Biomedical subjects
F McCormick
Publications and source records attributed to F McCormick.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
The COOH-terminal domain of the Rap1A (Krev-1) protein is isoprenylated and supports transformation by an H-Ras:Rap1A chimeric protein.
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.
Protein-tyrosine kinases regulate the phosphorylation, protein interactions, subcellular distribution, and activity of p21ras GTPase-activating protein.
The p21ras GTPase-activating protein (GAP) down-regulates p21ras by stimulating its intrinsic GTPase activity. GAP is found predominantly as a monomer in the cytosol of normal cells. However, in cells expressing an activated cytoplasmic protein-tyrosine kinase, p60v-src, or stimulated with epidermal growth factor, GAP becomes phosphorylated on tyrosine and serine and forms distinct complexes with two phosphoproteins of 62 and 190 kDa (p62 and p190). In v-src-transformed Rat-2 cells, a minor fraction of GAP associates with the highly tyrosine phosphorylated p62 to form a complex that is localized at the plasma membrane and in the cytosol. In contrast, the majority of GAP enters a distinct complex with p190 that is exclusively cytosolic and contains predominantly phosphoserine. Epidermal growth factor stimulation also induces a marked conversion of monomeric GAP to higher-molecular-weight species in rat fibroblasts. The GAP-p190 complex is dependent on phosphorylation and shows reduced GAP activity. These results indicate that protein-tyrosine kinases induce GAP to form multiple heteromeric complexes, which are strong candidates for regulators or targets of p21ras.
Regulators and effectors of ras proteins.
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GTP-binding proteins as oncogenes in human tumors.
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Molecular mechanisms of cancer.
Cancer is caused by specific DNA damage. Several common mechanisms that cause DNA damage result in specific malignant disorders: First, proto-oncogenes can be activated by translocations. For example, translocation of the c-myc proto-oncogene from chromosome 8 to one of the immunoglobulin loci on chromosomes 2, 14, or 22 results in Burkitt's lymphomas. Translocation of the c-abl proto-oncogene from chromosome 9 to the BCR gene located on chromosome 22 produces a hybrid BCR/ABL protein resulting in chronic myelogenous leukemia. Second, proto-oncogenes can be activated by point mutations. For example, point mutations of genes coding for guanosine triphosphate-binding proteins, such as H-, K-, or N-ras or G proteins, can be oncogenic as noted in a large variety of malignant neoplasms. Proteins from these mutated genes are constitutively active rather than being faithful second messengers of periodic extracellular signals. Third, mutations that inactivate a gene can result in tumors if the product of the gene normally constrains cellular proliferation. Functional loss of these "tumor suppressor genes" is found in many tumors such as colon and lung cancers. The diagnosis, classification, and treatment of cancers will be greatly enhanced by understanding their abnormalities at the molecular level.
Purification, characterization, and western blot analysis of human GTPase-activating protein from native and recombinant sources.
Human ras GTPase-activating protein (GAP) is a cytoplasmic factor that stimulates the GTPase activity of normal N-ras p21 while having no stimulatory effect on the GTPase activity of oncogenic variants of N-ras p21. We have purified two forms of native ras GAP from human placental tissue. In addition to the Mr = 120,000 type I GAP reported previously (1), an equivalent amount of an Mr = 95,000 molecule with GAP activity was recovered and shown to have the N-terminal sequence expected for type II GAP. The two GAP forms in placental extracts were resolved by molecular sieve chromatography and appeared to have a monomeric native structure. Human recombinant type I GAP was produced intracellularly in Sf9 insect cells using a baculovirus expression vector, and 10-mg quantities were purified to homogeneity in three steps. Comparison of the purified native and recombinant GAP molecules revealed that all three displayed similar biological specific activities in an in vitro GAP assay. A polyclonal antibody to purified recombinant GAP was prepared and shown to neutralize the activity of both native and recombinant GAPs. The antibody was also highly specific for the detection of native GAP by Western blot. Type I and II GAP species were detected in approximately equal amounts in cytoplasmic extracts of human placenta, but only type I GAP was observed when other human tissues were examined.