Expression of a mutated ras gene in Dictyostelium discoideum alters the binding of cyclic AMP to its chemotactic receptor

1988 ◽  
Vol 90 (4) ◽  
pp. 701-706
Author(s):  
M.E. Luderus ◽  
C.D. Reymond ◽  
P.J. Van Haastert ◽  
R. Van Driel
Keyword(s):  
Cyclic Amp ◽  
Dictyostelium Discoideum ◽  
Cell Types ◽  
Ras Proteins ◽  
Wild Type ◽  
Ras Gene ◽  
Physiological Level ◽  
Ras Protein ◽  
Type Gene

Dictyostelium discoideum cells contain a ras gene that codes for a polypeptide that is highly homologous to the human ras proteins. Extra copies of the wild-type gene or a gene carrying a missense mutation in codon 12 (ras-Gly12 and ras-Thr12, respectively) have been introduced into Dictyostelium cells by transformation. We have investigated the properties of the chemotactic cell surface cyclic AMP receptor in crude membrane preparations of wild-type Dictyostelium cells and ras-Gly12 and ras-Thr12 transformants. In vitro, an ATP- and Ca2+-dependent reduction of the number of cyclic AMP receptors was observed in membranes from all three cell types. The number of available receptors was decreased maximally by about 50%. In the presence of ATP the half-maximal Ca2+ concentration required for this process was about 10(−5) M in wild-type and ras-Gly12 membranes, and less than 10(−7) M in ras-Thr12 membranes. Addition of GTP (but not GDP) or the phorbol ester PMA (phorbol-12-myristate-13-acetate) reduced the Ca2+ requirement of the process in wild-type and ras-Gly12 membranes to the physiological level of less than 10(−7) M. In membranes derived from ras-Thr12 cells addition of GTP or PMA had no effect. The results indicate that D. discoideum cells contain a cyclic AMP receptor-controlling pathway that can be activated in vitro and involves a GTP-binding protein and a Ca2+ plus ATP-dependent activity, possibly protein kinase C. It is concluded that the ras protein specifically interacts with this pathway; the pathway appears to be constitutively activated by the mutated ras gene product.

scholarly journals Mutations in the SHR5 gene of Saccharomyces cerevisiae suppress Ras function and block membrane attachment and palmitoylation of Ras proteins.

1995 ◽  
Vol 15 (3) ◽  
pp. 1333-1342 ◽  
Author(s):  
V Jung ◽  
L Chen ◽  
S L Hofmann ◽  
M Wigler ◽  
S Powers
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Current Data ◽  
Ras Proteins ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Ras Protein ◽  
Membrane Attachment ◽  
Extragenic Suppressors

We have identified a gene, SHR5, in a screen for extragenic suppressors of the hyperactive RAS2Val-19 mutation in the budding yeast Saccharomyces cerevisiae. SHR5 was cloned, sequenced, and found to encode a 23-kDa protein not significantly homologous to other proteins in the current data bases. Genetic evidence arguing that Shr5 operates at the level of Ras is presented. We tested whether SHR5, like previously isolated suppressors of hyperactivated RAS2, acts by affecting the membrane attachment and/or posttranslational modification of Ras proteins. We found that less Ras protein is attached to the membrane in shr5 mutants than in wild-type cells and that the Ras proteins are markedly underpalmitoylated, suggesting that Shr5 is involved in palmitoylation of Ras proteins. However, shr5null mutants exhibit normal palmitoyltransferase activity measured in vitro. Further, shr5null mutations attenuate Ras function in cells containing mutant Ras2 proteins that are not palmitoylated or farnesylated. We conclude that SHR5 encodes a protein that participates in the membrane localization of Ras but also interacts in vivo with completely unprocessed and cytosolic Ras proteins.

Mechanism of Activation of the Caenorhabditis elegans ras Homologue let-60 by a Novel, Temperature-Sensitive, Gain-of-Function Mutation

Genetics ◽  
1997 ◽  
Vol 146 (2) ◽  
pp. 553-565 ◽  
Author(s):  
David M Eisenmann ◽  
Stuart K Kim
Keyword(s):  
Caenorhabditis Elegans ◽  
Intrinsic Rate ◽  
Temperature Sensitive ◽  
Ras Proteins ◽  
Temperature Dependent ◽  
Ras Gene ◽  
Gain Of Function ◽  
Gtp Hydrolysis ◽  
Ras Protein

The Caenorhabditis elegans let-60 gene encodes a Ras protein that mediates induction of the hermaphrodite vulva. To better understand how mutations constitutively activate Ras and cause unregulated cell division, we have characterized ga89, a temperature-sensitive, gain-of-function mutation in let-60 ras. At 25°, ga89 increases let-60 activity resulting in a multivulva phenotype. At 15°, ga89 decreases let-60 activity resulting in a vulvaless phenotype in let-60(ga89)/Df animals. The ga89 mutation causes a leucine (L) to phenylalanine (F) substitution at amino acid 19, a residue conserved in all Ras proteins. We introduced the L19F change into human H-Ras protein and found that the in vitro GTPase activity of H-Ras became temperature-dependent. Genetic experiments suggest that LET-60(L19F) interacts with GAP and GNEF, since mutations that decrease GAP and GNEF activity affect the multivulva phenotype of let-60(ga89) animals. These results suggest that the L19F mutation primarily affects the intrinsic rate of GTP hydrolysis by Ras, and that this effect may be sufficient to account for the activated-Ras phenotype caused by let-60(ga89). Our results suggest that a mutation in a human ras gene analogous to ga89 might contribute to oncogenic transformation.

scholarly journals Dictyostelium discoideum mutant synag 7 with altered G-protein-adenylate cyclase interaction

1988 ◽  
Vol 91 (2) ◽  
pp. 287-294
Author(s):  
B.E. Snaar-Jagalska ◽  
P.J. Van Haastert
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
G Protein ◽  
Dictyostelium Discoideum ◽  
High Speed ◽  
Wild Type ◽  
High Affinity ◽  
Gtp Binding

Previous results have shown that Dictyostelium discoideum mutant synag 7 is defective in the regulation of adenylate cyclase by receptor agonists in vivo and by GTP gamma S in vitro; the guanine nucleotide activation of adenylate cyclase is restored by the high-speed supernatant from wild-type cells. Here we report that in synag 7 membranes: (1) cyclic AMP receptors had normal levels and were regulated by guanine nucleotides as in wild-type; (2) GTP binding and high-affinity GTPase were reduced but still stimulated by cyclic AMP; (3) the supernatant from wild-type cells restored GTP binding to membranes of this mutant, and partly restored high-affinity GTPase activity; (4) the supernatant of synag 7 was ineffective in these reconstitutions and did not influence GTP binding and GTPase activities in mutant or wild-type membranes. These results suggest that the defect in mutant synag 7 is located between G-protein and adenylate cyclase, and not between receptor and G-protein. A factor in the supernatant is absent in synag 7 and appears to be essential for normal GTP binding, GTPase and activation of adenylate cyclase. This soluble heat-labile factor may represent a new molecule required for receptor- and G-protein-mediated activation of adenylate cyclase.

scholarly journals Toll-Like Receptor 2 Controls the Gamma Interferon Response to Francisella tularensis by Mouse Liver Lymphocytes

2007 ◽  
Vol 75 (11) ◽  
pp. 5338-5345 ◽  
Author(s):  
Kee-Jong Hong ◽  
Jason R. Wickstrum ◽  
Hung-Wen Yeh ◽  
Michael J. Parmely
Keyword(s):  
Francisella Tularensis ◽  
Cell Types ◽  
Gamma Interferon ◽  
Interferon Response ◽  
Interleukin 12 ◽  
Toll Like Receptor ◽  
Wild Type ◽  
Ifn Γ ◽  
Deficient Mice

ABSTRACT The production of gamma interferon (IFN-γ) is a key step in the protective innate immune response to Francisella tularensis. Natural killer cells and T cells in the liver are important sources of this cytokine during primary F. tularensis infections, and interleukin-12 (IL-12) appears to be an essential coactivating cytokine for hepatic IFN-γ expression. The present study was undertaken to determine whether or not macrophages (Mφ) or dendritic cells (DC) provide coactivating signals for the liver IFN-γ response in vitro, whether IL-12 mediates these effects, and whether Toll-like receptor (TLR) signaling is essential to induce this costimulatory activity. Both bone marrow-derived Mφ and DC significantly augmented the IFN-γ response of F. tularensis-challenged liver lymphocytes in vitro. While both cell types produced IL-12p40 in response to F. tularensis challenge, only DC secreted large quantities of IL-12p70. DC from both IL-12p35-deficient and TLR2-deficient mice failed to produce IL-12p70 and did not costimulate liver lymphocytes for IFN-γ production in response to viable F. tularensis organisms. Conversely, liver lymphocytes from TLR2-deficient mice cocultured with wild-type accessory cells produced IFN-γ at levels comparable to those for wild-type hepatic lymphocytes. These findings indicate that TLR2 controls hepatic lymphocyte IFN-γ responses to F. tularensis by regulating DC IL-12 production. While Mφ also coinduced hepatic IFN-γ production in response to F. tularensis, they did so in a fashion less dependent on TLR2.

scholarly journals Absence of the Macrophage Mannose Receptor in Mice Does Not Increase Susceptibility to Pneumocystis carinii Infection In Vivo

2003 ◽  
Vol 71 (11) ◽  
pp. 6213-6221 ◽  
Author(s):  
Steve D. Swain ◽  
Sena J. Lee ◽  
Michel C. Nussenzweig ◽  
Allen G. Harmsen
Keyword(s):  
Pneumocystis Carinii ◽  
Hydrolytic Enzymes ◽  
Mannose Receptor ◽  
Opportunistic Pathogen ◽  
Cell Types ◽  
Surface Expression ◽  
Wild Type ◽  
Macrophage Mannose Receptor

ABSTRACT Host defense against the opportunistic pathogen Pneumocystis carinii requires functional interactions of many cell types. Alveolar macrophages are presumed to be a vital host cell in the clearance of P. carinii, and the mechanisms of this interaction have come under scrutiny. The macrophage mannose receptor is believed to play an important role as a receptor involved in the binding and phagocytosis of P. carinii. Although there is in vitro evidence for this interaction, the in vivo role of this receptor in P. carinii clearance in unclear. Using a mouse model in which the mannose receptor has been deleted, we found that the absence of this receptor is not sufficient to allow infection by P. carinii in otherwise immunocompetent mice. Furthermore, when mice were rendered susceptible to P. carinii by CD4+ depletion, mannose receptor knockout mice (MR-KO) had pathogen loads equal to those of wild-type mice. However, the MR-KO mice exhibited a greater influx of phagocytes into the alveoli during infection. This was accompanied by increased pulmonary pathology in the MR-KO mice, as well as greater accumulation of glycoproteins in the alveoli (glycoproteins, including harmful hydrolytic enzymes, are normally cleared by the mannose receptor). We also found that the surface expression of the mannose receptor is not downregulated during P. carinii infection in wild-type mice. Our findings suggest that while the macrophage mannose receptor may be important in the recognition of P. carinii, in vivo, this mechanism may be redundant, and the absence of this receptor may be compensated for.

Action of a slowly hydrolysable cyclic AMP analogue on developing cells of Dictyostelium discoideum

1979 ◽  
Vol 35 (1) ◽  
pp. 321-338
Author(s):  
C. Rossier ◽  
G. Gerisch ◽  
D. Malchow
Keyword(s):  
Cyclic Amp ◽  
Dictyostelium Discoideum ◽  
Type Strain ◽  
Wild Type Strain ◽  
Agar Plate ◽  
Cell Aggregation ◽  
Fruiting Bodies ◽  
Wild Type ◽  
Camp Analogue ◽  
Order Of Magnitude

Adenosine 3′,5′-cyclic phosphorothioate (cAMP-S) is a cyclic AMP (cAMP) analogue which is only slowly hydrolysed by phosphodiesterases of Dictyostelium discoideum. The affinity of cAMP-S to cAMP receptors at the cell surface is only one order of magnitude lower than that of cAMP. cAMP-S can replace cAMP as a stimulant with respect to all receptor-mediated responses tested, including chemotaxis and the induction of cAMP pulses. cAMP-S does not affect growth of D. discoideum but it blocks cell aggregation at a uniform concentration of 5 × 10(−7) M in agar plate cultures of strain NC-4 as well as its axenically growing derivative, Ax-2. Another wild-type strain of D. discoideum, v-12, is able to aggregate on agar plates supplemented with 1 mM cAMP-S. The development of Polysphondylium pallidum and P. violaceum is also highly cAMP-S resistant. In Ax-2 both differentiation from the growth phase to the aggregation-competent stage and chemotaxis are cAMP-S sensitive, whereas in v-12 only chemotaxis is inhibited. v-12 can still form streams of cohering cells and fruiting bodies when chemotaxis is inhibited by cAMP-S. Whereas cAMP induces differentiation into stalk cells at concentrations of 10(−3) or 10(−4) M, cAMP-S has the same effect in strain v-12 at the much lower concentration of 10(−6) M.

Ammonia depletion and DIF trigger stalk cell intact Dictyostelium discoideum slugs

Development ◽  
1989 ◽  
Vol 105 (3) ◽  
pp. 569-574 ◽  
Author(s):  
M. Wang ◽  
P. Schaap
Keyword(s):  
Cell Differentiation ◽  
Cyclic Amp ◽  
Dictyostelium Discoideum ◽  
Stalk Cell

The differentiation-inducing factor, DIF, was induce stalk cell differentiation in Dictyostelium incubated as submerged monolayers. We investigated the regulates the differentiation of stalk cells in the was found that in migrating or submerged slugs DIF cell differentiation, which is most likely due to the antagonist. Cyclic AMP and ammonia were earlier antagonists in vitro. We show here that ammonia, but an antagonist for DIF-induced stalk cell can induce stalk cell differentiation when ammonia are enzymically depleted. However, depletion of cAMP increase the efficacy of DIF. We propose that the cell differentiation during early culmination may be drop in ammonia levels inside the organism.

scholarly journals Loss of a Protein Phosphatase 2A Regulatory Subunit (Cdc55p) Elicits Improper Regulation of Swe1p Degradation

2000 ◽  
Vol 20 (21) ◽  
pp. 8143-8156 ◽  
Author(s):  
Haifeng Yang ◽  
Wei Jiang ◽  
Matthew Gentry ◽  
Richard L. Hallberg
Keyword(s):  
Phosphatase Activity ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Cell Types ◽  
Regulatory Subunit ◽  
Cyclin Dependent Kinase ◽  
Wild Type ◽  
Phosphatase 2A

ABSTRACT CDC55 encodes a Saccharomyces cerevisiaeprotein phosphatase 2A (PP2A) regulatory subunit.cdc55-null cells growing at low temperature exhibit a failure of cytokinesis and produce abnormally elongated buds, butcdc55-null cells producing the cyclin-dependent kinase Cdc28-Y19F, which is unable to be inhibited by Y19 phosphorylation, show a loss of the abnormal morphology. Furthermore,cdc55-null cells exhibit a hyperphosphorylation of Y19. For these reasons, we have examined in wild-type and cdc55-null cells the levels and activities of the kinase (Swe1p) and phosphatase (Mih1p) that normally regulate the extent of Cdc28 Y19 phosphorylation. We find that Mih1p levels are comparable in the two strains, and an estimate of the in vivo and in vitro phosphatase activity of this enzyme in the two cell types indicates no marked differences. By contrast, while Swe1p levels are similar in unsynchronized and S-phase-arrested wild-type and cdc55-null cells, Swe1 kinase is found at elevated levels in mitosis-arrestedcdc55-null cells. This excess Swe1p incdc55-null cells is the result of ectopic stabilization of this protein during G2 and M, thereby accounting for the accumulation of Swe1p in mitosis-arrested cells. We also present evidence indicating that, in cdc55-null cells, misregulated PP2A phosphatase activity is the cause of both the ectopic stabilization of Swe1p and the production of the morphologically abnormal phenotype.

scholarly journals Impaired insulin-stimulated glucose transport in ATM-deficient mouse skeletal muscle

2013 ◽  
Vol 38 (6) ◽  
pp. 589-596 ◽  
Author(s):  
James Kain Ching ◽  
Larry D. Spears ◽  
Jennifer L. Armon ◽  
Allyson L. Renth ◽  
Stanley Andrisse ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Glucose Transport ◽  
Cell Types ◽  
Glucose Disposal ◽  
Wild Type ◽  
Phosphatidylinositol 3 Kinase ◽  
Ataxia Telangiectasia Mutated ◽  
Akt Phosphorylation ◽  
Atm Protein

There are reports that ataxia telangiectasia mutated (ATM) plays a role in insulin-stimulated Akt phosphorylation, although this is not the case in some cell types. Because Akt plays a key role in insulin signaling, which leads to glucose transport in skeletal muscle, the predominant tissue in insulin-stimulated glucose disposal, we examined whether insulin-stimulated Akt phosphorylation and (or) glucose transport would be decreased in skeletal muscle of mice lacking functional ATM, compared with muscle from wild-type mice. We found that in vitro insulin-stimulated Akt phosphorylation was normal in soleus muscle from mice with 1 nonfunctional allele of ATM (ATM+/−) and from mice with 2 nonfunctional alleles (ATM−/−). However, insulin did not stimulate glucose transport or the phosphorylation of AS160 in ATM−/− soleus. ATM protein level was markedly higher in wild-type extensor digitorum longus (EDL) than in wild-type soleus. In EDL from ATM−/− mice, insulin did not stimulate glucose transport. However, in contrast to findings for soleus, insulin-stimulated Akt phosphorylation was blunted in ATM−/− EDL, concomitant with a tendency for insulin-stimulated phosphatidylinositol 3-kinase activity to be decreased. Together, the findings suggest that ATM plays a role in insulin-stimulated glucose transport at the level of AS160 in muscle comprised of slow and fast oxidative-glycolytic fibers (soleus) and at the level of Akt in muscle containing fast glycolytic fibers (EDL).

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