Interplay between insulin and nutrients in the regulation of translation factors

2001 ◽  
Vol 29 (4) ◽  
pp. 541-547 ◽  
Author(s):  
C. G. Proud ◽  
X. Wang ◽  
J. V. Patel ◽  
L. E. Campbell ◽  
M. Kleijn ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Mammalian Target Of Rapamycin ◽  
Chinese Hamster ◽  
Elongation Factor ◽  
Nucleotide Exchange ◽  
S6 Kinase ◽  
P70 S6 Kinase ◽  
Eef2 Kinase

Protein synthesis in mammalian cells is regulated through alterations in the states of phosphorylation of eukaryotic initiation factors and elongation factors (eIFs and eEFs respectively) and of other regulatory proteins. This modulates their activities or their abilities to interact with one another. Insulin activates several of these proteins including the following: the guanine-nucleotide exchange factor eIF2B; the eIF4F complex, which (through eIF4E) interacts with the cap of the mRNA; p70 S6 kinase; and elongation factor eEF2, which mediates the translocation step of elongation. Control of the last three of these is linked to mTOR (mammalian target of rapamycin). In Chinese hamster ovary cells, regulation of all these proteins by insulin is modulated by the presence of amino acids and/or glucose in the medium. For example, p70 S6 kinase activity declines in the absence of amino acids and cannot be stimulated by insulin under this condition. The readdition of amino acids, especially leucine, restores activity and sensitivity to insulin. With eIF2B and eEF2, both amino acids and glucose must be provided for insulin to regulate their activities. In contrast, insulin-stimulation of the formation of eIF4F complexes requires glucose but not amino acids. Glucose metabolism is required for this permissive effect. Our recent studies have also identified the mechanism by which mTOR signalling regulates the phosphorylation of eEF2. eEF2 kinase is phosphorylated by p70 S6 kinase at Ser-366; this results in the inactivation of eEF2 kinase, especially at low (micromolar) Ca concentrations.

scholarly journals Nutrients differentially regulate multiple translation factors and their control by insulin

1999 ◽  
Vol 344 (2) ◽  
pp. 433-441 ◽  
Author(s):  
Linda E. CAMPBELL ◽  
Xuemin WANG ◽  
Christopher G. PROUD
Keyword(s):  
Amino Acids ◽  
Protein Kinase ◽  
Mammalian Cells ◽  
Glycogen Synthase ◽  
Protein Kinase B ◽  
Elongation Factor ◽  
Mrna Translation ◽  
Initiation Factor ◽  
S6 Kinase ◽  
P70 S6 Kinase

Eukaryotic initiation factor eIF2B and eukaryotic elongation factor eEF2 each mediate regulatory steps important for the overall regulation of mRNA translation in mammalian cells and are activated by insulin. Here, we demonstrate that their activation by insulin requires the presence, in the medium in which the cells are maintained, of both amino acids and glucose: insulin only induced activation of eIF2B and the dephosphorylation of eEF2 when cells were exposed to both types of nutrient. Other translational regulators, e.g. the 70 kDa ribosomal protein S6 kinase (p70 S6 kinase) and the eIF4E binding protein 1, 4E-BP1, are also regulated by insulin but their control does not require glucose, only amino acids. The effects of nutrients on the activation of eIF2B do not reflect changes in the phosphorylation of eIF2 (and, by inference, operation of a kinase analogous to yeast Gcn2p), or a requirement for nutrients for inactivation of glycogen synthase kinase-3 or dephosphorylation of eIF2B. Nutrients did not affect the ability of insulin to activate protein kinase B. These data show that activation by insulin of p70 S6 kinase, which modulates the translation of specific mRNAs, depends on the availability of amino acids whereas regulation of factors involved in overall activation of translation (eIF2B, eEF2) requires both amino acids and glucose. These results add substantially to the emerging evidence that nutrients themselves modulate functions of mammalian cells and indicate that (i) nutrients modulate the activation of eIF2B and eEF2 through as-yet unidentified mechanisms and (ii) regulation of p70 S6 kinase and 4E-BP1 by insulin requires other inputs in addition to protein kinase B.

scholarly journals Characterization of a cysteine-less human reduced folate carrier: localization of a substrate-binding domain by cysteine-scanning mutagenesis and cysteine accessibility methods

2003 ◽  
Vol 374 (1) ◽  
pp. 27-36 ◽  
Author(s):  
Wei CAO ◽  
Larry H. MATHERLY
Keyword(s):  
Amino Acids ◽  
Mammalian Cells ◽  
Transmembrane Domain ◽  
Chinese Hamster Ovary Cells ◽  
Substrate Binding ◽  
Chinese Hamster ◽  
Water Soluble ◽  
Cysteine Residues ◽  
Reduced Folate Carrier ◽  
Cysteine Scanning Mutagenesis

The human reduced folate carrier (hRFC) mediates the transport of reduced folates and classical anti-folates into mammalian cells. Whereas the functionally important domains in hRFC are poorly characterized, previous studies with anti-folate-resistant cells suggest critical roles for transmembrane domain (TMD) 1 and residues (Gly44, Glu45, Ser46 and Ile48) in or flanking this region. An hRFC mutant devoid of cysteine residues was prepared by deleting the C-terminal 56 amino acids, including four cysteine residues, and mutagenizing the remaining cysteine residues to serine residues. A fully functional cysteine-less hRFC protein was expressed in transport-impaired MtxRIIOuaR2-4 Chinese-hamster ovary cells. To explore the role of residues in or flanking TMD1 in transport, all 24 amino acids from Trp25 to Ile48 of hRFC were mutated individually to cysteine residues, and the mutant hRFCs were transfected into MtxRIIOuaR2-4 cells. All of the 24 cysteine mutants were expressed and, with the exception of R42C (Arg42→Cys), were capable of mediating methotrexate uptake above the low level in MtxRIIOuaR2-4 cells. We found that by treating the transfected cells with the small, water-soluble, thiol-reactive anionic reagent, sodium (2-sulphonatoethyl) methanethiosulphonate, methotrexate transport by several of the cysteine-substituted hRFC mutants was significantly inhibited, including Q40C, G44C, E45C and I48C. Sodium (2-sulphonatoethyl) methanethiosulphonate transport inhibition of the Q40C, G44C and I48C mutants was protected by leucovorin [(6R,S)-5-formyltetrahydrofolate], indicating that these residues lie at or near a substrate-binding site. Using surface-labelling reagents [N-biotinylaminoethyl methanethiosulphonate and 3-(N-maleimidylpropionyl)biocytin, combined with 4-acetamido-4′-maleimidylstilbene-2,2′-disulphonic acid] with cysteine mutants from positions 37–48, the extracellular TMD1 boundary was found to lie between residues 39 and 40, and amino acids 44–46 and 48 were localized to the TMD1 exofacial loop. Collectively, our results imply that amino acids 40, 44, 48 and, possibly, 42 serve important roles in hRFC transport, albeit not as structural components of the putative transmembrane channel for folate substrates.

scholarly journals Molecular cloning of the cDNA for a mutant mouse ribonucleotide reductase M1 that produces a dominant mutator phenotype in mammalian cells.

1988 ◽  
Vol 8 (7) ◽  
pp. 2698-2704 ◽  
Author(s):  
I W Caras ◽  
D W Martin
Keyword(s):  
Ribonucleotide Reductase ◽  
Mammalian Cells ◽  
Mutant Mouse ◽  
Chinese Hamster Ovary Cells ◽  
Spontaneous Mutation ◽  
Single Point ◽  
Chinese Hamster ◽  
Fold Increase ◽  
Single Point Mutation ◽  
Mutator Phenotype

Mammalian ribonucleotide reductase is regulated by the binding of dATP and other nucleotide effectors to allosteric sites on subunit M1. Using mRNA from a mutant mouse T-lymphoma (S49) cell line, we have isolated a cDNA which encodes an altered, dATP feedback-resistant subunit M1. The mutant cDNA contains a single point mutation (a G-to-A transition) at codon 57, converting aspartic acid to asparagine. Proof that this mutation is responsible for the phenotype of dATP feedback resistance is provided by the following evidence. (i) The mutation was detected only in mutant S49 cells containing dATP feedback-resistant ribonucleotide reductase and not in wild-type or other mutant S49 cells. (ii) Transfection of Chinese hamster ovary cells with an expression plasmid containing the mutant M1 cDNA resulted in the production of dATP feedback-resistant ribonucleotide reductase. Transfected CHO cells expressing the mutant M1 cDNA exhibited a 15- to 25-fold increase in the frequency of spontaneous mutation to 6-thioguanine resistance, confirming that dATP feedback-resistant ribonucleotide reductase produces a mutator phenotype in mammalian cells. The availability of a cDNA which encodes dATP feedback-resistant subunit M1 thus provides a means of manipulating by transfection the frequency of spontaneous mutation in mammalian cells.

scholarly journals Cell cycle-dependent effects on deoxyribonucleotide and DNA labeling by nucleoside precursors in mammalian cells.

1987 ◽  
Vol 7 (1) ◽  
pp. 532-534 ◽  
Author(s):  
J M Leeds ◽  
C K Mathews
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
G1 Phase ◽  
Ovary Cells ◽  
Dna Labeling ◽  
Metabolic Pool ◽  
Specific Activities ◽  
Cycle Dependent

dCTP pools equilibrated to equivalent specific activities in Chinese hamster ovary cells or in nuclei after incubation of cells with radiolabeled nucleosides, indicating that dCTP in nuclei does not constitute a distinct metabolic pool. In the G1 phase, [5-3H]deoxycytidine labeled dCTP to unexpectedly high specific activities. This may explain reports of replication-excluded DNA precursor pools.

Metabolism of peptide amino acids by Chinese hamster ovary cells grown in a complex medium

1999 ◽  
Vol 62 (3) ◽  
pp. 324-335 ◽  
Author(s):  
Gregg B. Nyberg ◽  
R. Robert Balcarcel ◽  
Brian D. Follstad ◽  
Gregory Stephanopoulos ◽  
Daniel I. C. Wang
Keyword(s):  
Amino Acids ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Complex Medium ◽  
Ovary Cells

scholarly journals A fluorescent lipid analogue can be used to monitor secretory activity and for isolation of mammalian secretion mutants.

1995 ◽  
Vol 6 (2) ◽  
pp. 135-150 ◽  
Author(s):  
N T Ktistakis ◽  
C Y Kao ◽  
R H Wang ◽  
M G Roth
Keyword(s):  
Chinese Hamster Ovary ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Brefeldin A ◽  
Secretory Activity ◽  
Specific Marker ◽  
Ovary Cells ◽  
Fluorescent Lipid

The use of reporter proteins to study the regulation of secretion has often been complicated by posttranslational processing events that influence the secretion of certain proteins, but are not part of the cellular mechanisms that specifically regulate secretion. This has been a particular limitation for the isolation of mammalian secretion mutants, which has typically been a slow process. To provide a reporter of secretory activity independent of protein processing events, cells were labeled with the fluorescent lipid analogue C5-DMB-ceramide (ceramide coupled to the fluorophore boron dipyrromethene difluoride) and its secretion was followed by fluorescence microscopy and fluorescence-activated cell sorting. Brefeldin A, which severely inhibits secretion in Chinese hamster ovary cells, blocked secretion of C5-DMB-ceramide. At high temperature, export of C5-DMB-ceramide was inhibited in HRP-1 cells, which have a conditional defect in secretion. Using C5-DMB-ceramide as a reporter of secretory activity, several different pulse-chase protocols were designed that selected mutant Chinese hamster ovary cells that were resistant to the drug brefeldin A and others that were defective in the transport of glycoproteins to the cell surface. Mutant cells of either type were identified in a mutagenized population at a frequency of 10(-6). Thus, the fluorescent lipid C5-DMB-ceramide can be used as a specific marker of secretory activity, providing an efficient, general approach for isolating mammalian cells with defects in the secretory pathway.

In vitro biosynthesis of diphthamide, studied with mutant Chinese hamster ovary cells resistant to diphtheria toxin

1984 ◽  
Vol 4 (4) ◽  
pp. 642-650
Author(s):  
T J Moehring ◽  
D E Danley ◽  
J M Moehring
Keyword(s):  
Chinese Hamster Ovary ◽  
Diphtheria Toxin ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Elongation Factor ◽  
Synthetase Activity ◽  
Post Translational Modification ◽  
Ovary Cells ◽  
Elongation Factor 2

Diphthamide, a unique amino acid, is a post-translational derivative of histidine that exists in protein synthesis elongation factor 2 at the site of diphtheria toxin-catalyzed ADP-ribosylation of elongation factor 2. We investigated steps in the biosynthesis of diphthamide with mutants of Chinese hamster ovary cells that were altered in different steps of this complex post-translational modification. Biochemical evidence indicates that this modification requires a minimum of three steps, two of which we accomplished in vitro. We identified a methyltransferase activity that transfers methyl groups from S-adenosyl methionine to an unmethylated form of diphthine (the deamidated form of diphthamide), and we tentatively identified an ATP-dependent synthetase activity involved in the biosynthesis of diphthamide from diphthine. Our results are in accord with the proposed structure of diphthamide (B. G. VanNess, et al., J. Biol. Chem. 255:10710-10716, 1980).

Further Evidence That the Majority of Primary Nuclear RNA Transcripts in Mammalian Cells Do Not Contribute to mRNA

1982 ◽  
Vol 2 (6) ◽  
pp. 701-707
Author(s):  
M. Salditt-Georgieff ◽  
J. E. Darnell
Keyword(s):  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cdna Clones ◽  
Ovary Cells ◽  
Rna Molecules ◽  
Rna Transcripts ◽  
Polyadenylic Acid ◽  
Nuclear Molecules ◽  
Nuclear Rna

Nuclear RNA from Chinese hamster ovary cells was effectively separated into polyadenylic acid [poly(A)]-containing [poly (A) + ] and non-poly(A)-containing [poly(A) − ] fractions so that ∼90% of the poly(A) was present in the (A) + fraction. Only 25% of the 5′-terminal caps of the large nuclear molecules were present in the (A) + class, but about 70% of the specific mRNA sequences (assayed with cDNA clones) were in the (A) + class. It appears that many long capped heterogeneous nuclear RNA molecules are of a different sequence category from those molecules that are successfully processed into mRNA.

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