments suggest that LET-60(L19F) interacts with GAP and GNEF, since mutations that decrease GAP and GNEF activity affect the multivulva phenotype of let-60(ga89) animals. These results suggest that the L19F mutation primarily affects the intrinsic rate of GTP hydrolysis by Ras, and that this effect may be sufficient to account for the activated-Ras phenotype caused by let-60(ga89). Our results suggest that a mutation in a human ras gene analogous to ga89 might contribute to oncogenic transformation.     

Constitutive JNK activation in NIH 3T3 fibroblasts induces a partially transformed phenotype

The c-Jun N-terminal kinases (JNKs) (also known as stress-activated protein kinases or SAPKs), members of the mitogen-activated protein kinase (MAPK) family, regulate gene expression in response to a variety of physiological and unphysiological stimuli. Gene knockout experiments and the use of dominant interfering mutants have pointed to a role for JNKs in the processes of cell differentiation and survival as well as oncogenic transformation. Direct analysis of the transforming potential of JNKs has been hampered so far by the lack of constitutively active forms of these kinases. Recently, such mutants have become available by fusion of the MAPK with its direct upstream activator kinase. We have generated a constitutively active SAPK beta-MKK7 hybrid protein and, using this constitutively active kinase, we are able to demonstrate the transforming potential of activated JNK, which is weaker than that of classical oncogenes such as Ras or Raf. The inducible expression of SAPK beta-MKK7 caused morphological transformation of NIH 3T3 fibroblasts. Additionally, these cells formed small foci of transformed cells and grew anchorage-independent in soft agar. Furthermore, similar to oncogenic Ras and Raf, the expression of activated SAPK beta resulted in the disassembly of F-actin stress fibers. Our data suggest that constitutive JNK activation elicits major aspects of cellular transformation but is unable to induce the complete set of changes which are required to establish the fully transformed phenotype.     

Cytosolic Ras supports eye development in Drosophila

Ras proteins associate with cellular membranes as a consequence of a series of posttranslational modifications of a C-terminal CAAX sequence that include prenylation and are thought to be required for biological activity. In Drosophila melanogaster, Ras1 is required for eye development. We found that Drosophila Ras1 is inefficiently prenylated as a consequence of a lysine in the A(1) position of its CAAX sequence such that a significant pool remains soluble in the cytosol. We used mosaic analysis with a repressible cell marker (MARCM) to assess if various Ras1 transgenes could restore photoreceptor fate to eye disc cells that are null for Ras1. Surprisingly, we found that whereas Ras1 with an enhanced efficiency of membrane targeting could not rescue the Ras1 null phenotype, Ras1 that was not at all membrane targeted by virtue of a mutation of the CAAX cysteine was able to fully rescue eye development. In addition, constitutively active Ras1(12V,C186S) not targeted to membranes produced a hypermorphic phenotype and stimulated mitogen-activated protein kinase (MAPK) signaling in S2 cells. We conclude that the membrane association of Drosophila Ras1 is not required for eye development.     

Differential regulation of cyclooxygenase 2 expression by small GTPases Ras, Rac1, and RhoA

Cyclooxygenase 2 (COX-2) is an immediate early gene induced by a variety of stimuli and its expression is stimulated by individual activation of Ras or Rho GTPases. Here we investigate the role of coordinate activation of Ras and Rho GTPases in the induction of COX-2. Individual expression of constitutively active Ras, RhoA, or Rac1 was capable of stimulating COX-2 expression in NIH3T3 cells, but co-expression of constitutively active RhoA with either constitutively active Ras or Rac1 was required for full stimulation of COX-2 expression. Serum growth factors differentially activated Ras, RhoA, and Rac1, which correlated with the activation of Raf-1, ERK, and c-Jun as well as with induction of COX-2. Inhibition of Ras significantly blocked the activation of Raf-1, ERK, and c-Jun and the stimulation of COX-2 expression in response to serum. In contrast, inhibition of Rho family GTPases partially blocked serum induction of ERK activation but had little effects on COX-2 expression. Both inhibitors of MEK (PD098059) and JNK (SP600125) inhibited serum induction of COX-2. PD98059 only inhibited constitutively active Ras-induced COX-2 expression, while SP600125 significantly inhibited both constitutively active Ras- and RhoA-induced COX-2 expression. Together, our data suggest that constitutively active oncogenic Ras and Rho coordinately stimulate COX-2 expression whereas transient activation of Ras but not RhoA or Rac1 mediates the induction of COX-2 in response to serum. Furthermore, ERK and JNK activation are both required for serum- and oncogenic Ras-mediated COX-2 expression whereas only JNK activation is required for oncogenic RhoA-mediated stimulation of COX-2 expression.     

Dominant active Rac and dominant negative Rac revert the dominant active Ras phenotype in Colletotrichum trifolii by distinct signalling pathways

The small G-protein superfamily is an evolutionarily conserved group of GTPases that regulate diverse signalling pathways including pathways for growth and development in eukaryotes. Previously, we showed that dominant active mutation in the unique Ras gene (DARas) of the fungal phytopathogen Colletotrichum trifolii displays a nutrient-dependent phenotype affecting polarity, growth and differentiation. Signalling via the MAP kinase pathway is significantly impaired in this mutant as well. Here we describe the cloning and functional characterization of Rac (Ct-Rac1), a member of the Rho family of G proteins. Ct-Rac1 expression is downregulated by DARas under limiting nutrition. Co-expression of DARas with dominant active Rac (DARac) stimulates MAPK activation and restores the wild-type phenotype. Inhibition of MAPK activation suppresses phenotypic restoration suggesting Rac-mediated MAPK activation is responsible for reversion to the wild-type phenotype. We also examined the role of reactive oxygen species (ROS) in these genetic backgrounds. The DARas mutant strain generates high levels of ROS as determined by DCFH-DA fluorescence. Co-expression with DNRac decreases ROS generation to wild-type levels and restores normal fungal growth and development. Pretreatment of DARas with antioxidants or a cytosolic phospholipase A2 inhibitor also restores the wild-type phenotype. These findings suggest that Ras-mediated ROS generation is dependent on a Rac-cPLA(2)-linked signalling pathway. Taken together, this study provides evidence that Rac functions to restore the hyphal morphology of DARas by regulating MAPK activation and intracellular ROS generation.     

Ral is both necessary and sufficient for the inhibition of myeloid differentiation mediated by Ras

Hyperactivation of Ras is one of the most common abnormalities in acute myeloid leukemia. In experimental models, Ras inhibits myeloid differentiation, which is characteristic of leukemia; however, the mechanism through which it disrupts hematopoiesis is poorly understood. In multipotent FDCP-mix cells, Ras inhibits terminal neutrophil differentiation, thereby indefinitely extending their proliferative potential. Ras also strongly promotes the sensitivity of these cells to granulocyte-macrophage colony-stimulating factor (GM-CSF). Using this model, we have dissected the signaling elements downstream of Ras to determine their relative contribution to the dysregulation of hematopoiesis. Cells expressing Ras mutants selectively activating Raf (Ras*T35S) or phosphatidylinositol 3-kinase (Ras*Y40C) did not significantly affect differentiation or proliferative capacity, whereas Ras*E37G (which selectively activates RalGEFs) perpetuated proliferation and blocked neutrophil development in a manner similar to that of Ras. Correspondingly, expression of constitutively active versions of these effectors confirmed the overriding importance of Ral guanine nucleotide exchange factors. Cells expressing Ras demonstrated hyperactivation of Ral, which itself was able to exactly mimic the phenotype of Ras, including hypersensitivity to GM-CSF. Conversely, dominant negative Ral promoted spontaneous neutrophil development. Ral, in turn, appears to influence differentiation through multiple effectors. These data show, for the first time, the importance of Ral in regulating differentiation and self-renewal in hematopoietic cells.     

Src tyrosine kinase-induced loss of luteinizing hormone responsiveness is via a Ras-dependent,phosphatidylinositol-3-kinase independent pathway

Gonadotropins stimulate gonadal cell steroid secretion primarily through activation of a cAMP-protein kinase A signal transduction pathway. Various growth factors have been shown to inhibit gonadotropin-stimulated steroidogenesis, however, the intracellular signaling cascades involved in growth factor inhibition have not been characterized. The present study investigated whether Src tyrosine kinase, a nonreceptor tyrosine kinase activated in response to growth factor stimulation and previously shown to inhibit LH-stimulated progesterone secretion, acts via activation of Ras stimulated pathways, phosphatidylinositol-3-kinase (PI3-kinase) stimulated pathways, or both in MA10 Leydig cells. Direct activation of Src in MA10 cells that express a temperature sensitive Src was associated with an increase in GTP-bound Ras, indicating increased Ras activity in response to Src activation. Direct activation of Ras by way of expression of a constitutively active Ras (Ras+) was associated with a decrease in LH responsiveness. Coexpression of a dominant negative Src, which by itself increases LH responsiveness in MA10 cells, had no effect on Ras+ inhibition on LH responsiveness, further demonstrating that Src is upstream of Ras. In addition, MA10(Ras+) cells were relatively unresponsive to cholera toxin or 8-bromo cAMP, indicating the effects of Ras are independent of cAMP generation. Wortmannin, a PI3-kinase inhibitor, did not restore LH responsiveness to cells expressing activated Src or constitutively active Ras. These results demonstrate that Src activates a Ras pathway in MA10 Leydig cells, and that activation of Ras is associated with a loss of LH responsiveness that is independent of PI3-kinase.     

Ras signaling contributes to survival of human T-cell leukemia/lymphoma virus type 1 (HTLV-1) Tax-positive T-cells

Ras signaling pathways play an important role in cellular proliferation and survival, and inappropriate activation of Ras frequently results in cell transformation and cancer. Human T-cell leukemia/lymphoma virus type 1 (HTLV-1) is the etiological agent of the adult T-cell leukemia/lymphoma (ATLL), a severe malignancy that has a poor prognosis and exhibits resistance to conventional chemotherapy. Although the mechanisms involved in cell transformation by HTLV-1 have not been completely clarified, it is generally thought that Tax plays a pivotal role in the process. We have previously proposed that a functionally active Ras protein is needed for efficient anti-apoptotic activity of Tax. In this study we report data indicating that the apoptotic resistance of cells expressing Tax, constitutively or transiently, is linked to the intracellular levels of Ras-GTP. Indeed, we found that Tax-positive cells have a high content of active Ras, and that inhibition of Ras signaling, using the antagonist farnesyl thyosalicylic acid (FTS), increases their sensitivity to apoptosis. FTS treatment was also accompanied by a decrease in ERK, but not Akt, phosphorylation. Thus, all together our data suggest that the interaction between Tax and Ras could be important to ATLL pathogenesis, and indicate Ras as a possible target for therapeutic intervention in ATLL patients.     

A genetic screen for novel components of the Ras/Mitogen-activated protein kinase signaling pathway that interact with the yan gene of Drosophila identifies split ends, a new RNA recognition motif-containing protein

The receptor tyrosine kinase (RTK) signaling pathway is used reiteratively during the development of all multicellular organisms. While the core RTK/Ras/MAPK signaling cassette has been studied extensively, little is known about the nature of the downstream targets of the pathway or how these effectors regulate the specificity of cellular responses. Drosophila yan is one of a few downstream components identified to date, functioning as an antagonist of the RTK/Ras/MAPK pathway. Previously, we have shown that ectopic expression of a constitutively active protein (yan(ACT)) inhibits the differentiation of multiple cell types. In an effort to identify new genes functioning downstream in the Ras/MAPK/yan pathway, we have performed a genetic screen to isolate dominant modifiers of the rough eye phenotype associated with eye-specific expression of yan(ACT). Approximately 190,000 mutagenized flies were screened, and 260 enhancers and 90 suppressors were obtained. Among the previously known genes we recovered are four RTK pathway components, rolled (MAPK), son-of-sevenless, Star, and pointed, and two genes, eyes absent and string, that have not been implicated previously in RTK signaling events. We also isolated mutations in five previously uncharacterized genes, one of which, split ends, we have characterized molecularly and have shown to encode a member of the RRM family of RNA-binding proteins.     

The chemotactic response to PDGF-BB: evidence of a role for Ras

The PDGF receptor-beta mediates both mitogenic and chemotactic responses to PDGF-BB. Although the role of Ras in tyrosine kinase- mediated mitogenesis has been characterized extensively, its role in PDGF-stimulated chemotaxis has not been defined. Using cells expressing a dominant-negative ras, we find that Ras inhibition suppresses migration toward PDGF-BB. Overexpression of either Ras-GTPase activating protein (Ras-GAP) or a Ras guanine releasing factor (GRF) also inhibited PDGF-stimulated chemotaxis. In addition, cells producing excess constitutively active Ras failed to migrate toward PDGF-BB, consistent with the observation that either excess ligand or excess signaling intermediate can suppress the chemotactic response. These results suggest that Ras can function in normal cells to support chemotaxis toward PDGF-BB and that either too little or too much Ras activity can abrogate the chemotactic response. In contrast to Ras overexpression, cells producing excess constitutively active Raf, a downstream effector of Ras, did migrate toward PDGF-BB. Cells expressing dominant-negative Ras were able to migrate toward soluble fibronectin demonstrating that these cells retained the ability to migrate. These results suggest that Ras is an intermediate in PDGF- stimulated chemotaxis but may not be required for fibronectin- stimulated cell motility.