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F McCormick

Publications and source records attributed to F McCormick.

At least 91 records · Page 5Linked to original sources
PubMed1994

The Ras-related protein R-ras interacts directly with Raf-1 in a GTP-dependent manner.

R-ras is a member of the ras family of small GTPases that associates with the apoptosis-suppressing proto-oncogene product Bcl-2. Using the yeast two-hybrid system we provide evidence for an interaction between R-ras and the Raf-1 kinase. This interaction requires only the N-terminal regulatory domain (amino acids 1-256) of Raf-1, and is observed with both the wild type and a constitutively active R-ras mutant, but not with a deletion mutant that lacks the potential effector domain or a mutant of R-ras impaired for GTP binding. Moreover, using an in vitro binding assay we show a direct GTP-dependent interaction of purified R-ras with a purified Raf-1 fragment corresponding to the proposed 81-amino-acid H-Ras-binding domain of Raf-1 (amino acids 51-131). Taken together, these data indicate that R-ras may exert its biological effect by means of modulating the activity of the Raf-1 kinase as its direct downstream effector.

Animals↗
PubMed1994

Loss of the normal NF1 allele from the bone marrow of children with type 1 neurofibromatosis and malignant myeloid disorders.

BACKGROUND: Children with type 1 neurofibromatosis (NF-1) are at increased risk for malignant myeloid disorders. Analysis of the NF-1 gene (NF1) suggests that the function of its product, neurofibromin, is reduced in affected persons and that NF1 belongs to the tumor-suppressor class of recessive cancer genes. This model is consistent with evidence that neurofibromin accelerates the intrinsic guanosine triphosphate-hydrolyzing activity of the Ras family of regulatory proteins. Loss of constitutional heterozygosity has not been reported in the benign tumors associated with NF-1, however, and has only been detected in a few malignant neural-crest tumors and in some tumor-derived cell lines. METHODS: We studied DNA extracted from the bone marrow of 11 children with NF-1 in whom malignant myeloid disorders developed and from parental leukocytes. We used a series of polymorphic markers within and near NF1 to determine whether leukemogenesis was associated with structural alterations of the gene. RESULTS: Bone marrow samples from five patients showed loss of heterozygosity. In each case, the NF1 allele was inherited from a parent with NF-1 and the normal allele was deleted. CONCLUSIONS: These data provide evidence of NF1 may function as a tumor-suppressor allele in malignant myeloid diseases in children with NF-1 and that neurofibromin is a regulator of ras in early myelopoiesis.

Alleles↗
PubMed1994

Analysis of the tumor suppressor activity of the K-rev-1 gene in human tumor cell lines.

Overexpression of the human K-rev-1 gene in v-Ki-ras-transformed NIH 3T3 cells has been reported to result in the reversal of transformation and tumor suppression. To address whether human K-rev-1 is a tumor suppressor gene of human tumor cells, we have systematically transfected epitope-tagged wild-type or activated mutant K-rev-1 complementary DNA expression vectors into a series human tumor cell lines that express an activated ras oncogene, namely HT1080, EJ, and SW480. Using the epitope-tag-specific monoclonal antibody, it is shown that the K-rev-1 protein localizes to the medial/trans-Golgi network. Ectopic expression of the wild-type or activated mutant K-rev-1 protein did not significantly affect the morphology or in vitro growth of any clones. Furthermore, all clones expressing the wild-type or activated mutant K-rev-1 protein were tumorigenic. Western blot analysis of tumor reconstitutes demonstrated that there was no decrease or loss of introduced K-rev-1 protein expression. The results in the present study demonstrate that expression of K-rev-1 does not reverse the transformed phenotype or significantly affect the tumorigenic phenotype of human tumor cell lines that express endogenous ras oncogenes.

Animals↗
PubMed1994

Genetic analysis of the catalytic domain of the GAP gene in human lung cancer cell lines.

Cell lines of non-small cell lung cancer (non-SCLC) have been shown to contain activating mutation of the K-ras oncogene in about 30% of cases, whereas no small cell lung cancer (SCLC) cell lines displayed these mutations. Biochemically, these mutations result in the ras gene product (p21) being constitutively activated in its GTP-bound form and insensitive to the hydrolytic action of the ras-specific GTPase-activating protein (ras GAP). We hypothesized that, if tumor development is related to the p21 ras being in the active GTP-bound state, then a similar malignant phenotype may result from an inactivating mutation in the ras GAP gene in the region that interacts with ras p21 (so-called catalytic domain). To test this hypothesis, we screened a panel of SCLC and non-SCLC cell lines for major genetic alterations in the catalytic domain of the GAP gene with the Southern blot technique, and for minor genetic abnormalities (e.g., point mutations) with denaturing gradient gel electrophoresis and single-strand conformation polymorphism. Mutations in the catalytic domain of the GAP gene could not be demonstrated by any technique in any cell line examined. We conclude that mutational inactivation of the catalytic domain of the GAP gene probably does not contribute to the development of lung cancer.

Blotting, Southern↗
PubMed1994

Activators and effectors of ras p21 proteins.

Over the past year, major advances have been made in understanding the key steps involved in signaling pathways--from receptor tyrosine kinases to ras p21, and on to a cascade of serine/threonine kinases. A chain of specific protein-protein interactions is responsible for signal transduction. Components of the pathway are highly conserved between flies, nematodes and mammals, and constitute a primary signaling device in most cell types.

Animals↗
PubMed1994

Raf-1 interferes with Ras and Rap1A effector functions in yeast.

Raf-1 is a serine/threonine kinase that acts downstream of Ras in mitogenic signal transduction pathways, but the mechanism by which Ras transmits signals to Raf-1 is not known. We have examined the interaction between Raf-1 and human H-ras in three different systems that utilize H-ras-induced phenotypes in Saccharomyces cerevisiae. In each system, the effects of H-ras depend on guanosine triphosphate and appear to be mediated through the H-ras effector binding region. H-ras effector function was blocked in each case by expression of the N-terminal regulatory domain of Raf-1. These inhibitory effects did not require the Raf-1 kinase domain. Raf-1 also blocked Rap1A effector function in S. cerevisiae. Raf-1, therefore, appears to interact with H-Ras and Rap1A in these in vivo systems with properties that suggest it is an immediate downstream effector.

Cell Cycle Proteins↗
PubMed1994

Platelet-derived growth factor-induced p21ras-mediated signaling is independent of platelet-derived growth factor receptor interaction with GTPase-activating protein or phosphatidylinositol-3-kinase.

Stimulation with platelet-derived growth factor (PDGF) results in the association of several SH2 domain-containing proteins with the activated PDGF receptor, including GAP, a GTPase-activating protein of p21ras, and phosphatidylinositol-3-kinase (PI-3K). To investigate the role of GAP-PI-3K receptor interaction in p21ras signaling, we have used cell lines expressing mutant PDGF receptors that either are impaired in GAP binding or fail to bind both GAP and PI-3K. In these cell lines, PDGF treatment resulted in activation of extracellular signal-regulated kinase 2 (ERK2), which could be blocked by the expression of a dominant-negative mutant of p21ras (p21ras(asn17)), indicating that these mutations in the PDGF receptor do not abolish p21ras-mediated activation of ERK2. In addition, the PDGF-induced increase in levels of p21rasGTP, as measured either in intact cells or in permeabilized cells, appears to be normal in the cell lines expressing the mutant PDGF receptors. These results indicate that binding of GAP and/or PI-3K to the PDGF receptor is not necessary for PDGF-induced p21ras activation and p21ras-mediated signaling to ERK2. We also show that, in contrast to the activation of ERK2, PDGF-induced GAP and PI-3K interaction with the PDGF receptor are not inhibited by p21ras(asn17) expression, indicating that these interactions do not require p21ras activation.

Animals↗
PubMed1994

The non-catalytic domain of ras-GAP inhibits transformation induced by G protein coupled receptors.

We have studied the relationship between ras-GAP and G protein coupled receptors in a proliferative setting comprised of NIH3T3 expressing transfected muscarinic receptors (mAChRs). GAP expression plasmids were engineered to encode wild-type GAP, its carboxyl-terminal catalytic domain, a mutant lacking a portion of the catalytic domain, and an amino-terminal domain which contained the hydrophobic region as well as SH2-SH3 domains. Cotransfection of each GAP expression plasmid into NIH3T3 cells did not affect the transforming ability of the v-mos oncogene, but plasmids encoding wild-type GAP or the mutant consisting of an intact catalytic domain inhibited transformation induced by normal c-ras. Wild-type GAP also prevented transformation by m1 mAChRs, whereas the mutant consisting of only its catalytic domain lacked any demonstrable effect. In contrast, the N-terminal non-catalytic domain of GAP effectively prevented m1-induced focus-formation. Cell lines coexpressing m1 receptors and each of the GAP constructs revealed that GAP proteins do not affect m1 receptor density, receptor ligand binding characteristics or coupling to the PI-PLC signal transduction pathway. Thus, our findings suggest a role for the N-terminal non-catalytic domain of GAP in regulating biological functions mediated by G protein-coupled receptors.

3T3 Cells↗
PubMed1993

Proteins regulating Ras and its relatives.

GTPases of the Ras superfamily regulate many aspects of cell growth, differentiation and action. Their functions depend on their ability to alternate between inactive and active forms, and on their cellular localization. Numerous proteins affecting the GTPase activity, nucleotide exchange rates and membrane localization of Ras superfamily members have now been identified. Many of these proteins are much larger and more complex than their targets, containing multiple domains capable of interacting with an intricate network of cellular enzymes and structures.

Amino Acid Sequence↗
PubMed1993

Inhibition by cAMP of Ras-dependent activation of Raf.

Activation of the Raf and extracellular signal-regulated kinases (ERKs) (or mitogen-activated protein kinases) are key events in mitogenic signalling, but little is known about interactions with other signaling pathways. Agents that raise levels of intracellular cyclic adenosine 3',5'-monophosphate (cAMP) blocked DNA synthesis and signal transduction in Rat1 cells exposed to epidermal growth factor (EGF) or lysophosphatidic acid. In the case of EGF, receptor tyrosine kinase activity and association with the signaling molecules Grb2 and Shc were unaffected by cAMP. Likewise, EGF-dependent accumulation of the guanosine 5'-triphosphate-bound form of Ras was unaffected. In contrast, activation of Raf-1 and ERK kinases was inhibited. Thus, cAMP appears to inhibit signal transmission from Ras by preventing Ras-dependent activation of Raf-1.

1-Methyl-3-isobutylxanthine↗
PubMed1993

N-ras mutations are associated with poor prognosis and increased risk of leukemia in myelodysplastic syndrome.

To evaluate the clinical significance of N-ras mutations in the myelodysplastic syndrome (MDS) archival bone marrow samples from 252 patients were studied for the presence of N-ras exon I mutations using polymerase chain reaction amplification and differential oligonucleotide hybridization. Subsequently, clinical information about these patients was obtained and analyzed. Of 220 evaluable patients, 20 (9%) had point mutation of N-ras involving codon 12. Individuals with N-ras mutation had a significantly shorter survival period than those who were N-ras negative (P = .02). An increased risk of acute myelogenous leukemia (AML) was also found in patients with N-ras mutations (P = .005). N-ras mutations were not associated with any French-American-British (FAB) subtype, with the presence of increased myeloblasts, or with chromosomal aberrations in the bone marrow. However, the presence of increased bone marrow blasts was strongly associated with poor survival rate and risk of AML (P < .001 for each). After stratifying for the percentage of blasts, N-ras mutations remained significantly associated with shorter survival period (P = .04) and increased risk of AML (P = .02). Bone marrow cytogenetic abnormalities, particularly when multiple abnormalities were present, were significantly associated with a poor prognosis (P < .001). In conclusion, N-ras mutation, although relatively infrequent in MDS, is associated with short survival period and increased probability of developing AML.

Bone Marrow↗
PubMed1993

Structural requirements for the interaction of p21ras with GAP, exchange factors, and its biological effector target.

From the multitude of mutations that have been tested in p21ras, a common theme emerges regarding its interaction with the effector target, GAPs, and the newly discovered exchange factors. Many of the mutations that result in dysfunction for all three types of interactions are localized to the switch 1 and switch 2 regions of the p21ras three-dimensional structure (Fig. 2). These two regions change conformation on GTP binding by p21ras and, accordingly, both GAP binding and Ras biological activity are GTP-dependent processes. In addition, certain mutations in the switch 1 and 2 regions alter the affinity of GAP for p21ras, again implicating this region in the binding interaction. On the other hand, the SDC25 exchange factor appears to promote dissociation of both GTP and GDP from p21ras, suggesting that the overall conformation of the switch 1 and 2 regions may not be important for recognition by SDC25. Moreover, none of the switch 1 or 2 mutations that impaired stimulation by SDC25 affected its binding to p21ras. This suggests that these residues are essential for the mechanism of activation by SDC25 but not for its binding to p21ras. Amino acids at positions 73, 75, and possibly 102 and 103 appear also to be involved in the activation of p21ras by exchange factors. Whether these are required for binding to the exchanger has not been reported.

Animals↗
PubMed1993

Targets of B lymphocyte antigen receptor signal transduction include the p21ras GTPase-activating protein (GAP) and two GAP-associated proteins.

Cross-linking membrane Ig (mIg) on B cells stimulates tyrosine phosphorylation of proteins involved in signal transduction including the mIg-associated proteins Ig-alpha and Ig-beta, the tyrosine kinases p53/p56lyn, p55blk, p59fyn, and PTK72, phosphatidylinositol 3-kinase, phospholipase C gamma 1 and gamma 2, and the mitogen-activated protein kinase. We now show that the p21ras GTPase-activating protein (GAP) is also a substrate for mIg-activated tyrosine kinases. p21ras is a key regulator of cell growth and GAP may act as both a regulator of p21ras activity and as a downstream effector of p21ras. We found that mIg cross-linking caused a rapid increase in tyrosine phosphorylation of GAP in the immature B cell line WEHI-231, the mature B cell lines BAL 17 and Daudi, and the IgG-bearing B cell line A20. In fibroblasts, tyrosine kinase activation causes GAP to associate with two other tyrosine-phosphorylated proteins, p62 and p190, which have homologies to an RNA-binding protein and a transcriptional repressor, respectively. Similarly, mlg cross-linking induced the association of GAP with a 62-kDa tyrosine-phosphorylated protein in BAL 17, WEHI-231, and Daudi cells. Anti-Ig treatment also increased the amount of a 190-kDa tyrosine-phosphorylated protein associated with GAP in WEHI-231 and Daudi cells. After separation by SDS-PAGE and transfer to nitrocellulose, the tyrosine-phosphorylated p62 and p190 present in anti-GAP immunoprecipitates from B cells were capable of binding radiolabeled recombinant GAP, as previously reported for the GAP-associated p62 and p190 from fibroblasts. The amount of p62 that could be detected in this way after immunoprecipitation with antiphosphotyrosine antibodies was much greater from anti-IgM-treated BAL 17 cells than from unstimulated BAL 17 cells. This probably reflects anti-Ig-induced tyrosine phosphorylation of p62. In any case, GAP, p62, and/or p190 may be involved in signal transduction by mIg in B cells.

Animals↗
PubMed1993

Characterization of full-length neurofibromin: tubulin inhibits Ras GAP activity.

Full-length neurofibromin is a GTPase activating protein (GAP) for the Ras proto-oncogene product. Regulation of neurofibromin activity therefore has important implications for cell growth. Neurofibromin co-purifies with tubulin when expressed in insect cells. The interaction between neurofibromin and tubulin is sensitive to the microtubule depolymerizing agent colchicine. Neurofibromin GAP activity is inhibited even at low concentrations of tubulin. However, maximal inhibition of GAP activity is only approximately 70%, suggesting that the neurofibromin-tubulin complex retains residual GAP activity. This decreased activity is reflected by a 4-fold decrease in its affinity for Ras. A truncated mutant of neurofibromin with reduced sensitivity to tubulin localizes some tubulin-binding determinants to an 80 residue segment immediately N-terminal to the GAP-related domain. Since tubulin is an abundant protein in eukaryotic cells, the tubulin-neurofibromin interaction may regulate the Ras signalling pathway.

Amino Acid Sequence↗