scholarly journals Aberrant function of the Ras-related protein TC21/R-Ras2 triggers malignant transformation.

1994 ◽  
Vol 14 (6) ◽  
pp. 4108-4115 ◽  
Author(s):  
S M Graham ◽  
A D Cox ◽  
G Drivas ◽  
M G Rush ◽  
P D'Eustachio ◽  
...  
Keyword(s):  
Amino Acid ◽  
Transformed Cells ◽  
Related Protein ◽  
Ras Proteins ◽  
Oncogenic Potential ◽  
Oncogenic Ras ◽  
Ras Genes ◽  
Transforming Activity ◽  
Related Proteins ◽  
Nih 3T3

Although the human Ras proteins are members of a large superfamily of Ras-related proteins, to date, only the proteins encoded by the three mammalian ras genes have been found to possess oncogenic potential. Among the known Ras-related proteins, TC21/R-Ras2 exhibits the most significant amino acid identity (55%) to Ras proteins. We have generated mutant forms of TC21 that possess amino acid substitutions analogous to those that activate Ras oncogenic potential [designated TC21(22V) and TC21(71L)] and compared the biological properties of TC21 with those of Ras proteins in NIH 3T3 and Rat-1 transformation assays. Whereas wild-type TC21 did not show any transforming potential in vitro, both TC21(22V) and TC21(71L) displayed surprisingly potent transforming activities that were comparable to the strong transforming activity of oncogenic Ras proteins. Like Ras-transformed cells, NIH 3T3 cells expressing mutant TC21 proteins formed foci of morphologically transformed cells in monolayer cultures, proliferated in low serum, formed colonies in soft agar, and developed progressive tumors in nude mice. Thus, TC21 is the first Ras-related protein to exhibit potent transforming activity equivalent to that of Ras. Furthermore, mutant TC21 proteins also stimulated constitutive activation of mitogen-activated protein kinases as well as transcriptional activation from Ras-responsive promoter elements (Ets/AP-1 and NF-kappa B). We conclude that aberrant TC21 function may trigger cellular transformation via a signal transduction pathway similar to that of oncogenic Ras and suggest that deregulated TC21 activity may contribute significantly to human oncogenesis.

scholarly journals Aberrant function of the Ras-related protein TC21/R-Ras2 triggers malignant transformation

1994 ◽  
Vol 14 (6) ◽  
pp. 4108-4115
Author(s):  
S M Graham ◽  
A D Cox ◽  
G Drivas ◽  
M G Rush ◽  
P D'Eustachio ◽  
...  
Keyword(s):  
Amino Acid ◽  
Transformed Cells ◽  
Related Protein ◽  
Ras Proteins ◽  
Oncogenic Potential ◽  
Oncogenic Ras ◽  
Ras Genes ◽  
Transforming Activity ◽  
Related Proteins ◽  
Nih 3T3

Although the human Ras proteins are members of a large superfamily of Ras-related proteins, to date, only the proteins encoded by the three mammalian ras genes have been found to possess oncogenic potential. Among the known Ras-related proteins, TC21/R-Ras2 exhibits the most significant amino acid identity (55%) to Ras proteins. We have generated mutant forms of TC21 that possess amino acid substitutions analogous to those that activate Ras oncogenic potential [designated TC21(22V) and TC21(71L)] and compared the biological properties of TC21 with those of Ras proteins in NIH 3T3 and Rat-1 transformation assays. Whereas wild-type TC21 did not show any transforming potential in vitro, both TC21(22V) and TC21(71L) displayed surprisingly potent transforming activities that were comparable to the strong transforming activity of oncogenic Ras proteins. Like Ras-transformed cells, NIH 3T3 cells expressing mutant TC21 proteins formed foci of morphologically transformed cells in monolayer cultures, proliferated in low serum, formed colonies in soft agar, and developed progressive tumors in nude mice. Thus, TC21 is the first Ras-related protein to exhibit potent transforming activity equivalent to that of Ras. Furthermore, mutant TC21 proteins also stimulated constitutive activation of mitogen-activated protein kinases as well as transcriptional activation from Ras-responsive promoter elements (Ets/AP-1 and NF-kappa B). We conclude that aberrant TC21 function may trigger cellular transformation via a signal transduction pathway similar to that of oncogenic Ras and suggest that deregulated TC21 activity may contribute significantly to human oncogenesis.

scholarly journals Specific isoprenoid modification is required for function of normal, but not oncogenic, Ras protein.

1992 ◽  
Vol 12 (6) ◽  
pp. 2606-2615 ◽  
Author(s):  
A D Cox ◽  
M M Hisaka ◽  
J E Buss ◽  
C J Der
Keyword(s):  
Mammalian Cells ◽  
Protein Modification ◽  
Biological Activities ◽  
Ras Proteins ◽  
Ras Protein ◽  
Oncogenic Ras ◽  
Transforming Activity ◽  
Nih 3T3 ◽  
Similar Growth ◽  
Growth Inhibiting

While the Ras C-terminal CAAX sequence signals modification by a 15-carbon farnesyl isoprenoid, the majority of isoprenylated proteins in mammalian cells are modified instead by a 20-carbon geranylgeranyl moiety. To determine the structural and functional basis for modification of proteins by a specific isoprenoid group, we have generated chimeric Ras proteins containing C-terminal CAAX sequences (CVLL and CAIL) from geranylgeranyl-modified proteins and a chimeric Krev-1 protein containing the H-Ras C-terminal CAAX sequence (CVLS). Our results demonstrate that both oncogenic Ras transforming activity and Krev-1 antagonism of Ras transforming activity can be promoted by either farnesyl or geranylgeranyl modification. Similarly, geranylgeranyl-modified normal Ras [Ras(WT)CVLL], when overexpressed, exhibited the same level of transforming activity as the authentic farnesyl-modified normal Ras protein. Therefore, farnesyl and geranylgeranyl moieties are functionally interchangeable for these biological activities. In contrast, expression of moderate levels of geranylgeranyl-modified normal Ras inhibited the growth of untransformed NIH 3T3 cells. This growth inhibition was overcome by coexpression of the mutant protein with oncogenic Ras or Raf, but not with oncogenic Src or normal Ras. The similar growth-inhibiting activities of Ras(WT)CVLL and the previously described Ras(17N) dominant inhibitory mutant suggest that geranylgeranyl-modified normal Ras may exert its growth-inhibiting action by perturbing endogenous Ras function. These results suggest that normal Ras function may specifically require protein modification by a farnesyl, but not a geranylgeranyl, isoprenoid.

Specific isoprenoid modification is required for function of normal, but not oncogenic, Ras protein

1992 ◽  
Vol 12 (6) ◽  
pp. 2606-2615
Author(s):  
A D Cox ◽  
M M Hisaka ◽  
J E Buss ◽  
C J Der
Keyword(s):  
Mammalian Cells ◽  
Protein Modification ◽  
Biological Activities ◽  
Ras Proteins ◽  
Ras Protein ◽  
Oncogenic Ras ◽  
Transforming Activity ◽  
Nih 3T3 ◽  
Similar Growth ◽  
Growth Inhibiting

While the Ras C-terminal CAAX sequence signals modification by a 15-carbon farnesyl isoprenoid, the majority of isoprenylated proteins in mammalian cells are modified instead by a 20-carbon geranylgeranyl moiety. To determine the structural and functional basis for modification of proteins by a specific isoprenoid group, we have generated chimeric Ras proteins containing C-terminal CAAX sequences (CVLL and CAIL) from geranylgeranyl-modified proteins and a chimeric Krev-1 protein containing the H-Ras C-terminal CAAX sequence (CVLS). Our results demonstrate that both oncogenic Ras transforming activity and Krev-1 antagonism of Ras transforming activity can be promoted by either farnesyl or geranylgeranyl modification. Similarly, geranylgeranyl-modified normal Ras [Ras(WT)CVLL], when overexpressed, exhibited the same level of transforming activity as the authentic farnesyl-modified normal Ras protein. Therefore, farnesyl and geranylgeranyl moieties are functionally interchangeable for these biological activities. In contrast, expression of moderate levels of geranylgeranyl-modified normal Ras inhibited the growth of untransformed NIH 3T3 cells. This growth inhibition was overcome by coexpression of the mutant protein with oncogenic Ras or Raf, but not with oncogenic Src or normal Ras. The similar growth-inhibiting activities of Ras(WT)CVLL and the previously described Ras(17N) dominant inhibitory mutant suggest that geranylgeranyl-modified normal Ras may exert its growth-inhibiting action by perturbing endogenous Ras function. These results suggest that normal Ras function may specifically require protein modification by a farnesyl, but not a geranylgeranyl, isoprenoid.

The Endocannabinoid/Endovanilloid N-Arachidonoyl Dopamine Suppresses the Plasma Membrane Translocation and Neoplastic Transforming Activity of NRAS and KRAS4A

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 434-434
Author(s):  
Min Wu ◽  
Bo Jiao ◽  
Ruibao Ren
Keyword(s):  
Plasma Membrane ◽  
Transformed Cells ◽  
Specific Cell ◽  
Ras Proteins ◽  
Membrane Translocation ◽  
Homologous Proteins ◽  
Ras Genes ◽  
Human Cancers ◽  
New Findings ◽  
Transforming Activity

Abstract The RAS family includes three RAS genes, which encode four highly homologous proteins: H-, N-, and KRAS4A and 4B, the latter two being alternatively spliced forms differing only at the carboxyl terminus. RAS oncogenic mutations, mostly KRAS and NRAS, are common in human cancers. Since the enzymatic activity of RAS is used to turn itself off and its GTP binding affinity is very high, RAS proteins have been difficult to target. Identification of alternative means to block the RAS oncogenic signaling is critical for developing therapies against RAS-driven cancer. We took a chemical biology approach to discover novel therapeutic strategies for RAS-related cancer. Since the biological activity of RAS proteins relies upon lipid modifications and RAS regulates lipid metabolisms in cancer cells, we used a RAS specific cell viability assay to screen a bioactive lipid library for potential therapeutics against RAS-driven cancer. We found that endocannabinoid/endovanilloid N-arachidonoyl dopamine (NADA) could effectively kill RAS-transformed cells. The structure-activity analysis showed that the anti-RAS activity of NADA relied on both the dopamine and arachidonoyl moieties in the same molecule and was independent of its ability to engage cannabinoid or vanilloid receptors. Further analysis showed that NADA was more active in killing cells transformed by oncogenic NRAS or KRAS4A than that of KRAS4B. Consistently, NADA was more active in inhibiting the phosphorylation of ERK in NRAS-transformed cells than that in KRAS4B-transformed cells. Interestingly, NADA blocked the plasma membrane translocation of NRAS and KRAS4A, but not that of KRAS4B. Significant effect of NADA on palmitoylated NRAS levels was not detected, suggesting that NADA inhibits NRAS and KRAS4A plasma membrane translocation through a novel mechanism. These results demonstrate that NADA possesses a novel property of inhibiting the plasma membrane translocation and neoplastic transforming activity of NRAS and KRAS4A. Since NRAS mutations are common in hematological malignancies as well as in solid tumors such as melanoma and colorectal carcinoma, and KRAS4A is required for carcinogen-induced lung cancer, the new findings would help to develop novel therapies for a broad range of human cancers. Disclosures No relevant conflicts of interest to declare.

The COOH-terminal domain of the Rap1A (Krev-1) protein is isoprenylated and supports transformation by an H-Ras:Rap1A chimeric protein

1991 ◽  
Vol 11 (3) ◽  
pp. 1523-1530
Author(s):  
J E Buss ◽  
L A Quilliam ◽  
K Kato ◽  
P J Casey ◽  
P A Solski ◽  
...  
Keyword(s):  
Biological Activities ◽  
Chimeric Protein ◽  
Cell System ◽  
Full Length ◽  
Ras Proteins ◽  
3T3 Cells ◽  
Farnesyl Transferase ◽  
Oncogenic Ras ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Although the Rap1A protein resembles the oncogenic Ras proteins both structurally and biochemically, Rap1A exhibits no oncogenic properties. Rather, overexpression of Rap1A can reverse Ras-induced transformation of NIH 3T3 cells. Because the greatest divergence in amino acid sequence between Ras and Rap1A occurs at the COOH terminus, the role of this domain in the opposing biological activities of these proteins was examined. COOH-terminal processing and membrane association of Rap1A were studied by constructing and expressing a chimeric protein (composed of residues 1 to 110 of an H-Ras activated by a Leu-61 mutation attached to residues 111 to 184 of Rap1A) in NIH 3T3 cells and a full-length human Rap1A protein in a baculovirus-Sf9 insect cell system. Both the chimeric protein and the full-length protein were synthesized as a 23-kDa cytosolic precursor that rapidly bound to membranes and was converted into a 22-kDa form that incorporated label derived from [3H]mevalonate. The mature 22-kDa form also contained a COOH-terminal methyl group. Full-length Rap1A, expressed in insect cells, was modified by a C20 (geranylgeranyl) isoprenoid. In contrast, H-Ras, expressed in either Sf9 insect or NIH 3T3 mouse cells contained a C15 (farnesyl) group. This suggests that the Rap1A COOH terminus is modified by a prenyl transferase that is distinct from the farnesyl transferase that modifies Ras proteins. Nevertheless, in NIH 3T3 cells the chimeric Ras:Rap1A protein retained the transforming activity conferred by the NH2-terminal Ras61L domain. This demonstrates that the modifications and localization signals of the COOH terminus of Rap1A can support the interactions between H-Ras and membranes that are required for transformation.

Expression of a gene family in the dimorphic fungus Mucor racemosus which exhibits striking similarity to human ras genes

1990 ◽  
Vol 10 (12) ◽  
pp. 6654-6663
Author(s):  
W L Casale ◽  
D G Mcconnell ◽  
S Y Wang ◽  
Y J Lee ◽  
J E Linz
Keyword(s):  
Amino Acid ◽  
Cell Development ◽  
Amino Acid Sequences ◽  
Model Systems ◽  
Striking Similarity ◽  
Ras Proteins ◽  
Mucor Racemosus ◽  
Dimorphic Fungus ◽  
Transcript Accumulation ◽  
Ras Genes

Sporulation, spore germination, and yeast-hypha dimorphism in the filamentous fungus Mucor racemosus provide useful model systems to study cell development in eucaryotic cells. Three RAS genes (MRAS1, MRAS2, and MRAS3) from M. racemosus have been cloned, and their nucleotide sequences have been determined. The predicted amino acid sequences and the sizes of the three MRAS proteins exhibit a high degree of similarity with other ras proteins, including that encoded by H-ras, which have been implicated in regulation of proliferation and development in eucaryotic cells by mediating signal transduction pathways. The MRAS proteins show conservation of functional domains proposed for ras proteins, including guanine nucleotide interaction domains, an effector domain, a binding epitope for neutralizing antibody Y13-259, and the COOH-terminal CAAX box, which is a site of thiocylation and membrane attachment. Amino acid sequences unique to each MRAS protein occur adjacent to the CAAX box, consistent with the location of the hypervariable region in other ras proteins. Northern (RNA) analysis was used to study expression of the three MRAS genes in relation to cell development. Gene-specific probes for two of these genes, MRAS1 and MRAS3, hybridized to different 1.3-kb mRNA transcripts. The accumulation of these transcripts depended on the developmental stage, and this pattern was different between the two MRAS genes. No transcript for MRAS2 was detected in the developmental stages examined. The unique patterns of MRAS transcript accumulation suggest that individual MRAS genes and proteins may play distinct roles in cell growth or development.

Dominant inhibitory Ras mutants selectively inhibit the activity of either cellular or oncogenic Ras

1991 ◽  
Vol 11 (8) ◽  
pp. 4053-4064 ◽  
Author(s):  
D W Stacey ◽  
L A Feig ◽  
J B Gibbs
Keyword(s):  
Soluble Form ◽  
Detectable Effect ◽  
Mutant Proteins ◽  
Ras Protein ◽  
Downstream Target ◽  
Oncogenic Ras ◽  
Binding Function ◽  
Transforming Activity ◽  
Nih 3T3 ◽  
Effector Binding

Two dominant inhibitory Ras mutant proteins were analyzed by microinjection. One, [Asn-17]Ras, had a substitution in the putative Mg(2+)-binding site of Ha-Ras. The other, RAST, had a mutation in a yeast RAS protein that impaired its GTPase activity and increased its affinity for GAP. RAST also had a mutation that blocked its localization to the plasma membrane. In NIH 3T3 cells [Asn-17]Ras inhibited the function of normal Ras much more efficiently than that of oncogenic Ras. In contrast, RAST interfered with the transforming activity of oncogenic Ras more efficiently than that of normal Ras. These conclusions were based on two separate types of analysis. The inhibitory Ras mutant proteins were first microinjected into cells stably transformed either by oncogenic Ras or by high levels of expression of cellular Ras. Results obtained in stably transformed cells were then verified by coinjection of the inhibitory Ras mutant proteins together with transforming concentrations of either oncogenic or normal Ras protein. Whereas RAST was active in soluble form. [Asn-17]Ras required membrane localization for activity. Furthermore, mutations in the GAP/effector-binding domain reduced or eliminated the inhibitory activity of RAST but had no detectable effect on [Asn-17]Ras. These results are consistent with the possibility that [Asn-17]Ras functions by blocking the activation of endogenous Ras proteins, while RAST functions by blocking the ability of activated Ras to stimulate a downstream target within the cells. The properties of RAST suggest that interference with the GAP/effector-binding function of RAS represents a strategy for the preferential inactivation of oncogenic Ras in cells.

scholarly journals Transformation properties of the E2a-Pbx1 chimeric oncoprotein: fusion with E2a is essential, but the Pbx1 homeodomain is dispensable.

1994 ◽  
Vol 14 (12) ◽  
pp. 8304-8314 ◽  
Author(s):  
K Monica ◽  
D P LeBrun ◽  
D A Dedera ◽  
R Brown ◽  
M L Cleary
Keyword(s):  
Transcriptional Activation ◽  
Binding Sites ◽  
Chromosomal Translocation ◽  
Wild Type ◽  
3T3 Cells ◽  
Homeodomain Proteins ◽  
Malignant Lymphomas ◽  
Oncogenic Potential ◽  
Related Proteins ◽  
Nih 3T3

The t(1;19) chromosomal translocation in acute lymphoblastic leukemias creates chimeric E2a-Pbx1 oncoproteins that can act as DNA-binding activators of transcription. A structural analysis of the functional domains of E2a-Pbx1 showed that portions of both E2a and Pbx1 were essential for transformation of NIH 3T3 cells and transcriptional activation of synthetic reporter genes containing PBX1 consensus binding sites. Hyperexpression of wild-type or experimentally truncated Pbx1 proteins was insufficient for transformation, consistent with their inability to activate transcription. When fused with E2a, the Pbx-related proteins Pbx2 and Pbx3 were also transformation competent, demonstrating that all known members of this highly similar subfamily of homeodomain proteins have latent oncogenic potential. The oncogenic contributions of E2a to the chimeras were localized to transactivation motifs AD1 and AD2, as their mutation significantly impaired transformation. Either the homeodomain or Pbx1 amino acids flanking this region could mediate transformation when fused to E2a. However, the homeodomain was not essential for transformation, since a mutant E2a-Pbx1 protein (E2a-Pbx delta HD) lacking the homeodomain efficiently transformed fibroblasts and induced malignant lymphomas in transgenic mice. Thus, transformation mediated by the chimeric oncoprotein E2a-Pbx1 is absolutely dependent on motifs acquired from E2a but the Pbx1 homeodomain is optional. The latter finding suggests that E2a-Pbx1 may interact with cellular proteins that assist or mediate alterations in gene expression responsible for oncogenesis even in the absence of homeodomain-DNA interactions.

Transformation properties of the E2a-Pbx1 chimeric oncoprotein: fusion with E2a is essential, but the Pbx1 homeodomain is dispensable

1994 ◽  
Vol 14 (12) ◽  
pp. 8304-8314
Author(s):  
K Monica ◽  
D P LeBrun ◽  
D A Dedera ◽  
R Brown ◽  
M L Cleary
Keyword(s):  
Transcriptional Activation ◽  
Binding Sites ◽  
Chromosomal Translocation ◽  
Wild Type ◽  
3T3 Cells ◽  
Homeodomain Proteins ◽  
Malignant Lymphomas ◽  
Oncogenic Potential ◽  
Related Proteins ◽  
Nih 3T3

The t(1;19) chromosomal translocation in acute lymphoblastic leukemias creates chimeric E2a-Pbx1 oncoproteins that can act as DNA-binding activators of transcription. A structural analysis of the functional domains of E2a-Pbx1 showed that portions of both E2a and Pbx1 were essential for transformation of NIH 3T3 cells and transcriptional activation of synthetic reporter genes containing PBX1 consensus binding sites. Hyperexpression of wild-type or experimentally truncated Pbx1 proteins was insufficient for transformation, consistent with their inability to activate transcription. When fused with E2a, the Pbx-related proteins Pbx2 and Pbx3 were also transformation competent, demonstrating that all known members of this highly similar subfamily of homeodomain proteins have latent oncogenic potential. The oncogenic contributions of E2a to the chimeras were localized to transactivation motifs AD1 and AD2, as their mutation significantly impaired transformation. Either the homeodomain or Pbx1 amino acids flanking this region could mediate transformation when fused to E2a. However, the homeodomain was not essential for transformation, since a mutant E2a-Pbx1 protein (E2a-Pbx delta HD) lacking the homeodomain efficiently transformed fibroblasts and induced malignant lymphomas in transgenic mice. Thus, transformation mediated by the chimeric oncoprotein E2a-Pbx1 is absolutely dependent on motifs acquired from E2a but the Pbx1 homeodomain is optional. The latter finding suggests that E2a-Pbx1 may interact with cellular proteins that assist or mediate alterations in gene expression responsible for oncogenesis even in the absence of homeodomain-DNA interactions.

scholarly journals The COOH-terminal domain of the Rap1A (Krev-1) protein is isoprenylated and supports transformation by an H-Ras:Rap1A chimeric protein.

1991 ◽  
Vol 11 (3) ◽  
pp. 1523-1530 ◽  
Author(s):  
J E Buss ◽  
L A Quilliam ◽  
K Kato ◽  
P J Casey ◽  
P A Solski ◽  
...  
Keyword(s):  
Biological Activities ◽  
Chimeric Protein ◽  
Cell System ◽  
Full Length ◽  
Ras Proteins ◽  
3T3 Cells ◽  
Farnesyl Transferase ◽  
Oncogenic Ras ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Although the Rap1A protein resembles the oncogenic Ras proteins both structurally and biochemically, Rap1A exhibits no oncogenic properties. Rather, overexpression of Rap1A can reverse Ras-induced transformation of NIH 3T3 cells. Because the greatest divergence in amino acid sequence between Ras and Rap1A occurs at the COOH terminus, the role of this domain in the opposing biological activities of these proteins was examined. COOH-terminal processing and membrane association of Rap1A were studied by constructing and expressing a chimeric protein (composed of residues 1 to 110 of an H-Ras activated by a Leu-61 mutation attached to residues 111 to 184 of Rap1A) in NIH 3T3 cells and a full-length human Rap1A protein in a baculovirus-Sf9 insect cell system. Both the chimeric protein and the full-length protein were synthesized as a 23-kDa cytosolic precursor that rapidly bound to membranes and was converted into a 22-kDa form that incorporated label derived from [3H]mevalonate. The mature 22-kDa form also contained a COOH-terminal methyl group. Full-length Rap1A, expressed in insect cells, was modified by a C20 (geranylgeranyl) isoprenoid. In contrast, H-Ras, expressed in either Sf9 insect or NIH 3T3 mouse cells contained a C15 (farnesyl) group. This suggests that the Rap1A COOH terminus is modified by a prenyl transferase that is distinct from the farnesyl transferase that modifies Ras proteins. Nevertheless, in NIH 3T3 cells the chimeric Ras:Rap1A protein retained the transforming activity conferred by the NH2-terminal Ras61L domain. This demonstrates that the modifications and localization signals of the COOH terminus of Rap1A can support the interactions between H-Ras and membranes that are required for transformation.

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