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L A Feig

Publications and source records attributed to L A Feig.

51 records · Page 3Linked to original sources
PubMed1991

Characterization of a GTPase-activating protein for the Ras-related Ral protein.

We have demonstrated the presence of a GTPase-activating protein (GAP) for the Ras-related Ral A protein in the cytosolic fraction of brain and testis. This protein, designated Ral-GAP, was distinguished from Ras-GAP by its behavior in two chromatography systems and by the fact that the two GAP proteins did not stimulate the GTPase activity of each others target GTP binding proteins. The lack of effect of Ral-GAP on Ras GTPase activity also distinguished it from the product of the neurofibromatosis gene NF-1. Ral-GAP also differed from Rho-GAP and Rap-GAP by virtue of its elution from a gel filtration column with proteins of Mr greater than 10(6). This was likely an overestimate of the protein's molecular mass, however, since it sedimented in sucrose gradients between standard proteins of 150 and 443 kDa. Ral-GAP failed to promote the GTPase activity of mutant Ral proteins containing amino acid substitutions that in Ras lead to GAP-insensitive proteins.

Animals↗
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

Dominant inhibitory mutations in the Mg(2+)-binding site of RasH prevent its activation by GTP.

We have previously demonstrated that substitution of Asn for Ser at position 17 of RasH yields a dominant inhibitory protein whose expression in cells interferes with endogenous Ras function (L. A. Feig, and G. M. Cooper, Mol. Cell. Biol. 8:3235-3243, 1988). Subsequent structural studies have shown that the hydroxyl group of Ser-17 contributes to the binding of Mg2+ associated with bound nucleotide. In this report, we show that more subtle amino acid substitutions at this site that would be expected to interfere with complexing Mg2+, such as Cys or Ala, also generated dominant inhibitory mutants. In contrast, a Thr substitution that conserves a reactive hydroxyl group maintained normal Ras function. These results argue that the defect responsible for the inhibitory activity is improper coordination of Mg2+. Preferential affinity for GDP, observed in the original Asn-17 mutant, was found exclusively in inhibitory mutants. However, this binding specificity did not completely block the mutant proteins from binding GTP in vivo since introduction of the autophosphorylation site, Thr-59, in 17N Ras resulted in the phosphorylation of the double mutant in cells. Furthermore, inhibitory mutants failed to activate a model downstream target, yeast adenylate cyclase, even when bound to GTP. Thus, the consequence of improper complexing of Mg2+ was to lock the protein in a constitutively inactive state. A model is presented to explain how these properties could cause the mutant protein to inhibit the activation of endogenous Ras by competing for a guanine nucleotide-releasing factor.

Adenylyl Cyclases↗
PubMed1991

A mutation in the putative Mg(2+)-binding site of Gs alpha prevents its activation by receptors.

The properties of a Gs alpha mutant with an Asn substituted for Ser at position 54, designated mutant 54Asn alpha s, were studied after expression in S49 alpha s-deficient (cyc-) cells. Ser-54 in alpha s is comparable to Ser-17 in Ras, which is involved in binding Mg2+ associated with bound nucleotide. 54Asn alpha s did not restore either hormone-induced cyclic AMP production in intact cyc- cells or hormone-induced adenylyl cyclase activation in membranes isolated from these cells. The defect was a failure of ligand-bound receptor to activate 54Asn alpha s, since the mutant protein retained the ability to activate adenylyl cyclase in isolated membranes in the presence of GTP or GTP gamma S. Guanine nucleotide regulation of mutant alpha s suggested that it has increased guanine nucleotide exchange rates and an increased preference for diphosphates over triphosphates. Hormone stimulation magnified the preference of 54Asn alpha s for diphosphates, which could account for its inability to be activated by receptor. The properties of this mutant are discussed in terms of similarities to and differences with the analogous RasH mutant, which has been shown to interfere with endogenous Ras function in cells.

Adenylyl Cyclases↗
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↗
PubMed1988

Relationship among guanine nucleotide exchange, GTP hydrolysis, and transforming potential of mutated ras proteins.

The effect of a series of mutations on the transforming potential of normal human rasH has been compared with their effects on GTPase and guanine nucleotide exchange rates of p21. The mutation Val-146 resulted in partial activation of transforming potential which could be attributed to a greater than 1,000-fold-increased rate of nucleotide exchange in the absence of an effect on GTPase. In contrast, the more modest enhancement of exchange rate (approximately 100-fold) which resulted from the mutation Met-14 did not affect biological activity. The partially activating mutation Thr-59 was found to result in both a 5-fold reduction in GTPase and a 10-fold increase in nucleotide exchange. However, the nontransforming mutant Ile-59 displayed a comparable decrease in GTPase without an effect on nucleotide exchange. The activating effect of the Thr-59 mutation may thus represent a combined effect of reduced GTPase and increased exchange. Similarly, the strongly activating mutation Leu-61 resulted in a fivefold increase in nucleotide exchange in addition to decreased GTPase, whereas weakly activating mutations at position 61 (Trp and Pro) resulted only in decreased GTPase without affecting nucleotide exchange rates. Finally, combining the two mutations Met-14 and Ile-59, which alone had no effect on biological activity, yielded a double mutant with a 20-fold increased transforming potential, demonstrating a synergistic effect of these two mutations. Overall, these results indicate that large increases in nucleotide exchange can activate ras transforming potential in the absence of decreased GTPase and that relatively modest increases in nucleotide exchange can act synergistically with decreased GTPase to contribute to ras activation.

Fibroblasts↗
PubMed1988

Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP.

Substitution of asparagine for serine at position 17 decreased the affinity of rasH p21 for GTP 20- to 40-fold without significantly affecting its affinity for GDP. Transfection of NIH 3T3 cells with a mammalian expression vector containing the Asn-17 rasH gene and a Neor gene under the control of the same promoter yielded only a small fraction of the expected number of G418-resistant colonies, indicating that expression of Asn-17 p21 inhibited cell proliferation. The inhibitory effect of Asn-17 p21 required its localization to the plasma membrane and was reversed by coexpression of an activated ras gene, indicating that the mutant p21 blocked the endogenous ras function required for NIH 3T3 cell proliferation. NIH 3T3 cells transformed by v-mos and v-raf, but not v-src, were resistant to inhibition by Asn-17 p21, indicating that the requirement for normal ras function can be bypassed by these cytoplasmic oncogenes. The Asn-17 mutant represents a novel reagent for the study of ras function by virtue of its ability to inhibit cellular ras activity in vivo. Since this phenotype is likely associated with the preferential affinity of the mutant protein for GDP, analogous mutations might also yield inhibitors of other proteins whose activities are regulated by guanine nucleotide binding.

Animals↗
PubMed1987

Structure/function analysis of ras using random mutagenesis coupled with functional screening assays.

We review the use of functional assays for the ras protein, p21, that have allowed us to screen for mutant ras genes encoding proteins defective in either interactions with guanine nucleotides or transforming activity. GTP binding and GTP-dependent autokinase activities were assayed directly on lysed bacterial colonies expressing p21. Mutants encoding ras proteins deficient in these activities were isolated after randomly mutagenizing a v-rasH expression vector. Transformation defective mutants were isolated by randomly mutagenizing a v-rasH retroviral shuttle vector. NIH cells were then infected with a stock of nonreplicating mutagenized retroviruses and nontransformed infected colonies were isolated. The mutant ras genes were then rescued from these cells for analysis. Characterization of these mutants defines domains of p21 involved in both biochemical and biological activities and addresses the role of guanine nucleotide binding in p21 function.

Animals↗
PubMed1986

Isolation of ras GTP-binding mutants using an in situ colony-binding assay.

We have developed a strategy to isolate mutant ras genes encoding proteins defective in GTP binding. Random in vitro mutagenesis of a v-Harvey (Ha)-ras expression vector was followed by an in situ GTP-binding assay on lysed bacterial colonies. Single amino acid substitutions at ras codon 83, 119, or 144 decreased the affinity of p21 for GTP by a factor of 25-100 primarily as a consequence of increased rates of dissociation of GTP from p21. Nevertheless, these mutant genes induced transformation of NIH 3T3 cells with efficiencies comparable to wild-type v-Ha-ras. In transformed cells, mutant p21s were phosphorylated to a degree similar to that of wild-type v-Ha-ras p21, suggesting that a decrease in affinity by a factor of 100 did not prevent the mutant ras protein from binding GTP in vivo. These results are discussed with respect to the role of GTP in the regulation of p21 function.

Amino Acid Sequence↗
PubMed1984

Somatic activation of rasK gene in a human ovarian carcinoma.

A tumor isolate from a patient with serous cystadenocarcinoma of the ovary contained an activated rasK gene detected hy transfection of NIH/3T3 cells. In contrast, DNA from normal cells of the same patient lacked transforming activity, indicating that activation of this transforming gene was the consequence of somatic mutation in the neoplastic cells. The transforming gene product displayed an electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels that differed from the mobilities of rasK transforming proteins in other tumors, indicating that a previously undescribed mutation was responsible for activation of rasK in this ovarian carcinoma.

Animals↗
PubMed1983

Mitogenic polypeptide of the mammalian seminiferous epithelium: biochemical characterization and partial purification.

A mitogenic polypeptide, previously identified in Sertoli cells of the prepuberal mouse (Feig, L. A., A. R. Bellvé, N. Horbach-Erickson, and M. Klagsbrun, 1980, Proc. Natl. Acad. Sci. USA., 77:4774-4778), now has been shown to exist in Sertoli cells of the adult mouse and in the seminiferous epithelium of several other mammalian species, including the rat, guinea pig, and calf. The levels of this seminiferous growth factor (SGF) are not appreciably reduced in adult mouse testes following hypophysectomy. SGF purified from either the adult mouse or newborn calf seminiferous epithelium has a molecular weight (Mr) of 15,700 and a pl between 4.8 and 5.8, when exposed to denaturing conditions. Furthermore, SGF from these two mammalian species probably has few exposed hydrophobic domains and has a strong propensity to aggregate into multiple, high Mr species. A purification sequence based on these biochemical properties has enabled a greater than 350-fold enrichment of SGF activity from the calf seminiferous epithelium. The protocol involves a sequence of: (a) ammonium sulfate precipitation, (b) DEAE-cellulose ion exchange chromatography, (c) gel filtration chromatography on Bio-Gel P150 in 1.0 M ammonium acetate, (d) hydrophobic chromatography on dodecyl agarose, and (e) gel filtration chromatography in 6.0 M guanidine hydrochloride. Subsequent analysis of this purified preparation by SDS PAGE, followed by silver staining, reveals approximately 7 polypeptides with Mr between 14,000 and 20,000.

Animals↗
PubMed1982

The effect of angiotensin II on in vivo albumin transport in normal rabbit aortic tissue.

Angiotensin II and other vasoactive amines may have a direct effect on the permeability of the arterial wall. We have investigated the effect of angiotensin II in vivo albumin transport across the aortic wall in rabbits following intravenous injection of [125I]albumin. Transmural concentration profiles of 125I-labeled albumin across the intima and media of the aorta, generated during 25 min of either angiotensin or saline infusion, were measured by a serial-sectioning technique. The uptake of labeled albumin through the aortic wall was found to be dependent on position and to increase from the descending thoracic up to the arch. Angiotensin infusion increased albumin uptake in the region of the aorta proximal to the first pair of intercostal arteries and magnified the position dependence. Angiotensin infusion did not change the uptake of albumin in the descending thoracic aorta between intercostal arteries. The arterial blood pressure elevation associated with angiotensin infusion was not of prime importance in producing the uptake patterns described above.

Angiotensin II↗
PubMed1980

Sertoli cells contain a mitogenic polypeptide.

The seminiferous epithelium of the mouse testis contains a potent mitogen that induces DNA synthesis and cell division in cutures of confluent, quiescent BALB/c 3T3 cells. Homogenates of adult mouse seminiferous tubules, when added to 3T3 cells for 48 hr, produce a dose-dependent increase in DNA synthesis with half-maximal activity at 400 micrograms of protein per ml. Maximal stimulation causes a 70- to 100-fold increase in [methyl-3H]thymidine incorporation into DNA, a labeling of > 99% of 3T3 cell nuclei, and a doubling in cell number. The level of mitogenic activity in seminiferous tubules isolated from newborn mice is five times greater than that in tubules of adult mice. Furthermore, prepuberal Sertoli cells contain substantially greater levels of mitogenic activity than any other cell type purified from the seminiferous epithelium. A homogenate of prepuberal Sertoli cells induces half-maximal DNA synthesis at 20 micrograms of protein per ml. The mitogenic factor is sensitive to proteases and to heating at 100 degrees C for 2 min but not to dithiothreitol. Gel-filtration chromatography reveals that the factor is a polypeptide with a Mr of approximately 15,500. These observations provide evidence for the presence of a growth factor in the mammalian testis.

Animals↗