A ras effector domain mutant which is temperature sensitive for cellular transformation: interactions with GTPase-activating protein and NF-1.

A series of v-rasH effector domain mutants were analyzed for their ability to transform rat 2 cells at either low or high temperatures. Three mutants were found to be significantly temperature sensitive: Ile-36 changed to Leu, Ser-39 changed to Cys (S39C), and Arg-41 changed to Leu. Of these, the codon 39 mutant (S39C) showed the greatest degree of temperature sensitivity. When the same mutation was analyzed in the proto-oncogene form of ras(c-rasH), this gene was also found to be temperature sensitive for transformation. Biochemical analysis of the proteins encoded by v-rasH(S39C) and c-rasH(S39C) demonstrated that the encoded p21ras proteins were stable and bound guanine nucleotides in vivo at permissive and nonpermissive temperatures. On the basis of these findings, it is likely that the temperature-sensitive phenotype results from an inability of the mutant (S39C) p21ras to interact properly with the ras target effector molecule(s) at the nonpermissive temperature. We therefore analyzed the interaction between the c-rasH(S39C) protein and the potential target molecules GTPase-activating protein (GAP) and the GAP-related domain of NF-1, on the basis of stimulation of the mutant p21ras GTPase activity by these molecules in vitro. Assays conducted across a range of temperatures revealed no temperature sensitivity for stimulation of the mutant protein, compared with that of authentic c-rasH protein. We conclude that for this mutant, there is a dissociation between the stimulation of p21ras GTPase activity by GAP and the GAP-related domain NF-1 and their potential target function. Our results are also consistent with the existence of a distinct, as-yet-unidentified effector for mammalian ras proteins.     

Plasma membrane-targeted ras GTPase-activating protein is a potent suppressor of p21ras function.

Although p21ras is localized to the plasma membrane, proteins it interacts with, such as the GTPase-activating proteins (GAPs) ras GAP and neurofibromin (NF1), are not, suggesting that one function of p21ras GTP may be to target such proteins to the plasma membrane. To investigate the effects of targeting ras GAP to the plasma membrane, ras C-terminal motifs sufficient for plasma membrane localization of p21ras were cloned onto the C terminus of ras GAP. Plasma membrane-targeted ras GAP is growth inhibitory to NIH 3T3 fibroblasts and COS cells. This growth inhibition correlates with GAP catalytic activity, since the plasma membrane-targeted C-terminal catalytic domain or the GAP-related domain of neurofibromin is inhibitory, whereas the similarly targeted N-terminal domain is not. Moreover, the inhibition is abrogated by the inactivating mutation L902I, which abolishes ras GAP catalytic activity. Coexpression of oncogenic mutant ras rescues cell viability, but the majority of rescued colonies are phenotypically untransformed. Furthermore, in focus assays, targeted ras GAP suppresses transformation by oncogenic mutant ras, and in reversion assays, targeted ras GAP can revert cells transformed by oncogenic mutant ras. Neither the targeted or nontargeted N-terminal domain nor the L902I mutant of ras GAP has any transforming activity. These data demonstrate that ras GAP can function as a negative regulator of ras and that plasma membrane localization potentiates this activity. However, if ras GAP is involved in the effector functions of p21ras, it can only be part of the effector complex for cell transformation.     

ralGDS family members interact with the effector loop of ras p21.

Using a yeast two-hybrid system, we identified a novel protein which interacts with ras p21. This protein shares 69% amino acid homology with ral guanine nucleotide dissociation stimulator (ralGDS), a GDP/GTP exchange protein for ral p24. We designated this protein RGL, for ralGDS-like. Using the yeast two-hybrid system, we found that an effector loop mutant of ras p21 was defective in interacting with the ras p21-interacting domain of RGL, suggesting that this domain binds to ras p21 through the effector loop of ras p21. Since ralGDS contained a region highly homologous with the ras p21-interacting domain of RGL, we examined whether ralGDS could interact with ras p21. In the yeast two-hybrid system, ralGDS failed to interact with an effector loop mutant of ras p21. In insect cells, ralGDS made a complex with v-ras p21 but not with a dominant negative mutant of ras p21. ralGDS interacted with the GTP-bound form of ras p21 but not with the GDP-bound form in vitro. ralGDS inhibited both the GTPase-activating activity of the neurofibromatosis gene product (NF1) for ras p21 and the interaction of Raf with ras p21 in vitro. These results demonstrate that ralGDS specifically interacts with the active form of ras p21 and that ralGDS can compete with NF1 and Raf for binding to the effector loop of ras p21. Therefore, ralGDS family members may be effector proteins of ras p21 or may inhibit interactions between ras p21 and its effectors.     

Tissue and subcellular distributions of the smg-21/rap1/Krev-1 proteins which are partly distinct from those of c-ras p21s.

We have made a specific antiserum recognizing both smg p21A (the rap1A/Krev-1 protein) and -B (the rap1B protein), ras p21-like GTP-binding proteins having the same putative effector domain as ras p21s and have used this antiserum to study the tissue and subcellular distributions of smg p21s by immunoblot and immunocytochemical analyses. By immunoblot analysis, smg p21s were detected in various rat tissues and at the highest level in brain. By light microscopic immunocytochemical analysis, smg p21s were also detected in various rat tissues. Particularly, smg p21s in brain were found abundantly in the cytoplasmic region of most types of neuronal cell bodies and moderately in neuropil, whereas c-ras p21s were found more abundantly in neuropil than in the cytoplasmic region of most types of neuronal cell bodies. smg p21s in testis were found in spermatogenic cells, in which c-ras p21s were not significantly detected. By subcellular fractionation analysis of cerebrum, smg p21s were detected in all of the particulate fractions but not in the cytosol fraction. Among the particulate fractions, approximately 70% of smg p21s was recovered with the highest specific content in the fraction containing mainly synaptosomes, mitochondria, and myelin. In further fractionation of this fraction, approximately 40% of smg p21s was recovered in each of the synaptosome fraction and the mitochondrial fraction. This subcellular distribution of smg p21s in cerebrum was partly distinct from that of c-ras p21s, which were mainly recovered in the synaptosome and microsome fractions but present at very low levels in the mitochondrial fraction. These tissue and subcellular distributions of smg p 21s together with the fact that smg p21s have the same putative effector domain as ras p21s exert their own specific actions in addition to the actions similar or antagonistic to those of c-ras p21s.     

Identification of distinct cytoplasmic targets for ras/R-ras and rho regulatory proteins

The protein products of the mammalian ras genes, p21ras, are regulatory guanine nucleotide binding proteins that are involved in the control of cell proliferation, though the exact biochemical processes regulated are unknown. Recently a cytoplasmic protein has been identified that interacts with and increases the GTPase activity of p21ras. It has been shown that this GTPase-activating protein, or GAP, interacts with the effector domain of ras, leading us and others to propose that GAP may be the target for regulation by p21ras. It has become apparent that ras is part of a much larger family of proteins, and at least 15 ras-related genes have now been identified in the mammalian genome. Each encodes a small (about 21 kDa) guanine nucleotide binding protein, but the functions of none of these regulatory molecules are known. We report here that mammalian cytoplasmic extracts contain GAP-like activity toward the products of two other ras-related genes, R-ras and rho. It appears that p23R-ras interacts with the same 125-kDa GAP protein as p21ras whereas p21rho interacts with a distinct 29-kDa protein, rho GAP.     

How does p21ras transform cells?

Oncogenic forms of p21ras are found in a wide range of human tumors. However, the mechanism by which p21ras transforms remains obscure. Genetic evidence has identified a domain of p21ras that is involved with interaction with an effector molecule required for transformation. Two proteins, GAP and the tumor suppressor NF1, interact with p21ras in this region but it is an unresolved puzzle whether either of these is the an unresolved puzzle whether either of these is the effector. After interaction with an effector, two downstream events--activation of protein kinase C and another pathway--are necessary for induction of DNA synthesis by oncogenic p21ras; however, morphological transformation does not require activation of protein kinase C.     

Identification of amino acid residues required for Ras p21 target activation.

The Krev-1 gene has been shown to suppress ras-mediated transformation in vitro. Both ras and Krev-1 proteins have identical effector domains (ras residues 32 to 40), which are required for biological activity and for the interaction of Ras p21 with Ras GTPase-activating protein (GAP). In this study, five amino acid residues flanking the ras effector domain, which are not conserved with the Krev-1 protein, were shown to be required for normal protein-protein interactions and biological activity. The substitution of Krev-1 p21 residues 26, 27, 30, 31, and 45 with the corresponding amino acid residues from Ras p21 resulted in a Krev-1 protein which had ras function in both mammalian and yeast biological assays. Replacement of these residues in Ras p21 with the corresponding Krev-1 p21 amino acids resulted in ras proteins which were impaired biologically or reduced in their affinity for in vitro GAP binding. Evaluation of these mutant ras proteins have implications for Ras p21-GAP interactions in vivo.     

IQGAP1, a calmodulin-binding protein with a rasGAP-related domain, is a potential effector for cdc42Hs.

Proteins that associate with the GTP-bound forms of the Ras superfamily of proteins are potential effector targets for these molecular switches. A 195 kDa protein was purified from cell lysates by affinity chromatography on immobilized cdc42Hs-GTP and a corresponding cDNA was isolated. Sequence analysis revealed localized identities to calponin, the WW domain, unconventional myosins and to the rasGAP-related domain (GRD) contained in IRA, NF-1, SAR1 and rasGAP. p195 was found to be identical to IQGAP1, a protein previously reported to bind ras. Purified recombinant p195/IQGAP1 bound to and inhibited the GTPase activity of cdc42Hs and rac whereas no interaction with ras was detected. The C-terminal half of IQGAP1 containing the GRD bound to cdc42 and rac in a GRD-dependent fashion, but a smaller fragment containing only the GRD did not. Cdc42 was also co-immunoprecipitated from cell lysates with antibody specific to p195/IQGAP1. Calmodulin also co-immunoprecipitated with p195/IQGAP1 and was found to associate with fragments containing the IQ domain. Expression of a cDNA fragment encoding the GRD inhibited the CDC24/CDC42 pathway in yeast, but no effect on ras was observed. In mammalian cells, both endogenous and ectopically expressed p195/IQGAP1 were localized to lamellipodia and ruffling cell membranes, where co-localization with actin was apparent. These results suggest that IQGAP1 is an effector target for cdc42Hs and may mediate the effects of this GTPase on cell morphology.     

Evidence from sequence information that the interleukin-1 receptor is a transmembrane GTPase.

Evidence is presented that the cytoplasmic domain of the type I interleukin-1 receptor (IL-1R) may be a GTPase. This domain conserves segments of hydrophobic amino acids that suggest a structural relatedness to the ras protooncogene protein and other members of the GTPase superfamily, despite a lack of significant detectable sequence homology. When the hydrophobic segments of the IL-1R were aligned with similar segments of the GTPases, it became apparent that the IL-1Rs possess a number of conserved amino acids that represent plausible functional residues for base-specific binding of GTP, magnesium chelation, and phosphate ester hydrolysis. Furthermore, a segment of five contiguous residues were found that is identical between ras and the IL-1R, and which is positioned to form part of the guanine base binding pocket. If this model is correct, then the IL-1Rs possess a highly conserved effector protein binding region, but one that is entirely unrelated to the effector regions of other superfamily members. Therefore, if the IL-1R is indeed a GTPase, then its activation function may be directed to as-yet unrecognized effector target proteins, as part of a unique cellular signal transduction pathway.     

Ras-induced transformation and signaling pathway.

Ras is a signal-transducing, guanine nucleotide-binding protein for various membrane receptors including tyrosine kinase receptors. Ras participates in the regulation of cell proliferation, differentiation, and morphology. Activated ras oncogenes have been identified in various forms of human cancer including epithelial carcinomas of the lung, colon, and pancreas. The cells of these cancers, as well as those that have been experimentally transformed by the activated ras gene, exhibit abnormal growth, morphological changes and alterations of cell adhesions. Although the main effector protein has been thought to be Raf serine/threonine kinase, research has revealed that the Ras-induced signaling pathway is mediated by multiple effector proteins and has the crosstalk with various factors containing other small GTPases. In this review, we summarize the involvement of each effector protein for Ras and the crosstalk with other small GTPases in Ras-induced transformation.     

Effector domain mutations dissociate p21ras effector function and GTPase-activating protein interaction.

The GTPase activity of p21ras is stimulated by GTPase-activating proteins (GAPs) such as p120GAP and the product of the neurofibromatosis 1 gene, which may negatively regulate p21 function. GAPs are also proposed effectors of ras. We have sought activating substitutions in c-H-ras in the region encoding the effector domain, on the rationale that such mutations would dissociate effector function from negative regulation by GAP. One such activating mutation, Pro-34-->Arg, encodes protein that is substantially bound to GTP in vivo. In vitro, this protein is not stimulated by GAPs, and its binding to p120GAP is grossly impaired. The results support the idea that the p21 structural requirements for effector function and GAP interaction are quite different and suggest that some molecule(s) other than p120GAP serves as the ras effector. In contrast to the results obtained with p120GAP, the Pro-34-->Arg p21 species is effectively coupled to the raf-1 product, as judged from electrophoretic mobility shifts of the Raf-1 phosphoprotein.     

ras proteins enhance the phosphorylation of a 38 kDa protein (p38) in rat liver plasma membrane

Phosphorylation of a 38 kDa protein (p38) present in rat liver plasma membrane has been shown for the first time to be enhanced by ras proteins. This increase in phosphorylation is about 3-16-fold depending on the incubation time and the type of ras protein used. Acid treatment removes phosphate from this protein suggesting that the phosphorylation involves phosphoramidate derivatives of basic amino acids. Experiments carried out in the presence of diethylpyrocarbonate suggest that the phosphorylation occurs on (a) histidine residue(s). It is probable that the function of p38 in the cell is modulated by ras proteins through phosphorylation. The significance of phosphorylation of p38 in terms of malignant transformation is presently known.     

Mutational analysis of a ras catalytic domain.

We used linker insertion-deletion mutagenesis to study the catalytic domain of the Harvey murine sarcoma virus v-rasH transforming protein, which is closely related to the cellular rasH protein. The mutants displayed a wide range of in vitro biological activity, from those that induced focal transformation of NIH 3T3 cells with approximately the same efficiency as the wild-type v-rasH gene to those that failed to induce any detectable morphologic changes. Correlation of transforming activity with the location of the mutations enabled us to identify three nonoverlapping segments within the catalytic domain that were dispensable for transformation and six other segments that were required for transformation. Segments that were necessary for guanosine nucleotide (GDP) binding corresponded to three of the segments that were essential for transformation; two of the three segments share strong sequence homology with other purine nucleotide-binding proteins. Loss of GDP binding was associated with apparent instability of the protein. Lesions in two of the three other required regions significantly reduced GDP binding, while small lesions in the last required region did not impair GDP binding or membrane localization. We speculate that this latter region interacts with the putative cellular target of ras. The results suggest that transforming ras proteins require membrane localization, guanosine nucleotide binding, and an additional undefined function that may represent interaction with their target.     

Mutant p53 tumor suppressor alleles release ras-induced cell cycle growth arrest.

Overexpression of an activated ras gene in the rat embryo fibroblast line REF52 results in growth arrest at either the G1/S or G2/M boundary of the cell cycle. Both the DNA tumor virus proteins simian virus 40 large T antigen and adenovirus 5 E1a are able to rescue ras induced lethality and cooperate with ras to fully transform REF52 cells. In this report, we present evidence that the wild-type activity of the tumor suppressor gene p53 is involved in the negative growth regulation of this model system. p53 genes encoding either a p53Val-135 or p53Pro-193 mutation express a highly stable p53 protein with a conformation-dependent loss of wild-type activity and the ability to eliminate any endogenous wild-type p53 activity in a dominant negative manner. In cotransfection assays, these mutant p53 genes are able to rescue REF52 cells from ras-induced growth arrest, resulting in established cell lines which express elevated levels of the ras oncoprotein and show morphological transformation. Full transformation, as assayed by tumor formation in nude mice, is found only in the p53Pro-193-plus-ras transfectants. These cells express higher levels of the ras protein than do the p53Val-135-plus-ras-transfected cells. Transfection of REF52 cells with ras alone or a full-length genomic wild-type p53 plus ras results in growth arrest and lethality. Therefore, the selective event for p53 inactivation or loss during tumor progression may be to overcome a cell cycle restriction induced by oncogene overexpression (ras). These results suggest that a normal function of p53 may be to mediate negative growth regulation in response to ras or other proliferative inducing signals.     

The catalytic domain of the neurofibromatosis type 1 gene product stimulates ras GTPase and complements ira mutants of S. cerevisiae

Sequencing of the neurofibromatosis gene (NF1) revealed a striking similarity among NF1, yeast IRA proteins, and mammalian GAP (GTPase-activating protein). Using both genetic and biochemical assays, we demonstrate that this homology domain of the NF1 protein interacts with ras proteins. First, expression of this NF1 domain suppressed the heat shock-sensitive phenotype of yeast ira1 and ira2 mutants. Second, this NF1 domain, after purification as a glutathione S-transferase (GST) fusion protein, strongly stimulated the GTPase activity of yeast RAS2 and human H-ras proteins. The GST-NF1 protein, however, did not stimulate the GTPase activity of oncogenic mutant ras proteins, H-rasVal-12 and yeast RAS2Val-19 mutants, or a yeast RAS2 effector mutant. These results establish that this NF1 domain has ras GAP activity similar to that found with IRA2 protein and mammalian GAP, and therefore may also regulate ras function in vivo.     

The recruitment of Fas-associated death domain/caspase-8 in Ras-induced apoptosis.

Oncogenic Ras induces cells to undergo apoptosis after inhibition of protein kinase C (PKC) activity. The integration of differential signaling pathways is required for full execution of apoptosis. In this study, we used Jurkat as well as Fas/FADD-defective cell lines expressing v-ras to determine the upstream elements required for activation of the caspase cascade in PKC/Ras-mediated apoptosis. During this Ras-induced apoptotic process, caspase-8 was activated, possibly through its binding to Fas-associated death domain (FADD), in Jurkat/ras and Jurkat/Fas(m)/ras cells but not in Jurkat/FADD(m)/ras cells. c-Jun NH(2)-terminal kinase (JNK) was activated in all three cell lines expressing ras in response to apoptotic stimulation. Suppression of JNK by dn-JNK1 blocked the interaction of FADD and caspase-8 and partially protected Jurkat/ras and Jurkat/Fas(m)/ras cells from apoptosis. However, dn-JNK1 had no effect on PKC/Ras-induced apoptosis in Jurkat/FADD(m)/ras cells. The results indicate that FADD/caspase-8 signaling is involved in PKC/Ras-mediated apoptosis, and JNK may be an upstream effector of caspase activation.     

Identification of a novel ligand-receptor pair constitutively activated by ras oncogenes

The Ras signaling pathway is thought to control the expression of a subset of yet to be defined genes that are crucial for cell growth and differentiation. Here we have identified by differential display a novel oncogenic Ras target, mob-5, encoding a 23-kDa cytokine-like secreted protein. Mob-5 expression could be induced by oncogenic Ha-ras and Ki-ras, but not by normal ras activation. Inhibitors of both Ha-Ras and mitogen-activated protein kinase kinase completely abolished the mob-5 expression in ras transformed cells, with concomitant loss of the transformation phenotype. Using an alkaline phosphatase-tagged Mob-5 as ligand, a putative Mob-5 receptor was identified on the cell surface of oncogenic ras transformed cells. Thus, the Mob-5/Mob-5 receptor may represent a novel putative autocrine loop coordinately activated by ras oncogenes.     

Model systems of carcinogenesis in vitro: the interaction of the ras oncogene with immortalized cells

Gene transfer experiments have shown that ras effector functions are sufficient to transform cells from a variety of established lines (e. g., mouse NIH3T3 cells). In contrast, primary cells and early passage rodent cells can be transformed by ras oncogenes only at low frequencies, unless cotransfected with collaborating genes such as adenovirus early region IA (EIA) or myc retroviral oncogene homologue. Primary rat embryo fibroblasts (REF) were chosen as a model for the analysis of multistep cellular transformation. Transfection of REF, immortalized by early region of simian adenovirus SA7 with c-Ha-ras oncogene cannot induce their morphological transformation. This phenomenon is observed only after second transfection with the same oncogene. These different cell lines can be used for further analysis of the mechanisms of carcinogenesis.     

The gene for the rat heat-shock cognate, hsc70, can suppress oncogene-mediated transformation.

In cells transformed by mutant mouse p53 plus ras, the former protein is found to be complexed with the heat-shock protein cognate hsc70. To determine whether hsc70 can directly affect neoplastic transformation, nonestablished rat embryo fibroblasts (REF) were transfected with rat genomic hsc70 DNA in conjunction with various oncogenes. We report here that the hsc70 gene could efficiently suppress focus induction by mutant p53 plus ras, as well as by myc plus ras. No inhibitory effect of hsc70 was detectable in assays monitoring the ability of REF to be immortalized by mutant p53, arguing against a nonspecific deleterious effect of the hsc70 genomic clone on REF survival and proliferation. Lines generated in the presence of the hsc70 plasmid produced augmented levels of hsc70. Plasmids encoding only short NH2-terminal fragments of hsc70 could also, in some cases, partially reduce oncogene-mediated focus formation. However, a maximal inhibitory effect required the production of a functional hsc70 protein. The data presented here raise the possibility that hsc70 may be directly involved in the modulation of oncogene-mediated transformation.