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C J Der

Publications and source records attributed to C J Der.

At least 109 records · Page 6Linked to original sources
PubMed1995

Biological and structural characterization of a Ras transforming mutation at the phenylalanine-156 residue, which is conserved in all members of the Ras superfamily.

Although Ras residue phenylalanine-156 (F156) is strictly conserved in all members of the Ras superfamily of proteins, it is located outside of the consensus GDP/GTP-binding pocket. Its location within the hydrophobic core of Ras suggests that its strict conservation reflects a crucial role in structural stability. However, mutation of the equivalent residue (F157L) in the Drosophila Ras-related protein Rap results in a gain-of-function phenotype, suggesting an alternative role for this residue. Therefore, we have introduced an F156L mutation into Ras to evaluate the role of this residue in Ras structure and function. Whereas introduction of this mutation activated the transforming potential of wild-type Ras, it did not impair that of oncogenic Ras. Further, Ras (156L) exhibited an extremely rapid off rate for bound GDP/GTP in vitro and showed increased levels of Ras.GTP in vivo. To determine the structural basis for these altered properties, we used high-resolution nuclear magnetic resonance spectroscopy. The F156L mutation caused loss of contact with residues 6, 23, 55, and 79, resulting in disruption of secondary structure in alpha-helix 1 and in beta-sheets 1-5. These major structural changes contrast with the isolated alterations induced by oncogenic mutation (residues 12 or 61) that perturb GTPase activity, and instead, weaken Ras contacts with Mg2+ and its guanine nucleotide substrate and result in increased rates of GDP/GTP dissociation. Altogether, these observations demonstrate the essential role of this conserved residue in Ras structure and its function as a regulated GDP/GTP switch.

3T3 Cells↗
PubMed1995

Guanine nucleotide exchange factors: activators of the Ras superfamily of proteins.

Ras proteins function as critical relay switches that regulate diverse signaling pathways between cell surface receptors and the nucleus. Over the past 2-3 years researchers have identified many components of these pathways that mediate Ras activation and effector function. Among these proteins are several guanine nucleotide exchange factors (GEFs), which are responsible for directly interacting with and activating Ras in response to extracellular stimuli. Analogous GEFs regulate Ras-related proteins that serve other diverse cellular functions. In particular, a growing family of proteins (Dbl homology proteins) has recently been identified, which may function as GEFs for the Rho family of Ras-related proteins. This review summarizes our current knowledge of the structure, biochemistry and biology of Ras and Rho family GEFs. Additionally, we describe mechanisms of GEF activation of Ras in signal transduction and address the potential that deregulated GEFs might contribute to malignant transformation through chronic Ras protein activation.

Animals↗
PubMed1995

Guanine nucleotide exchange factors: activators of Ras superfamily proteins.

Members of the Ras superfamily of proteins function as regulated GDP/GTP switches that cycle between active GTP-complexed and inactive GDP-complexed states. Guanine nucleotide exchange factors (GEFs) stimulate formation of the GTP-bound state, whereas GTPase activating proteins (GAPs) catalyze the formation of the GDP-bound state. We describe three studies that evaluate the mechanism of action of GEFs for Ras (SOS1 and RasGRF/CDC25) or Ras-related Rho (Dbl and Vav) proteins. Growth factor-mediated activation of Ras is believed to be mediated by activation of Ras GEFs (CDC25/GRF and SOS1/2). Although the mechanisms of Ras GEF regulation are unclear, recent studies suggest that translocation of SOS1 to the plasma membrane, where Ras is located, might be responsible for Ras activation. Our observation that the addition of the Ras plasma membrane-targeting sequence to the catalytic domains of CDC25 and SOS1 greatly enhanced their transforming and transactivation activities (10-50 fold and 5-10 fold, respectively) suggests that membrane translocation alone is sufficient to potentiate GEF activation of Ras. We have determined that two Ras-related proteins, designated R-Ras and R-Ras2/TC21, can trigger the malignant transformation of NIH 3T3 cells via activation of the Ras signal transduction pathway. Furthermore, like Ras and R-Ras, we observed that TC21 GTPase activity was stimulated by Ras GAPs. However, we observed that both SOS1 and CDC25 were activators of normal TC21, but not R-Ras, transforming activities. Therefore, TC21, but not R-Ras, may be activated by the same extracellular signaling events that activate Ras proteins. Dbl family proteins are believed to function as GEFs and activators of the Ras-related Rho family of proteins. However, one Dbl family oncogene, designated Vav, has been reported to be a GEF for Ras proteins. Therefore we were interested in determining whether Dbl family oncogenes cause transformation by triggering the constitutive activation of Rho or Ras proteins. Our results suggest that Dbl oncogenes cause transformation via a Ras-independent activation of MAP kinases and Rho family proteins.

Amino Acid Sequence↗
PubMed1995

Aberrant function of the Ras signal transduction pathway in human breast cancer.

Although ras mutations are infrequent (approximately 5%) in breast cancers, there is considerable evidence that suggests that the pathways which Ras services may still be deregulated in breast cancer cells. The recent identification of many of the components of the Ras signal transduction pathway has defined a network of proto-oncogene proteins controlling diverse signaling events that regulate cell growth and differentiation. Consequently, mutations that perturb the function of any one component of this signal pathway may trigger the same oncogenic events as mutation of ras itself. Moreover, several Ras-related proteins have recently been demonstrated to possess the ability to trigger malignant transformation via signaling pathways shared with Ras proteins. Thus, it is possible that the aberrant function of Ras-related proteins may contribute to breast cancer development. Consequently, it is important not to dismiss the Ras pathway in the development of breast cancer merely because of the infrequent detection of mutations in ras itself, but rather to consider the influence of aberrations upstream or downstream of Ras and of certain Ras-related proteins in the development of breast cancer. Finally, the critical importance of components upstream and downstream of Ras provides additional targets for rational drug design approaches to block the aberrant function of Ras signaling in human tumors.

Apoptosis↗
PubMed1995

Targeting proteins to membranes using signal sequences for lipid modification.

Covalent attachment of lipids appears to be an important mechanism by which many proteins interact with membranes. As we learn more about how lipids and adjacent amino acids participate in addressing proteins to specific membranes within the cell, it should be possible to design more elegant and precise membrane targeting systems that can be used to guide proteins to functionally relevant destinations.

Amino Acid Sequence↗
PubMed1995

Biological assays for Ras transformation.

The rodent fibroblast systems described above have provided sensitive and rapid biological assays to characterize the properties of normal and mutated Ras proteins. Furthermore, these assays have provided in vitro systems to measure the ability of other cellular components to modulate Ras signal transduction and transformation. However, while these assays provide an excellent measure of Ras-transforming activity, the fact that these cells are of fibroblastic origin, and can be transformed by a single hit, indicates that caution should be used in extrapolating observations from NIH 3T3 transformation assays to the situation in human tumors. Therefore, using human epithelial cell-based assays that more closely approximate the cell types where mutated ras alleles are most frequently detected may provide more realistic assays for examining the biochemical and biological consequences of aberrant Ras function in human tumors. Nevertheless, despite these cautions, these rodent transformation assays will continue to be the best and most widely applied assays for Ras biological activity.

3T3 Cells↗
PubMed1995

R-Ras promotes apoptosis caused by growth factor deprivation via a Bcl-2 suppressible mechanism.

The Bcl-2 protein is an important regulator of programmed cell death, but the biochemical mechanism by which this protein prevents apoptosis remains enigmatic. Recently, Bcl-2 has been reported to physically interact with a member of the Ras superfamily of small GTPases, p23-R-Ras. To examine the functional significance of R-Ras for regulation of cell death pathways, the IL-3-dependent cells 32D.3 and FL5.12 were stably transfected with expression plasmids encoding an activated form (38 Glycine-->Valine) of R-Ras protein. R-Ras(38V)-producing 32D.3 and FL5.12 cells experienced increased rates of apoptotic cell death relative to control transfected cells when deprived of IL-3. Analysis of several independent clones of transfected 32D.3 cells revealed a correlation between higher levels of R-Ras protein and faster rates of cell death upon withdrawal of IL-3 from cultures. 32D.3 cells cotransfected with R-Ras(38V) and Bcl-2 exhibited prolonged cell survival in the absence of IL-3, equivalent to 32D.3 cells transfected with Bcl-2 expression plasmids alone. R-Ras(38V) also increased rates of cell death in serum-deprived NIH-3T3 cells, and Bcl-2 again abrogated most of this effect. The ratio of GTP and GDP bound to R-Ras(38V) was not significantly different in control 32D.3 cells vs those that overexpressed Bcl-2, indicating that Bcl-2 does not abrogate R-Ras-mediated effects on cell death by altering R-Ras GDP/GTP regulation. Moreover, purified Bcl-2 protein had no effect on the GTPase activity of recombinant wild-type R-Ras in vitro. When expressed in Sf9 cells using recombinant baculoviruses, R-Ras(38V) bound to and induced activation of Raf-1 kinase irrespective of whether Bcl-2 was coproduced in these cells, suggesting that Bcl-2 does not nullify R-Ras effects by interfering with R-Ras-mediated activation of Raf-1 kinase. Taken together, these findings suggest that R-Ras enhances the activity of a cell death pathway in growth factor-deprived cells and imply that Bcl-2 acts downstream of R-Ras to promote cell survival.

3T3 Cells↗
PubMed1995

Tyrosine phosphorylation regulates the adhesions of ras-transformed breast epithelia.

Transformed epithelial cells often are characterized by a fibroblastic or mesenchymal morphology. These cells exhibit altered cell-cell and cell-substrate interactions. Here we have identified changes in the adhesions and cytoskeletal interactions of transformed epithelial cells that contribute to their altered morphology. Using MCF-10A human breast epithelial cells as a model system, we have found that transformation by an activated form of ras is characterized by less developed adherens-type junctions between cells but increased focal adhesions. Contributing to the modified adherens junctions of the transformed cells are decreased interactions among beta-catenin, E-cadherin, and the actin cytoskeleton. The ras-transformed cells reveal elevated phosphotyrosine in many proteins, including beta-catenin and p120 Cas. Whereas in the normal cells beta-catenin is found in association with E-cadherin, p120 Cas is not. In the ras-transformed cells, the situation is reversed; tyrosine-phosphorylated p120 Cas, but not tyrosine-phosphorylated beta-catenin, now is detected in E-cadherin complexes. The tyrosine-phosphorylated beta-catenin also shows increased detergent solubility, suggesting a decreased association with the actin cytoskeleton. p120 Cas, whether tyrosine phosphorylated or not, partitions into the detergent soluble fraction, suggesting that it is not tightly bound to the actin cytoskeleton in either the normal or ras-transformed cells. Inhibitors of tyrosine kinases decrease the level of tyrosine phosphorylation and restore a normal epithelial morphology to the ras-transformed cells. In particular, decreased tyrosine phosphorylation of beta-catenin is accompanied by increased interaction with both E-cadherin and the detergent insoluble cytoskeletal fraction. These results suggest that elevated tyrosine phosphorylation of proteins such as beta-catenin and p120 Cas contribute to the altered adherens junctions of ras-transformed epithelia.

Breast↗
PubMed1995

Activation of Rac1, RhoA, and mitogen-activated protein kinases is required for Ras transformation.

Although substantial evidence supports a critical role for the activation of Raf-1 and mitogen-activated protein kinases (MAPKs) in oncogenic Ras-mediated transformation, recent evidence suggests that Ras may activate a second signaling pathway which involves the Ras-related proteins Rac1 and RhoA. Consequently, we used three complementary approaches to determine the contribution of Rac1 and RhoA function to oncogenic Ras-mediated transformation. First, whereas constitutively activated mutants of Rac1 and RhoA showed very weak transforming activity when transfected alone, their coexpression with a weakly transforming Raf-1 mutant caused a greater than 35-fold enhancement of transforming activity. Second, we observed that coexpression of dominant negative mutants of Rac1 and RhoA reduced oncogenic Ras transforming activity. Third, activated Rac1 and RhoA further enhanced oncogenic Ras-triggered morphologic transformation, as well as growth in soft agar and cell motility. Finally, we also observed that kinase-deficient MAPKs inhibited Ras transformation. Taken together, these data support the possibility that oncogenic Ras activation of Rac1 and RhoA, coupled with activation of the Raf/MAPK pathway, is required to trigger the full morphogenic and mitogenic consequences of oncogenic Ras transformation.

3T3 Cells↗
PubMed1994

Polylysine domain of K-ras 4B protein is crucial for malignant transformation.

Previous studies have shown that posttranslational modifications are required for both oncogenic K-ras 4B protein membrane binding and transforming activity. In addition, Hancock et al. [Hancock, J. F., Patterson, H. & Marshall, C. J. (1990) Cell 63, 133-139] found that a polylysine domain contained at the C terminus of K-ras 4B was also absolutely essential for K-ras 4B membrane binding but, surprisingly, neither the polylysine domain nor membrane binding was required for transforming activity. We have performed similar studies, but our results are distinctly different. Our studies indicate that the polylysine domain is crucial for K-ras 4B transforming activity. Moreover, we demonstrate that although the polylysine domain increases K-ras 4B membrane binding, significant amounts of membrane binding can occur in the absence of this domain. Finally, while our studies are consistent with the notion that membrane binding is required for K-ras 4B transforming activity, we show that membrane binding, in and of itself, is not sufficient for efficient K-ras 4B transforming activity.

3T3 Cells↗
PubMed1994

Identification and characterization of Src SH3 ligands from phage-displayed random peptide libraries.

We have used the Src homology 3 (SH3) domain to screen two phage-displayed random peptide libraries, each containing 2 x 10(8) unique members, and have identified a series of high affinity peptide ligands. The peptides possess similar proline-rich regions, which yield a consensus Src SH3-binding motif of RPLPPLP. We have confirmed this motif by screening a phage-displayed peptide library biased for SH3 ligands and identifying the same consensus sequence. Binding studies using synthetic peptides suggest that the RPLPPLP motif is important for SH3 binding and confers specificity for the Src SH3 domain, and that residues which flank the motif may also contribute to binding. Peptides that contain the RPLPPLP motif compete Src, but not Abl or phospholipase C gamma, SH3 interactions with SH3-binding proteins from cell lysates (IC50 = 1-5 microM). Furthermore, RPLPPLP-related peptides are able to accelerate progesterone-induced maturation of Xenopus laevis oocytes. A similar acceleration has been observed in oocytes treated with activated, but not normal, Xenopus Src, suggesting the possibility that the peptides are able to antagonize the negative regulation of Src activity by Src SH3 in vivo.

3T3 Cells↗
PubMed1994

Membrane-targeting potentiates guanine nucleotide exchange factor CDC25 and SOS1 activation of Ras transforming activity.

Growth factor-triggered activation of Ras proteins is believed to be mediated by guanine nucleotide exchange factors (CDC25/GRF and SOS1/2) that promote formation of the active Ras GTP-bound state. Although the mechanism(s) of guanine nucleotide exchange factor regulation is unclear, recent studies suggest that translocation of SOS1 to the plasma membrane, where Ras is located, might be responsible for Ras activation. To evaluate this model, we generated constructs that encode the catalytic domains of human CDC25 or mouse SOS1, either alone (designated cCDC25 and cSOS1, respectively) or terminating in the carboxyl-terminal CAAX membrane-targeting sequence from K-Ras4B (designated cCDC25-CAAX and cSOS1-CAAX, respectively; in CAAX, C is Cys, A is an aliphatic amino acid, and X is Ser or Met). We then compared the transforming potential of cCDC25 and cSOS1 with their membrane-targeted counterparts. We observed that addition of the Ras plasma membrane-targeting sequence to the catalytic domains of CDC25 and SOS1 greatly enhanced their focus-forming activity (10- to 50-fold) in NIH 3T3 transfection assays. Similarly, we observed that the membrane-targeted versions showed a 5- to 10-fold enhanced ability to induce transcriptional activation from the Ets/AP-1 Ras-responsive element. Furthermore, whereas cells that stably expressed cCDC25 or cSOS1 exhibited the same morphologies as untransformed NIH 3T3 cells, cells expressing cCDC25-CAAX or cSOS1-CAAX displayed transformed morphologies that were indistinguishable from the elongated and refractile morphology of oncogenic Ras-transformed cells. Thus, these results suggest that membrane translocation alone is sufficient to potentiate guanine nucleotide exchange factor activation of Ras.

3T3 Cells↗
PubMed1994

The CAAX peptidomimetic compound B581 specifically blocks farnesylated, but not geranylgeranylated or myristylated, oncogenic ras signaling and transformation.

Recently developed CAAX peptidomimetic compounds have been shown to be potent and specific inhibitors of farnesyl protein transferase activity and to block the growth of Ras-transformed cells. However, whether this growth inhibitory action is specifically a consequence of blocking oncogenic Ras signaling has not been determined. To address this question, we have utilized mutants of the normally farnesylated oncogenic Ras protein (Ras-F) that are modified by alternative lipids, a geranylgeranyl isoprenoid (Ras-GG) or the fatty acid myristate (Myr-Ras), to determine the specificity of the CAAX peptidomimetic compound, B581. Like Ras-F, both Ras-GG and Myr-Ras are membrane-associated and transforming. Unexpectedly, NIH 3T3 cells transformed by each of the three Ras mutants underwent morphological alteration to a less transformed, but not normal, morphology. However, B581 inhibited the ability of only Ras-F-transformed cells, but not Ras-GG- or Myr-Ras- (or Raf-) transformed cells, to grow in soft agar. Furthermore, although all three lipid-modified versions of Ras stimulated mitogen-activated protein kinase activation, and both Jun and Elk-1 transcriptional activity, B581 inhibited only farnesylated Ras activation of these three downstream components of Ras signaling. Therefore, B581 prevents the growth of Ras-transformed cells by specifically antagonizing Ras-mediated signaling.

3T3 Cells↗
PubMed1994

Activated or dominant inhibitory mutants of Rap1A decrease the oxidative burst of Epstein-Barr virus-transformed human B lymphocytes.

Rap1A is a GTP-binding protein of the Ras superfamily that is highly abundant in phagocyte membranes. Although Rap1A copurifies with cytochrome b558, a component of the superoxide-generating NADPH oxidase complex of human phagocytes and B lymphocytes, the involvement of Rap1A in the regulation of the oxidative burst in these cells has not been clearly established. Therefore, we have stably transfected human Epstein-Barr virus-transformed B lymphocytes that possess an activable NADPH oxidase complex with cDNAs for mutants of Rap1A "locked" in a GTP-bound (63E) and GDP-bound (17N) state. Both the 17N and 63E mutants of Rap1A inhibited phorbol ester-stimulated O2-. production by 50 and 80%, respectively, while transfection with cDNA for wild-type Rap1A had no effect on the respiratory burst. No effects of the Rap1A mutants on cell viability, proliferation, expression of cell-surface markers, or phorbol 12-myristate 13-acetate-stimulated interleukin-8 generation were detected. These data demonstrate that Rap1A is a regulator of O2-. formation in intact cells. Furthermore, the inhibitory effect of both GTP- as well as GDP-bound mutants indicates that Rap1A functions in a dynamic cycle as opposed to a unidirectional pathway, as is the case for the other NADPH oxidase regulatory GTP-binding protein, Rac.

Antigens, Surface↗
PubMed1994

Oncogenic Ras activates c-Jun via a separate pathway from the activation of extracellular signal-regulated kinases.

c-Jun transcriptional activity is augmented by expression of oncogenic Ras and Raf proteins. This study demonstrates a direct correlation between Ras transforming activity and c-Jun activation, supporting an important role for c-Jun in transformation by Ras. Since we observed that Ras activated c-Jun transcriptional activity by increasing phosphorylation of the c-Jun activation domain at residues Ser-63/Ser-73 and that oncogenic Ras proteins activated extracellular signal-regulated protein kinases (ERK1 and ERK2) (also known as mitogen-activated protein kinases), we evaluated the possibility that ERKs were directly responsible for c-Jun activation. Coexpression of wild-type ERKs with oncogenic Ras proteins potentiated, while kinase-defective ERKs inhibited, Ras-induced transcriptional activation from the Ras-responsive element (Ets-1/AP-1) present in the NVL-3 enhancer and the serum-response element in the c-fos promoter. In contrast, coexpression of either wild-type or kinase-defective ERKs inhibited Ras and Raf activation of c-Jun transcriptional activity. Thus, although activation of both ERK and c-Jun are downstream consequences of activation of the Ras signal transduction pathway, our results suggest that Ras-induced c-Jun phosphorylation and transcriptional activation are not a direct consequence of ERK1 and ERK2 activation.

3T3 Cells↗
PubMed1994

Oncogene activation of human keratin 18 transcription via the Ras signal transduction pathway.

Keratin 8 (K8) and keratin 18 (K18) are intermediate filament proteins normally expressed in simple epithelial tissues and persistently expressed in a wide variety of carcinomas. Ectopic expression of K8 and K18 occurs in some epidermal and murine skin carcinomas induced by chemical carcinogenesis or oncogenic ras expression. We show here that K18 is a direct target of the Ras signal transduction pathway, by demonstrating that activated Ha-Ras, as well as activated Src, Lck, or Raf, stimulates the transcription of K18. This activation is mediated by an enhancer element containing essential and closely spaced Ets and AP-1 transcription factor binding sites. Oncogene activation of K18 transcription provides a molecular explanation for the persistent and sometimes unexpected expression of K18 in such a wide variety of tumors.

Animals↗