Biomedical subjects

D W Stacey

Publications and source records attributed to D W Stacey.

At least 19 recordsLinked to original sources

PubMed1996

Prostaglandin A2 blocks the activation of G1 phase cyclin-dependent kinase without altering mitogen-activated protein kinase stimulation.

Prostaglandin A2 (PGA2) reversibly blocked the cell cycle progression of NIH 3T3 cells at G1 and G2/M phase. When it was applied to cells synchronized in G0 or S phase, cells were blocked at G1 and G2/M, respectively. The G2/M blockage was transient. Microinjected oncogenic leucine 61 Ras protein could not override the PGA2 induced G1 blockage, nor could previous transformation with the v-raf oncogene. The serum-induced activation of mitogen-activated protein kinase was not inhibited by PGA2 treatment. These data suggest that PGA2 blocks cell cycle progression without interfering with the cytosolic proliferative signaling pathway. Combined microinjection of E2F-1 and DP-1 proteins or microinjected adenovirus E1A protein, however, could induce S phase in cells arrested in G1 by PGA2, indicating that PGA2 does not directly inhibit the process of DNA synthesis. In quiescent cells, PGA2 blocked the normal hyperphosphorylation of the retinoblastoma susceptible gene product and the activation of cyclin-dependent kinase (CDK) 2 and CDK4, in response to serum stimulation. PGA2 treatment elevated the p21Waf1/Cip1/Sdi1 protein expression level. These data indicate that PGA2 may arrest the cell cycle in G1 by interfering with the activation of G1 phase CDKs.

3T3 Cells↗

PubMed1995

A conserved region of c-Ha-Ras is required for efficient GTPase stimulation by GTPase activating protein but not neurofibromin.

The effector binding domain and the switch II region of c-Ha-Ras are necessary for p120GAP-stimulated GTP hydrolysis. We report a third region of c-Ha-Ras located within the alpha 3 helix (amino acids 101-103) which is also required for efficient p120GAP, but not neurofibromin-mediated hydrolysis. This highly conserved region of the Ras protein was investigated using an insertion-deletion mutant (Ras-100LIR104) originally characterized by Willumsen et al. (Willumsen, B. M., Adari, H., Zhang, K., Papageorge, A. G., Stone, J. C., McCormick, F., and Lowy, D. R (1989) in The Guanine Nucleotide Binding Proteins; Common Structural and Functional Properties (Bosch, L., Kraal, B., and Parmeggiani, A., eds) pp. 165-178, Plenum Press, New York). The 100LIR104 substitution did not alter the intrinsic hydrolytic rate of the protein. The p120GAP-stimulated hydrolysis of Ras-100LIR104, however, was decreased by 2-3-fold compared to wild type Ras. This decrease in p120GAP-stimulated hydrolysis was not due to its inability to physically associate with Ras-100LIR104. GTP (as determined by competitive binding assays). Surprisingly, neurofibromin-stimulated GTP hydrolysis was unaltered by the mutation. Finally, no differences were observed in the ability of either the p120GAP catalytic domain or the neurofibromin GRD to accelerate Ras-100LIR104 GTPase activity, indicating that the amino-terminal noncatalytic GAP region is critical for p120GAP-stimulated GTP hydrolysis. This is the first report of a Ras mutation which differentiates between p120GAP and neurofibromin activity.

Amino Acid Sequence↗

PubMed1994

The adenovirus E1A protein overrides the requirement for cellular ras in initiating DNA synthesis.

The adenovirus E1A protein can induce cellular DNA synthesis in growth-arrested cells by interacting with the cellular protein p300 or pRb. In addition, serum- and growth factor-dependent cells require ras activity to initiate DNA synthesis and recently we have shown that Balb/c 3T3 cells can be blocked in either early or late G1 following microinjection of an anti-ras antibody. In this study, the E1A 243 amino acid protein is shown through microinjection not only to shorten the G0 to S phase interval but, what is more important, to override the inhibitory effects exerted by the anti-ras antibody in either early or late G1. Specifically, whether E1A is co-injected with anti-ras into quiescent cells or injected 18 h following a separate injection of anti-ras after serum stimulation, it efficiently induces cellular DNA synthesis in cells that would otherwise be blocked in G0/G1. Moreover, injection of a mutant form of E1A that can no longer associate with p300 is just as efficient as wild-type E1A in stimulating DNA synthesis in cells whose ras activity has been neutralized by anti-ras. The results presented here show that E1A is capable of overriding the requirement of cellular ras activity in promoting the entry of cells into S phase. Moreover, the results suggest the possibility that pRb and/or pRb-related proteins may function in a ras-dependent pathway that enables E1A to achieve this activity.

3T3 Cells↗

PubMed1994

Cellular ras activity is required for passage through multiple points of the G0/G1 phase in BALB/c 3T3 cells.

Microinjection experiments demonstrated a requirement for cellular ras activity late in G1. In this study, we used two separate methods to identify an additional requirement for cellular ras activity early in the G0/G1 phase of the cell cycle. Quiescent BALB/c cells were injected with anti-ras antibody prior to stimulation with serum. The cells would therefore be inhibited in progression through the cell cycle at the earliest point requiring ras function. Alternatively, cells were inhibited in late G1 as in previous studies by injecting anti-ras several hours after serum addition to quiescent cells. The injected cultures were then treated with chemical cell cycle inhibitors known to function in mid-G1. Cells injected with anti-ras prior to serum stimulation were retained at a point of ras requirement prior to the execution point of the chemical inhibitor, while cells injected 3 to 5 h after serum stimulation were retained at a point of ras requirement downstream of the execution point of the chemical inhibitor. To confirm these results, quiescent BALB/c cells were injected with anti-ras antibody prior to or several hours following serum addition. In this case, however, second injections of oncogenic ras or adenoviral E1A protein were performed to overcome the inhibitory effects of the anti-ras antibody. Cells injected prior to serum addition were clearly inhibited at an early point of Ras requirement since they required 5 or 6 h longer to enter S phase than cells injected with anti-ras antibody after serum addition.

3T3 Cells↗

PubMed1994

The regulation of endothelial cell motility by p21 ras.

Directed endothelial cell (EC) movement is required for the development and repair of blood vessels and plays a critical role in angiogenic processes obligatory for large tumor formation. We now report that ras proteins have a critical role in regulation of movement of normal mammalian cells. Bovine aortic EC microinjected with oncogenic Ha-ras enter further into an artificial wound than uninjected cells. Treatment with oncogenic Ha-ras also converts the cell paths from nearly linear in control cells to apparent 'random-walk' trajectories in treated cells, suggesting that oncogenic ras alters the normal control processes regulating cell motility. Botulinum toxin C blocks ras-stimulated motility indicating that a member of the p21 rho family is a downstream participant in the motile pathway. In related experiments we have observed that microinjection of the neutralizing, ras-specific, Y13-259 monoclonal antibody completely blocks both basal and basic fibroblast growth factor-stimulated movement of aortic EC. Y13-259 blocks the initiation of EC movement, as well as the continued progress of cells already in motion, suggesting that ras activity is continuously required throughout the motile process. Together these data indicate that ras is an integral component of the signaling pathway regulating cell movement.

Animals↗

PubMed1994

An E2F dominant negative mutant blocks E1A induced cell cycle progression.

E2F is a cellular transcription factor that is regulated during the cell cycle through interactions with the product of the retinoblastoma susceptibility gene (RB1) and the pRb-like p107 and p130 proteins. Analysis of mutations within both adenovirus E1A and pRb, which affected their ability to regulate cellular proliferation and alter E2F activity, suggested that E2F may play a role in cell cycle progression. Microinjection of a GST-E2F-1 fusion protein into quiescent Balb/c 3T3 cells induced DNA synthesis whereas co-injection of GST-E2F-1 and GST-E2F(95-191) protein, encoding only the DNA binding domain of E2F-1, blocked the induction of S-phase. While E1A likely targets multiple cellular pathways, co-injection of the GST-E2F(95-191) dominant inhibitory protein with 12S E1A protein blocked E1A-mediated induction of DNA synthesis, suggesting that the E2F-dependent pathway is dominant. Analysis of the interval required for microinjected quiescent cells to enter S-phase indicated that E2F-1 acted faster than either E1A or serum.

3T3 Cells↗

PubMed1993

Release from G1 growth arrest by transforming growth factor beta 1 requires cellular ras activity.

Transforming growth factor beta 1 (TGF beta 1) is a potent inhibitor of epithelial cell growth, although the mechanism of growth inhibition remains unknown. We report here a critical relationship between cellular p21ras activity and TGF beta 1 action. Microinjection of oncogenic Ha-ras protein into TGF beta 1-arrested mink lung epithelial cells overcomes TGF beta 1 growth inhibition and allows progression into S phase. Cells released from TGF beta 1 inhibition following microinjection with anti-p21ras antibody, on the other hand, remain TGF beta 1-arrested and do not enter S phase, indicating a requirement for p21ras activity. These biological data are substantiated biochemically in that TGF beta 1 is shown to decrease the activation state of endogenous p21ras, as measured by the level of GTP-bound p21ras. In addition, the phosphorylation and kinase activity of mitogen-activated protein kinase, which depends upon cellular ras activity, is elevated in cells which have been released from growth arrest by TGF beta 1. Together these data demonstrate the involvement of p21ras activity in TGF beta 1-induced growth inhibition and suggest that the inhibitor controls proliferation by modulating the activity of p21ras.

Animals↗

PubMed1993

Inhibitory effect of arachidonic acid on GTPase activating protein is antagonized by 1-stearoyl, 2-arachidonoyl glycerol.

The intrinsic GTPase activity of cellular protein p21ras is strongly increased by cytosolic GTPase activating protein (GAP). The activity of this enzyme has been shown to be inhibited by arachidonic acid. We report here that this inhibition is antagonized by the presence of 1-stearoyl, 2-arachidonoyl glycerol. This effect is structure specific and dose dependent, being maximum at 200 micrograms/ml of diacylglycerol (DG). These results suggest that production of DG in response to hormones or growth factors stimulation could indirectly modulate the interaction between p21ras and GAP.

Animals↗

PubMed1993

Inhibitory effect of arachidonic acid on GTPase activating protein is antagonized by 1-stearoyl, 2-arachidonoyl glycerol.

Arachidonic Acid↗

PubMed1993

Functional role of GTPase-activating protein in cell transformation by pp60v-src.

Morphological transformation of NIH 3T3 cells was observed following coexpression of a portion of the ras GTPase-activating protein (GAP) comprising the amino terminus (GAP-N) and a mutant of v-src (MDSRC) lacking the membrane-localizing sequence. Cells expressing either of these genes alone remained nontransformed. Coexpression of GAP-N with MDSRC did not alter the subcellular localization, kinase activity, or pattern of cellular substrates phosphorylated by the MDSRC product. In contrast to SHC, phospholipase C-gamma 1, and the p85 alpha phosphatidylinositol 3'-kinase subunit, the endogenous GAP product (p120GAP) was highly tyrosine-phosphorylated only in cells transformed by wild-type v-src. Furthermore, for transformation induced by wild-type v-src as well as by coexpression of MDSRC and GAP-N, a strict correlation was observed between cell transformation, elevated tyrosine phosphorylation of p62, p190, and a novel protein of 150 kDa, and complex formation between these proteins and p120GAP. As with cells transformed by wild-type v-src, the MDSRC plus GAP-N transformants remained dependent on endogenous Ras. The results suggest that tyrosine phosphorylation and complex formation involving p120GAP represent critical elements of cell transformation by v-src and that complementation of the cytosolic v-src mutant by GAP-N results, at least in part, from the formation of these complexes.

3T3 Cells↗

PubMed1992

Catalytic properties, tissue and intracellular distribution of neurofibromin.

The neurofibromatosis type 1 (NF1) gene encodes a protein, neurofibromin, that shows homology with members of the GTPase-activating protein (GAP) family. To study neurofibromin, rabbit polyclonal antisera were raised against two synthetic peptides. These antisera immunoprecipitated a specific protein of about 240 kDa in lysates of adult murine and rat tissues both in the soluble (S100) and to a lesser degree in the particulate (P100) fractions. The neurofibromin immunoprecipitated from the lysates of several murine organs stimulated the intrinsic GTPase activity of p21 c-Ha-ras protein. Based on immunoblotting, immunoprecipitation and GTPase assays, neurofibromin appears to be at least 10-fold more abundant in the brain than in the other murine organs. The GTPase-stimulatory activity of full-length neurofibromin, like the catalytic GAP-related domain, is inhibited by arachidonic acid and the detergent dodecyl maltoside, while phosphatidic acid, containing arachidonic and stearic acid, is non-inhibitory. Immunofluorescence analysis with anti-neurofibromin sera in NIH3T3 cells suggests that at least some of the cellular protein associates with cytoplasmic structures that are distinct from actin or tubulin filaments.

Amino Acid Sequence↗

PubMed1991

Preferential inhibition of the oncogenic form of RasH by mutations in the GAP binding/"effector" domain.

The double mutation, D33H/P34S, reduced the transforming activity of oncogenic RasH proteins, G12V and Q61L, 400- and 20-fold, respectively. Remarkably, this same mutation did not reduce the transforming activity of normal RasH, nor did it impair the ability of the protein to restore a functional Ras pathway in cells whose endogenous Ras proteins were inhibited. Another mutation in this region, D38N, had similar effects. The mutations reduced downstream coupling efficiency of normal Ras as assessed by yeast adenylyl cyclase stimulation. However, this was offset by decreased GTPase activating protein (GAP) binding, since the latter resulted in elevated GTP-bound mutant Ras in cells. The mutations produced a similar decrease in downstream coupling efficiency of oncogenic Ras, but decreased GAP binding did not compensate because the GTPase activity of oncogenic Ras is not stimulated by GAP. These results imply that preferential inactivation of oncogenic Ras in human tumors may be achieved by reagents designed to inhibit the GAP-binding/"effector" domain of Ras proteins.

Animals↗

PubMed1991

The GTPase stimulatory activities of the neurofibromatosis type 1 and the yeast IRA2 proteins are inhibited by arachidonic acid.

Three proteins, GTPase activating protein (GAP), neurofibromatosis 1 (NF1) and the yeast inhibitory regulator of the RAS-cAMP pathway (IRA2), have the ability to stimulate the GTPase activity of Ras proteins from higher animals or yeast. Previous studies indicate that certain lipids are able to inhibit this activity associated with the mammalian GAP protein. Inhibition of GAP would be expected to biologically activate Ras protein. In these studies arachidonic acid is shown also to inhibit the activity of the catalytic fragments of the other two proteins, mammalian NF1 and the yeast IRA2 proteins. In addition, phosphatidic acid (containing arachidonic and stearic acid) was inhibitory for the catalytic fragment of NF1 protein, but did not inhibit the catalytic fragments of GAP or IRA2 proteins. These observations emphasize the biochemical similarity of these proteins and provide support for the suggestion that lipids might play an important role in their biological control, and therefore also in the control of Ras activity and cellular proliferation.

Arachidonic Acid↗

PubMed1991

Ras GTPase-activating protein physically associates with mitogenically active phospholipids.

The physical interaction between GTPase-activating protein (GAP) and lipids has been characterized by two separate analyses. First, bacterially synthesized GAP molecules were found to associate with detergent-mixed micelles containing arachidonic but not with those containing arachidic acid. This association was detected by a faster elution time during molecular exclusion chromatography. Second, GAP molecules within a crude cellular lysate were specifically retained by a column on which certain lipids had been immobilized. The lipids able to retain GAP on such columns were identical to those which were shown previously to be most active in blocking GAP activity. The association between lipids and GAP was dependent upon magnesium ions. Lipids unable to inhibit GAP activity were also unable to physically associate with GAP. The tight association of GAP with these lipids was predicted by and helps to rationalize their ability to inhibit GAP activity.

Animals↗

PubMed1991

Dominant inhibitory Ras mutants selectively inhibit the activity of either cellular or oncogenic Ras.

Two dominant inhibitory Ras mutant proteins were analyzed by microinjection. One, [Asn-17]Ras, had a substitution in the putative Mg(2+)-binding site of Ha-Ras. The other, RAST, had a mutation in a yeast RAS protein that impaired its GTPase activity and increased its affinity for GAP. RAST also had a mutation that blocked its localization to the plasma membrane. In NIH 3T3 cells [Asn-17]Ras inhibited the function of normal Ras much more efficiently than that of oncogenic Ras. In contrast, RAST interfered with the transforming activity of oncogenic Ras more efficiently than that of normal Ras. These conclusions were based on two separate types of analysis. The inhibitory Ras mutant proteins were first microinjected into cells stably transformed either by oncogenic Ras or by high levels of expression of cellular Ras. Results obtained in stably transformed cells were then verified by coinjection of the inhibitory Ras mutant proteins together with transforming concentrations of either oncogenic or normal Ras protein. Whereas RAST was active in soluble form. [Asn-17]Ras required membrane localization for activity. Furthermore, mutations in the GAP/effector-binding domain reduced or eliminated the inhibitory activity of RAST but had no detectable effect on [Asn-17]Ras. These results are consistent with the possibility that [Asn-17]Ras functions by blocking the activation of endogenous Ras proteins, while RAST functions by blocking the ability of activated Ras to stimulate a downstream target within the cells. The properties of RAST suggest that interference with the GAP/effector-binding function of RAS represents a strategy for the preferential inactivation of oncogenic Ras in cells.

Animals↗

PubMed1991

Dominant inhibitory Ras mutants demonstrate the requirement for Ras activity in the action of tyrosine kinase oncogenes.

Two inhibitory Ras mutant proteins [(Asn 17) Ras and RAST] were microinjected into NIH3T3 cells in order to compare their inhibitory activity with that of a neutralizing anti-ras antibody. Both mutants were able to block efficiently the mitogenic effects of serum added to quiescent NIH3T3 cells. Furthermore, each of the inhibitors blocked cell cycle progression at the same point as the injected anti-ras antibody, just prior to the initiation of a new round of DNA synthesis. Finally, as with the injected anti-ras antibody, each of the inhibitors was efficiently able to block proliferation and reverse the transformed morphology of cells transformed by tyrosine kinase oncogenes, while cells transformed by serine kinase oncogenes were unaffected. Therefore, results with all three reagents clearly indicate that cellular Ras activity is required in the late G1 phase of the cell cycle and is essential for the maintenance of the transformed phenotype induced by tyrosine but not serine kinase oncogenes. These studies demonstrate the utility of dominant inhibitory mutants as a means of interfering with the activity of cellular oncogenes.

3T3 Cells↗

PubMed1990

A cytoplasmic protein inhibits the GTPase activity of H-Ras in a phospholipid-dependent manner.

A cytoplasmic protein has been identified that inhibits the guanosine triphosphatase (GTPase) activity of bacterially synthesized, cellular H-Ras protein. This GTPase inhibiting protein is able to counteract the activity of GTPase activating protein (GAP), which has been postulated to function as a negative regulator of Ras activity. The potential biological importance of the GTPase inhibiting protein is further supported by its interaction with lipids. Phospholipids produced in cells as a consequence of mitogenic stimulation increase the activity of the GTPase inhibiting protein, as well as inhibit the activity of GAP. The interaction of such lipids with each of these two regulatory proteins would, therefore, tend to increase the biological activity of Ras and stimulate cell proliferation.

Animals↗

PubMed1990

Serum stimulation of NIH 3T3 cells induces the production of lipids able to inhibit GTPase-activating protein activity.

Quiescent NIH 3T3 cells were stimulated with serum prior to the extraction of total cellular lipids. These lipids were fractionated on thin-layer chromatography plates, and individual fractions were tested for the ability to inhibit GTPase-activating protein (GAP) activity. Two separate GAP inhibitory lipids were produced. One behaved similarly to arachidonic acid during silica gel chromatography, whereas the other was related to a phosphoinositide. Further study of the arachidonic acid-related material indicated that it was produced between 1 and 5 min after serum addition but was never observed in high-density, contact-inhibited cultures. The identity of these lipids is under investigation. The possibility raised by these results, that a metabolite of arachidonic acid is involved in mitogenic signaling, was supported by the finding that several lipoxygenase products of arachidonic acid efficiently inhibited GAP activity. These results provide further support for the hypothesis that lipids, GAP, and ras activity function together in the control of cellular proliferation.

Animals↗