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F McCormick

Publications and source records attributed to F McCormick.

At least 127 records · Page 7Linked to original sources
PubMed1992

Interactions between p21ras proteins and their GTPase activating proteins.

Two proteins that regulate p21ras GTPase activity have been identified. These proteins interact with a region of ras p21 that is necessary for p21ras function and may themselves be components of signalling complexes. The first of these proteins to be identified, GAP, contains domains that interact with receptor tyrosine kinases and other tyrosine phosphoproteins, providing a direct link between signalling pathways involving these proteins and p21ras. The second, the product of the NF1 gene, is less well characterized but seems to connect p21ras to other signalling pathways which are perturbed in the NF1 disease. The ability of p21ras to interact with GAP may be compromised by competitive binding to the product of the Ki-rev1 gene, p21rap1. This competition for binding to GAP, or other proteins that interact with the effector site of ras p21, may explain the ability of Ki-rev1 to suppress cellular transformation by ras oncogenes.

GTPase-Activating Proteins↗
PubMed1992

GTPase activating proteins.

Since Ras proteins negotiate many signalling pathways leading to cell growth or differentiation, the regulation of Ras activity is vital to cellular health. Ras activity, which derives from a collaboration between Ras and GTP, is terminated by the GTPase activating protein (GAP)-catalyzed hydrolysis of the GTP. Hence, a simple regulatory scheme emerges: extracellular signals control Ras activity via membrane receptors and GAPs. However, the signalling scenario is probably not so simple. In looking for factors which interpret Ras activity, researchers have been led to the same factors which also regulate Ras activity, namely the GAPs. Therefore, it may be that Ras proteins are actually regulators of GAPs.

Animals↗
PubMed1992

Prevalence of N-ras mutations in children with myelodysplastic syndromes and acute myeloid leukemia.

The ras proto-oncogene family encodes a group of 21 kDa nucleotide-binding proteins. Activating mutations of ras genes are associated with certain types of malignancies, indicating that they are related in some way to the malignant process. We have examined bone marrow cells from nine children with myelodysplastic syndromes (MDS) and 35 with acute myeloid leukemia (AML) for activating point mutations of ras genes by in vitro amplification using polymerase chain reaction (PCR), oligonucleotide hybridization and sequencing of PCR products. We found N-ras mutations in cells from 3 of 9 children (33%) with MDS and only 2 of 35 children with AML (6%; 95% confidence interval is 0.7-19%). All mutations the second nucleotide of codon 12 or the first nucleotide of codon 61 of N-ras. There was no apparent correlation with clinical or laboratory characteristics, including karyotype; however, an association of N-ras activation with the most aggressive type of MDS was noted. Among the patients with MDS, 2 of 6 with monosomy 7 had N-ras mutations; however, three children with monosomy 7 which presented with AML lacked ras mutations. One patient was studied at time of diagnosis of MDS and again after progression to AML. At the preleukemic stage of disease, an N-ras mutation was identified; however, after development of AML this mutation was not present in the leukemic clone. In conclusion, these data show that ras mutations, while not necessary for leukemic transformation, may be important for the initiation of preleukemias evolving into overt AML.

Adolescent↗
PubMed1992

Association of a tyrosine kinase activity with GAP complexes in v-src transformed fibroblasts.

p21ras GAP is phosphorylated on tyrosine residues and associates with 62 kDa and 190 kDa tyrosine phosphorylated proteins in v-src-transformed fibroblasts. We were interested in identifying the tyrosine kinase responsible for phosphorylation of GAP and the two associated proteins. Here, we report that GAP-immunoprecipitates from v-src transformed cells contain a tyrosine kinase activity that phosphorylates GAP, p62 and p190. Tryptic peptide analysis indicated that the sites phosphorylated in vitro and in vivo are indistinguishable, suggesting that the precipitated kinase could be responsible for tyrosine phosphorylation of GAP in vivo. The GAP-associated kinase activity might be due to v-src itself, because pp60v-src is able to associate with GAP in vitro and GAP can be phosphorylated by pp60v-src immunecomplexes.

3T3 Cells↗
PubMed1991

Heterogeneous amino acids in Ras and Rap1A specifying sensitivity to GAP proteins.

Guanosine triphosphatase (GTPase) activity of Ras is increased by interaction with Ras-GAP (GTPase-activating protein) or with the GAP-related domain of the type 1 neurofibromatosis protein (NF1-GRD), but Ras is not affected by interaction with cytoplasmic and membrane forms of Rap-GAP; Rap1A, whose effector function can suppress transformation by Ras, is sensitive to both forms of Rap-GAP and resistant to Ras-GAP and NF1-GRD. A series of chimeric proteins composed of portions of Ras and Rap were constructed; some were sensitive to Ras-GAP but resistant to NF1-GRD, and others were sensitive to cytoplasmic Rap-GAP but resistant to membrane Rap-GAP. Sensitivity of chimeras to Ras-GAP and cytoplasmic Rap-GAP was mediated by amino acids that are carboxyl-terminal to the effector region. Residues 61 to 65 of Ras conferred Ras-GAP sensitivity, but a larger number of Rap1A residues were required for sensitivity to cytoplasmic Rap-GAP. Chimeras carrying the Ras effector region that were sensitive only to Ras-GAP or only to cytoplasmic Rap-GAP transformed NIH 3T3 cells poorly. Thus, distinct amino acids of Ras and Rap1A mediate sensitivity to each of the proteins with GAP activity, and transforming potential of Ras and sensitivity of Ras to Ras-GAP are at least partially independent properties.

Cell Membrane↗
PubMed1991

Evidence for regulation of the human ABL tyrosine kinase by a cellular inhibitor.

Phosphotyrosine cannot be detected on normal human ABL protein-tyrosine kinases, but activated oncogenic forms of the human ABL protein are phosphorylated on tyrosine in vivo. Activation of ABL can occur by substitution of the ABL first exon with breakpoint cluster region (BCR) sequences or by deletion of the noncatalytic SH3 (src homology region 3) domain. An alternative mode for the activation of the ABL kinases is hyperexpression at greater than 500-fold over endogenous levels. This is not a consequence of transphosphorylation of the hyperexpressed ABL molecules. ABL proteins translated in vitro lack phosphotyrosine, but tyrosine kinase activity is uncovered after immunoprecipitation and removal of lysate components. The rates of dephosphorylation of ABL and BCR-ABL fusion protein by phosphotyrosine-specific phosphatases are approximately the same. These combined results indicate that inhibition of ABL activity is reversible and suggest that a cellular component interacts noncovalently with ABL to inhibit its autophosphorylation.

Animals↗
PubMed1991

Molecular cloning of a GTPase activating protein specific for the Krev-1 protein p21rap1.

The rap1/Krev-1 gene encodes a ras-related protein that suppresses transformation by ras oncogenes. We have purified an 88 kd GTPase activating protein (GAP), specific for the rap1/Krev-1 gene product, from bovine brain. Based on partial amino acid sequences obtained from this protein, a 3.3 kb cDNA was isolated from a human brain library. Expression of the cDNA in insect Sf9 cells resulted in high level production of an 85-95 kd rap1GAP that specifically stimulated the GTPase activity of p21rap1. The complete deduced amino acid sequence is not homologous to any known protein sequences, including GAPs specific for p21ras. Northern and Western blotting analysis indicate that rap1GAP is not ubiquitously expressed and appears most abundant in fetal tissues and certain tumor cell lines, particularly the Wilms' kidney tumor, SK-NEP-1, and the melanoma, SK-MEL-3, cell lines.

Amino Acid Sequence↗
PubMed1991

Differential regulation of rasGAP and neurofibromatosis gene product activities.

The ras-encoded p21ras proteins bind GTP very tightly, but catalyse hydrolysis to GDP very slowly. In humans, two genes encode proteins that stimulate this GTPase activity (GAP, or GTPase-activating proteins), one of relative molecular mass 120,000, referred to as p120-GAP, and another NF1-GAP, which is encoded by the neurofibromatosis type-1 gene. Both GAPs are widely expressed in mammalian tissues. Here we show that although they will both bind oncogenic mutants of p21ras, neither will stimulate their GTPase activity. NF1-GAP binds to the p21ras proteins up to 300 times more efficiently than p120-GAP. The two GAPs are inhibited to different extents by certain lipids: micromolar concentrations of arachidonate, phosphatidate and phosphatidylinositol-4,5-bisphosphate affect only NF1-GAP. This inhibition does not compete with p21ras, and lipid-inactivated NF1-GAP can still bind p21ras. We used the detergent dodecyl maltoside, which inhibits only NF1-GAP, to distinguish between the two activities in cell extracts and found both types present together in several mammalian cell lines. In contrast, GAP activity in extracts of Xenopus oocytes was not affected by dodecyl maltoside. By these criteria, the mammalian cells contain both GAP activities and the oocytes have only p120-like GAP activity. These results indicate that more than one GAP regulates p21ras in the same cell.

Animals↗
PubMed1991

Ras mutations in United Kingdom examples of oral malignancies are infrequent.

Point mutations in codons 12, 13 or 61 of the oncogenes Ha-ras, Ki-ras or N-ras have been identified in human malignancies of many types. Using the PCR (polymerase chain reaction) technique for DNA amplification in vitro and stringent probing of the amplified DNA on dot blots with a library of specific oligonucleotides, we have screened for the presence of ras mutations in oral and para-oral malignancies and some associated lesions. The material, from UK patients, consisted of 22 oral squamous-cell carcinomas including 5 neck metastases, 1 oral mucosal dysplasia, 1 proliferative verrucous leukoplakia, 1 antral and 1 tonsillar carcinoma, 1 basal-cell carcinoma, 1 salivary adenocarcinoma, 1 salivary adenoid cystic carcinoma and 1 lung adenocarcinoma metastatic to the gingiva. Genomic DNA was extracted from tissues which were fresh or preserved in liquid nitrogen. Two DNA samples contained point mutations in codon 61 of Ki-ras. One of these mutations was in the lymphocytes infiltrating a retromolar SCC. The other mutation (CAA to CAU; substitution of glutamine by histidine) was in the lung adenocarcinoma metastasis. The absence of ras mutations in the epithelium of primary oral squamous-cell carcinomas is of considerable interest as other work in our Department on Indian cases of oral carcinomas associated with chewing tobacco (quid) revealed that 35% of these had a codon 12, 13 or 61 mutation in Ha-ras. While ras activations arising from point mutations may occur in a high proportion of oral malignancies associated with chewing tobacco (quid), this was not the case in UK oral malignancies, even where tobacco was smoked.

Aged↗
PubMed1991

Regulation of Ras-GAP and the neurofibromatosis-1 gene product by eicosanoids.

Ras-GAP (GTPase activating protein) is a regulatory protein that stimulates the intrinsic guanosine triphosphatase (GTPase) activity of the proto-oncogene product p21ras. A domain of the neurofibromatosis gene product (NF1) that has sequence similarity to the catalytic domain of Ras-GAP and to yeast IRA gene products also has a specific stimulatory activity toward p21ras GTPase. Arachidonic acid and phosphatidic acid inactivate GAP, but no agents have been identified that stimulate GAP and thereby switch p21ras off. With the use of recombinant Ha-c-Ras and Ras-GAP, NF1, and GAP catalytic domains, it was found that prostaglandins PGF2 alpha and PGA2 stimulated Ras-GAP and that prostacyclin PGI2 inhibited Ras-GAP. The stimulatory effect of PGF2 alpha was saturable and structure-specific and competed with the inhibitory effect of arachidonic acid. Arachidonic acid also inhibited the catalytic activity of NF1, but prostaglandins were not stimulatory. These results suggest a mechanism for the allosteric control of Ras function through the modulation of arachidonate metabolism.

Arachidonic Acid↗
PubMed1991

The GTPase superfamily: conserved structure and molecular mechanism.

GTPases are conserved molecular switches, built according to a common structural design. Rapidly accruing knowledge of individual GTPases--crystal structures, biochemical properties, or results of molecular genetic experiments--support and generate hypotheses relating structure to function in other members of the diverse family of GTPases.

Amino Acid Sequence↗
PubMed1991

Purification of a plasma membrane-associated GTPase-activating protein specific for rap1/Krev-1 from HL60 cells.

rap1/Krev-1 is a p21ras-related GTP-binding protein that has been implicated in the reversion of the ras-transformed cell phenotype. We have identified a GTPase-activating protein (GAP) specific for rap in plasma membranes isolated from differentiated HL60 cells. The rap GAP activity remained quantitatively associated with the membrane following washes with buffered 1 M LiCl containing 20 mM EDTA but was solubilized with the detergents Nonidet P-40 and deoxycholate. On the basis of size-exclusion chromatography, the membrane-associated rap GAP (rap GAPm) appeared distinct from the rap GAP detected in the cytosolic fraction from HL60 cells. The molecular sizes of the membrane and cytosolic forms were estimated to be 36 and 54 A, respectively. rap GAPm was solubilized and purified to near homogeneity by successive column chromatographies in the presence of detergent. The rap GAPm activity corresponded to a single polypeptide that migrated with a molecular mass of approximately 88 kDa on SDS/polyacrylamide gels. The purified rap GAPm was inactive toward the GTP-bound forms of p21ras, rho, G25K, and rac-1 and did not stimulate dissociation of guanine nucleotide from rap.

Amino Acid Sequence↗
PubMed1991

Role of GTPase activating protein in mitogenic signalling through phosphatidylcholine-hydrolysing phospholipase C.

Recent evidence has accumulated showing that activation of PLC-catalysed hydrolysis of phosphatidylcholine (PC-PLC) is a critical step in mitogenic signal transduction both in fibroblasts and in oocytes from Xenopus laevis. The products of ras genes activate PC-PLC, bind guanine nucleotides, have intrinsic GTPase activity, and are regulated by a GTPase-activating protein (GAP). It has been suggested that, in addition to its regulatory properties, GAP may also be necessary for ras function as a downstream effector molecule. In this study, evidence is presented that strongly suggests that the functional interaction between ras p21 and GAP is sufficient and necessary for activation of maturation promoting factor (MPF) H1-kinase activity in oocytes, and that PC hydrolysis is critically involved in this mechanism. Therefore, we identify GAP as a further step required for signalling through PC-PLC, and necessary for the control of oocyte maturation in response to ras p21/insulin but not to progesterone.

Animals↗
PubMed1991

Insulin stimulation of gene expression mediated by p21ras activation.

In fibroblasts, insulin is a weak mitogen and does not induce expression of c-fos, c-jun or p33. However, increasing the expression levels of either normal p21Hras or the insulin receptor, but not mutant p21Hras, enables insulin to induce the expression of these genes. In cells expressing elevated levels of insulin receptor, this process involves a rapid increase in p21rasGTP levels (from 20% to 70% GTP as a percentage of total guanine nucleotides). No increase in p21rasGTP levels was observed after PDGF and EGF stimulation of cells expressing high levels of the cognate receptor, stressing the specificity of the insulin-induced increase. We conclude that in fibroblasts, p21ras is an intermediate of the insulin signal transduction pathway involved in the regulation of gene expression and mitogenicity.

Fibroblasts↗
PubMed1991

Regulation of ras p21 by GTPase activating proteins.

We propose a model for dual effector functions of the known ras GAPs p120-GAP and NF1-GAP. This model is consistent with known biological and biochemical effects of GAPs in mammalian cells, but it is clearly not a proven hypothesis, and several difficulties remain in making this model convincing. One is the apparent difference between mammalian cells and yeasts, in which GAPs do not have a demonstrable effector function. The other is the difficulty of eliminating the possibility that other effectors exist that do not have GAP activity and do not bind ras p21 sufficiently tightly to allow detection through physical association. We hope that further analysis of GAP function will clarify the roles of these proteins, allowing at least a partial description of ras action in normal and malignant mammalian cells.

Animals↗
PubMed1991

A synthetic peptide corresponding to a sequence in the GTPase activating protein inhibits p21ras stimulation and promotes guanine nucleotide exchange.

Amino acid sequence homology between the GTPase Activating Protein (GAP) and the GTP-binding regulatory protein, Gs alpha, suggests that a specific region of GAP primary structure (residues 891-898) may be involved in its stimulation of p21ras GTP hydrolytic activity (McCormick, F. [1989] Nature 340, 678-679). A peptide, designated p891, corresponding to GAP residues 891-906 (M891RTRVVSGFVFLRLIC906) was synthesized and tested for its ability to inhibit GAP-stimulated p21ras GTPase activity. At a concentration of 25 microM, p891 inhibited GAP activity approximately 50%. Unexpectedly, p891 also stimulated GTP binding to p21N-ras independent of GAP. This stimulation correlated with an enhancement of p21N-ras.GDP dissociation; an approximate 15-fold increase in the presence of 10 microM p891. In contrast, dissociation of the p21N-ras.GTP gamma S complex was unaffected by 10 microM p891. The p21N-ras.GDP complex was unresponsive to 100 microM mastoparan, a peptide toxin shown previously to accelerate GDP dissociation from the guanine nucleotide regulatory proteins, Gi and Go. p21H-ras, as well as the two p21H-ras effector mutants, Ala-38, and Ala-35, Leu-36, also exhibited increased rates of GDP dissociation in the presence of p891. Also tested were three ras-related GTP-binding proteins; rap, G25K and rac. The rap.-GDP complex was unaffected by 10 microM p891. Dissociation of the G25K- and rac.GDP complexes were enhanced slightly; approximately 1.3- and 1.8-fold over control, respectively. Thus, the inhibitory effect of p891 on GAP stimulation of p21ras suggests that amino acids within the region 891-906 of GAP may be essential for interaction with p21ras. In addition, p891 independently affects the nucleotide exchange properties of p21ras.

Amino Acid Sequence↗