Biomedical subjects

C J Der

Publications and source records attributed to C J Der.

At least 127 records · Page 7Linked to original sources

PubMed1994

The Ras signal transduction pathway.

Considerable progress has been made over the past year in elucidating the mechanisms by which extracellular signals are transduced via cell surface receptors to trigger changes in gene expression which determine the growth and differentiated state of a cell. In particular, Ras proteins have been implicated as key intermediates that mediate the signal from upstream tyrosine kinases to a downstream cascade of serine/threonine kinases, which then activate nuclear factors that control gene expression and protein synthesis. How Ras proteins function is regulated in this role as a molecular switch, and how the signal is transmitted between the various components of the pathway, are now being determined. Finally, the Rho family of Ras-related proteins, which regulate the actin cytoskeleton, have also been implicated as mediators of oncogenic Ras transformation. The brisk pace at which the key components of Ras-mediated signal transduction pathways are being identified hold great promise that new targets for therapeutic intervention in cancer may now be identified.

Animals↗

PubMed1994

Transcriptional activation analysis of oncogene function.

Because no single assay provides a complete analysis of the transformed phenotype, transactivation assays complement the cell growth and tumorigenicity analyses of oncogene function. Transactivation of ORE-containing genes is such a common feature of a diverse variety of viral and cellular oncogenes that it can be considered one aspect of the oncogene-induced phenotype. After the initial identification of oncogenes that activate transcription, studies of the mechanisms of activation and the identification of the downstream target genes should lead to a better understanding of the events leading to cellular transformation. The fact that cell type specificity of transactivation and transformation can be similar means that the transactivation assay may be a useful tool in dissecting cell type-specific transformation. The transactivation assay of oncogene function also has the advantage that it is easy to perform and significantly more rapid than assays based on altered cell growth. This is of particular advantage when one wishes to examine the function of a large number of oncogene mutants generated in vitro. Overall, transactivation assays provide another tool for examining transforming potential and a starting point for the analysis of the downstream targets of oncogenes.

3T3 Cells↗

PubMed1994

Biological assays for cellular transformation.

A number of standard and widely applied procedures have been used to determine whether expression of a particular gene triggers the growth alterations that are characteristic of most oncogenes. The assays have been used extensively to evaluate the transforming potential of a wide variety of genes that encode tyrosine or serine/threonine kinases, small and heterotrimeric GTP-binding signal transduction regulators, and nuclear transcription factors, among others. Therefore, the growth-promoting characteristics of a particular gene can be compared with the properties of other genes that have been characterized by the same assays. The assays described do not represent a complete evaluation of the transforming activity of a gene. Failure to detect growth-promoting activity in any of the assays does not definitively eliminate the possibility that a particular gene is an oncogene. Specialized assays that use (nonfibroblast) recipient cells more closely approximating the likely environment of the gene of interest may provide better approaches for subsequent studies. Other biological assays for transforming potential include measurements of the adhesion properties of cells on different substrata, the ability to grow on confluent monolayers of normal cells, the ability to invade into various artificial tissue matrices, and transgenic animal models. Finally, more specific assays for biochemical alterations that reflect the transformed state can also be employed. For example, as discussed in [23] in this volume, one widely used biochemical measure of transforming potential employs transcriptional activation of genes whose promoters contain so-called oncogene-responsive elements. This, as well as other biochemical assays, can be applied to complement the biological studies described in this chapter.

3T3 Cells↗

PubMed1994

Dbl and Vav mediate transformation via mitogen-activated protein kinase pathways that are distinct from those activated by oncogenic Ras.

Vav and Dbl are members of a novel class of oncogene proteins that share significant sequence identity in a approximately 250-amino-acid domain, designated the Dbl homology domain. Although Dbl functions as a guanine nucleotide exchange factor (GEF) and activator of Rho family proteins, recent evidence has demonstrated that Vav functions as a GEF for Ras proteins. Thus, transformation by Vav and Dbl may be a consequence of constitutive activation of Ras and Rho proteins, respectively. To address this possibility, we have compared the transforming activities of Vav and Dbl with that of the Ras GEF, GRF/CDC25. As expected, GRF-transformed cells exhibited the same reduction in actin stress fibers and focal adhesions as Ras-transformed cells. In contrast, Vav- and Dbl-transformed cells showed the same well-developed stress fibers and focal adhesions observed in normal or RhoA(63L)-transformed NIH 3T3 cells. Furthermore, neither Vav- or Dbl-transformed cells exhibited the elevated levels of Ras-GTP (60%) observed with GRF-transformed cells. Finally, GRF, but not Vav or Dbl, induced transcriptional activation from Ras-responsive DNA elements (ets/AP-1, fos promoter, and kappa B). However, like Ras- and GRF-transformed cells, both Vav- and Dbl-transformed cells exhibited constitutively activated mitogen-activated protein kinases (MAPKs) (primarily p42MAPK/ERK2). Since kinase-deficient forms of p42MAPK/ERK2 and p44MAPK/ERK1 inhibited Dbl transformation, MAPK activation may be an important component of its transforming activity. Taken together, our observations indicate that Vav and Dbl transformation is not a consequence of Ras activation and instead may involve the constitutive activation of MAPKs.

3T3 Cells↗

PubMed1994

Identification of residues critical for Ras(17N) growth-inhibitory phenotype and for Ras interaction with guanine nucleotide exchange factors.

The Ras(17N) dominant negative antagonizes endogenous Ras function by forming stable, inactive complexes with Ras guanine nucleotide exchange factors (GEFs; e.g., SOS1). We have used the growth-inhibitory phenotype of Ras(17N) to characterize two aspects of Ras interaction with GEFs. First, we used a nonprenylated version of Ras(17N), designated Ras(17N/186S), which no longer associates with the plasma membrane and lacks the growth-inhibitory phenotype, to address the importance of Ras subcellular location and posttranslational modification for its interaction with GEFs. We observed that addition of an N-terminal myristylation signal to Ras(17N/186S) restored the growth-inhibitory activity of nonprenylated Ras(17N). Thus, membrane association, rather than prenylation, is critical for Ras interaction with Ras GEFs. Second, we used a biological selection approach to identify Ras residues which are critical for Ras(17N) growth inhibition and hence for interaction with Ras GEFs. We identified mutations at residues 75, 76, and 78 that abolished the growth-inhibitory activity of Ras(17N). Since GEF interaction is dispensable for oncogenic but not normal Ras function, our demonstration that single-amino-acid substitutions at these three positions impaired the transforming activity of normal but not oncogenic Ras provides further support for the role of these residues in Ras-GEF interactions. Finally, Ras(WT) proteins with mutations at these residues were no longer activated by mammalian SOS1. Altogether, these results suggest that the Ras intracellular location and Ras residues 75 to 78 are critical for Ras-GEF interaction.

3T3 Cells↗

PubMed1994

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

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.

3T3 Cells↗

PubMed1994

R-Ras induces malignant, but not morphologic, transformation of NIH3T3 cells.

Although previous studies have not identified transforming properties of the Ras-related protein R-Ras, two recent observations have prompted our further evaluation of R-Ras function. First, we observed that mutant forms of the closely related R-Ras2/TC21 protein (approximately 70% identity) exhibited the same potent transforming activity as oncogenic Ras proteins. Second, R-Ras association with Bcl-2 suggested a possible role for R-Ras in apoptotic growth control. Therefore, we have performed a detailed analysis of R-Ras transforming potential in NIH3T3 cells. Whereas expression of a mutant R-Ras protein (38V; analogous to the 12V activating Ras mutation) did not induce morphologic transformation of NIH3T3 cells, R-Ras(38V)-expressing cells proliferated in low serum, formed colonies in soft agar, and formed progressive tumors in nude mice. Like Ras-transformed cells, R-Ras(38V)-transformed cells exhibited constitutively activated mitogen activated protein kinases. Furthermore, R-Ras(38V) stimulated transcriptional activation of Ras-responsive promoter elements, and this activity (and transformation) was blocked by Raf dominant negative proteins. Finally, whereas co-expression of Bcl-2 did not cause significant alteration in wild type or mutant R-Ras transforming activity, coexpression of v-Myc and R-Ras(38V) induced a striking morphologic transformation of NIH3T3 cells. Taken together, these observations suggest that aberrant R-Ras function may stimulate malignant transformation, in the absence of morphologic transformation, via up-regulation of part of the Ras signal transduction pathway.

3T3 Cells↗

PubMed1994

Ras-15A protein shares highly similar dominant-negative biological properties with Ras-17N and forms a stable, guanine-nucleotide resistant complex with CDC25 exchange factor.

We show that expression of Ras-15A, previously shown to be a dominant-negative mutant in yeast, is a potent inhibitor of endogenous Ras protein function in mammalian cells. Expression of Ras-15A did not inhibit the growth of cells containing an oncogenic ras gene nor did it interfere with the ability of transiently expressed oncogenic ras or raf genes to activate transcription from a Ras-responsive ets1/AP-1 promoter. In contrast, expression of Ras-15A completely blocked growth of normal cells and activation of the ets1/AP-1 promoter by transiently overexpressed SOS1 and normal Ras proteins. These results suggest that Ras-15A, like Ras-17N, blocks endogenous Ras function by interfering with upstream activation of Ras proteins rather than downstream effects. To test whether Ras-15A and Ras-17N interfere with Ras function by blocking GDP-GTP exchange proteins, we examined their physical interaction with the CDC25 exchange protein. All three proteins formed stable complexes with CDC25 in the absence of guanine-nucleotides, but only Ras-15A was not released from CDC25 by physiological concentrations of GDP or GTP. These results establish that Ras-15A blocks the activation of normal Ras proteins by sequestering GDP-GTP exchange factors into non-productive complexes. In contrast, it would appear that the similar biological properties of Ras-17N are mediated by a reversible, competitive sequestration of exchange factors.

3T3 Cells↗

PubMed1994

Oncogenic Ras can induce transcriptional activation through a variety of promoter elements, including tandem c-Ets-2 binding sites.

Oncogenic Ras activates the transcription of a variety of viral and cellular genes through promoter elements consisting of two closely linked binding sites for transcription factors from several distinct families. To better understand what constitutes a promoter oncogene response element (ORE), various transcription factor binding site configurations were inserted into a reporter gene, and transactivation by oncogenic Ras was measured by cotransfection assays in NIH3T3 cells. We show that a single copy of two closely linked binding sites for either AP-1, Ets, NF-kappa B, or single closely linked Ets and AP-1 binding sites, are sufficient to confer at least 10-fold transactivation by similar amounts of oncogenic Ras. Single binding sites for these factors, or several other pairings of binding sites, are not sufficient to confer Ras responsiveness. The effect of altered ORE binding site spacing and orientation was systematically analysed, and limited flexibility was observed. The novel observation that two adjacent c-Ets-2 binding sites are sufficient to act as an ORE, indicates that Ets family proteins are a target of the Ras pathway distinct from AP-1. This ORE also mediates equivalent transactivation by c-Ets-2, and mutant OREs show a parallel decrease in Ras and c-Ets-2 responsiveness. Together, these data help to define transcriptional targets of the Ras signal transduction pathway.

3T3 Cells↗

PubMed1993

BCR-ABL-induced oncogenesis is mediated by direct interaction with the SH2 domain of the GRB-2 adaptor protein.

BCR-ABL is a chimeric oncoprotein that exhibits deregulated tyrosine kinase activity and is implicated in the pathogenesis of Philadelphia chromosome (Ph1)-positive human leukemias. Sequences within the first exon of BCR are required to activate the transforming potential of BCR-ABL. The SH2/SH3 domain-containing GRB-2 protein links tyrosine kinases to Ras signaling. We demonstrate that BCR-ABL exists in a complex with GRB-2 in vivo. Binding of GRB-2 to BCR-ABL is mediated by the direct interaction of the GRB-2 SH2 domain with a phosphorylated tyrosine, Y177, within the BCR first exon. The BCR-ABL-GRB-2 interaction is required for activation of the Ras signaling pathway. Mutation of Y177 to phenylalanine (Y177F) abolishes GRB-2 binding and abrogates BCR-ABL-induced Ras activation. The BCR-ABL (Y177F) mutant is unable to transform primary bone marrow cultures and is impaired in its ability to transform Rat1 fibroblasts. These findings implicate activation of Ras function as an important component in BCR-ABL-mediated transformation and demonstrate that GRB-2 not only functions in normal development and mitogenesis but also plays a role in oncogenesis.

Adaptor Proteins, Signal Transducing↗

PubMed1993

The carboxyl-terminal CXXX sequence of Gi alpha, but not Rab5 or Rab11, supports Ras processing and transforming activity.

Although the heterotrimeric Gi alpha subunit terminates in an apparent CXXX prenylation signal (CGLF), it is not modified by isoprenylation. To determine if the Gi alpha CXXX sequence can signal prenylation when placed at the carboxyl termini of normally prenylated proteins, we have characterized the processing and biological activity of chimeric oncogenic Ras proteins that terminate in the Gi alpha CXXX sequence (Ras/Gi alpha). Surprisingly, these chimeras were prenylated both in vivo and in vitro, demonstrated significant membrane association, exhibited transforming activity, and induced transcriptional transactivation from Ras-responsive elements. We then extended these studies to determine if, unlike the CC or CXC carboxyl-terminal sequences of other Rab proteins, the carboxyl-terminal CXXX sequences of the Ras-related Rab5 and Rab11 proteins represent conventional CXXX prenylation signals that can support Ras processing and transforming activity. Unexpectedly, these Ras/Rab chimeras were nonprenylated, were cytosolic, and lacked detectable transforming or transcriptional transactivation activity. Taken together, these results suggest that the context within which a CXXX sequence occurs may also critically control the modification of a protein by prenylation, and that the Rab5 and Rab11 carboxyl termini do not possess conventional CXXX sequences. Instead, their CCXX and CCXXX motifs may represent additional classes of protein prenylation signals.

3T3 Cells↗

PubMed1993

Differential antagonism of Ras biological activity by catalytic and Src homology domains of Ras GTPase activation protein.

Ras p120 GTPase activation protein (GAP), a cytosolic protein, is a negative mediator and potential downstream effector of Ras function. Since membrane association is critical for Ras function, we introduced the Ras membrane-targeting signal (a 19-residue peptide ending in CAAX, where C = cysteine, A = aliphatic amino acid, and X = any amino acid) onto the GAP N-terminal Src homology 2 and 3 and the C-terminal catalytic domains (designated nGAP/CAAX and cGAP/CAAX, respectively) to determine the role of membrane association in GAP function. cGAP/CAAX and full-length GAP/CAAX, but not GAP or nGAP/CAAX, exhibited potent growth inhibitory activity. Whereas both oncogenic and normal Ras activity were inhibited by cGAP/CAAX, nGAP/CAAX, despite lacking the Ras binding domain, inhibited the activity of oncogenic Ras without affecting the action of normal Ras. Altogether, these results demonstrate that membrane association potentiates GAP catalytic activity, support an effector function for GAP, and suggest that normal and oncogenic Ras possess different downstream interactions.

3T3 Cells↗

PubMed1993

Emerging concepts in the Ras superfamily of GTP-binding proteins.

The Ras superfamily of GTP-binding proteins (> 50 members) regulates a diverse spectrum of intracellular processes. These include cellular proliferation and differentiation, intracellular vesicular trafficking, cytoskeletal control, NADPH oxidase function, as well as others. In this review, we describe recent progress and emerging themes in the action and regulation of these important cellular regulatory molecules. Structural studies have provided insight into the function of low molecular weight GTP-binding proteins (LMWGs) as molecular switches, and are defining modes of interaction with associated regulatory molecules. Details of the enzymatic processes involved in the posttranslational processing of LMWGs, and how this processing is important for protein function, are being elucidated. A variety of GTPase activating proteins, GDP/GTP dissociation stimulators, and GDP dissociation inhibitors have been identified, and their ability to determine the activity of LMWG-regulated systems is being worked out. The discovery of multifunctional regulatory molecules has indicated that substantial "crosstalk" between various LMWG may occur. The continuing emergence of additional cellular functions that are regulated by LMWGs, and particularly the recent availability of in vitro analytical systems for studies of the mechanism (or mechanisms) of action of LMWGs, is resulting in a wealth of information about the regulation and integration of cellular signaling, form, and function.

Dimethylallyltranstransferase↗

PubMed1992

Ras (CXXX) and Rab (CC/CXC) prenylation signal sequences are unique and functionally distinct.

Rab proteins typically lack the consensus carboxyl-terminal CXXX motif that signals isoprenoid modification of Ras and other isoprenylated proteins and, instead, terminate in either CC or CXC sequences (C = cysteine, X = any amino acid). To compare the functional relationship between the Ras CXXX and the Rab CC/CXC motifs, we have generated chimeric Ras proteins terminating in Rab carboxyl-terminal CC or CXC sequences. These mutant Ras proteins were not isoprenylated in vitro or in vivo, demonstrating that the CC and CXC sequences alone are not sufficient to replace a CXXX sequence to signal Ras isoprenoid modification. Surprisingly, chimeric Ras/Rab proteins terminating in significant lengths of carboxyl-terminal sequences from Rab1b (7-139 residues), Rab2 (5-151 residues), or Rab3a (12 residues) were also not isoprenylated. These results demonstrate that the sequence requirements for isoprenoid modification of Rab proteins are more complex than the simple tetrapeptide CXXX sequence for isoprenoid modification of Ras proteins and suggest that the Rab geranylgeranyl transferase(s) requires recognition of protein conformation to signal the addition of geranylgeranyl groups. Finally, competition studies demonstrate that a common geranylgeranyl transferase activity is responsible for the modification of Rab proteins terminating in CC or CXC motifs.

3T3 Cells↗

PubMed1992

Isoprenoid addition to Ras protein is the critical modification for its membrane association and transforming activity.

We have introduced a variety of amino acid substitutions into carboxyl-terminal CA1A2X sequence (C = cysteine; A = aliphatic; X = any amino acid) of the oncogenic [Val12]Ki-Ras4B protein to identify the amino acids that permit Ras processing (isoprenylation, proteolysis, and carboxyl methylation), membrane association, and transformation in cultured mammalian cells. While all substitutions were tolerated at the A1 position, substitutions at A2 and X reduced transforming activity. The A2 residue was important for both isoprenylation and AAX proteolysis, whereas the X residue dictated the extent and specificity of isoprenoid modification only. Differences were observed between Ras processing in living cells and farnesylation efficiency in a cell-free system. Finally, one farnesylated mutant did not undergo either proteolysis or carboxyl methylation but still displayed efficient membrane association (approximately 50%) and transforming activity, indicating that farnesylation alone can support Ras transforming activity. Since both farnesylation and carboxyl methylation are critical for yeast a-factor biological activity, the three CAAX-signaled modifications may have different contributions to the function of different CAAX-containing proteins.

3T3 Cells↗

PubMed1992

Structure and biological effects of lipid modifications on proteins.

Both the prevalence of lipid modifications of proteins and their importance for protein function and cellular localization have been widely observed. The advances made during the past year in defining the enzymology of lipid addition and in understanding the biological consequences of these modifications on protein function are discussed.

Acyltransferases↗

PubMed1992

Protein prenylation: more than just glue?

As with other lipid modifications of proteins, prenylation now appears to be critically important in the regulation of protein function. Recent research has led to an explosion of information concerning prenylation signals, prenyl transferase enzymes and the role of prenylation in protein-membrane interactions. Experiments have examined the role of prenylation in protein function and the results suggest that protein prenylation may be involved in facilitating proper subcellular localization, promoting protein-protein and protein-membrane interactions and regulating protein function.

Amino Acid Sequence↗

PubMed1992

GTP-binding mutants of rab1 and rab2 are potent inhibitors of vesicular transport from the endoplasmic reticulum to the Golgi complex.

We have examined the role of ras-related rab proteins in transport from the ER to the Golgi complex in vivo using a vaccinia recombinant T7 RNA polymerase virus to express site-directed rab mutants. These mutations are within highly conserved domains involved in guanine nucleotide binding and hydrolysis found in ras and all members of the ras superfamily. Substitutions in the GTP-binding domains of rab1a and rab1b (equivalent to the ras 17N and 116I mutants) resulted in proteins which were potent trans dominant inhibitors of vesicular stomatitis virus glycoprotein (VSV-G protein) transport between the ER and cis Golgi complex. Immunofluorescence analysis indicated that expression of rab1b121I prevented delivery of VSV-G protein to the Golgi stack, which resulted in VSV-G protein accumulation in pre-Golgi punctate structures. Mutants in guanine nucleotide exchange or hydrolysis of the rab2 protein were also strong trans dominant transport inhibitors. Analogous mutations in rab3a, rab5, rab6, and H-ras did not inhibit processing of VSV-G to the complex, sialic acid containing form diagnostic of transport to the trans Golgi compartment. We suggest that at least three members of the rab family (rab1a, rab1b, and rab2) use GTP hydrolysis to regulate components of the transport machinery involved in vesicle traffic between early compartments of the secretory pathway.

Amino Acid Sequence↗