epidermal-growth-factor and queuine

The eukaryotic tRNA:guanine transglycosylase (TGT) catalyses the base-for-base exchange of guanine for queuine (the q-base)--a nutrition factor for eukaryotes--at position 34 of the anticodon of tRNAsGUN (where 'N' represents one of the four canonical tRNA nucleosides), yielding the modified tRNA nucleoside queuosine (Q). This unique tRNA modification process was investigated in HeLa cells grown under either aerobic (21% O2) or hypoxic conditions (7% O2) after addition of chemically synthesized q-base to q-deficient cells. While the q-base was always inserted into tRNA under aerobic conditions, HeLa cells lost this ability under hypoxic conditions, however, only when serum factors became depleted from the culture medium. The inability to insert q into tRNA did not result from a lack of substrate, because the q-base accumulated within these cells against the concentration gradient, suggesting the presence of an active transport system for this base in HeLa cells. The activity of the TGT enzyme was restored after treatment of the cells with the protein kinase C activator, TPA, even in the presence of mRNA or protein synthesis inhibitors. The results indicate that the eukaryotic tRNA modifying enzyme, TGT, is a downstream target of activated protein kinase C.

Topics: Anticodon; Blood; Culture Media; Enzyme Reactivators; Epidermal Growth Factor; Guanine; HeLa Cells; Humans; Kinetics; Oxygen; Pentosyltransferases; Platelet-Derived Growth Factor; Protein Kinase C; RNA, Transfer; Tetradecanoylphorbol Acetate

The nutrition factor queuine (q; the q-base) is a modulator of mammalian cell proliferation: it can be inhibitory or stimulatory, depending on the metabolic state of the cell. The mechanism underlying this growth-modulating activity was investigated. It was found that the q-base acts antagonistic to epidermal growth factor (EGF) but synergistic with platelet-derived growth factor (PDGF) in HeLa cells. Binding of either growth factor to its receptor resulted in the activation of distinct cellular kinases. The activities of these kinases were profoundly affected by q. The results suggest that the q-base is involved in the homologous regulation of signalling by receptor tyrosine kinases.

Topics: Calcium-Calmodulin-Dependent Protein Kinases; Drug Interactions; Epidermal Growth Factor; Guanine; HeLa Cells; Humans; Kinetics; Platelet-Derived Growth Factor; Receptor Protein-Tyrosine Kinases; Signal Transduction

The guanine analogue, queuine (q), is a nutrition factor for eukaryotes and occurs as a free base or as modified nucleoside queuosine (Q) inserted into the anticodon of tRNAs(GUN) in place of guanosine (G). The free q-base and the corresponding Q-deficient tRNAs accumulate in fast proliferating tumors and in embryonic cells. The present data show that treatment of Q-deficient HeLa cells with queuine resulted in reduced in vitro phosphorylation of two membrane-associated proteins, pp110 and pp18. Reduced phosphorylation of pp110 and pp18 was also observed when a membrane-containing particulate fraction from queuine starved HeLa cells was labelled with [gamma-32P]ATP in the presence of queuine. Incorporated phosphate into pp110 was alkali-stable, suggesting that this protein became phosphorylated at tyrosine residues. After stimulation of intact Q-deficient cells with epidermal growth factor, pp110 was rapidly phosphorylated at tyrosine residues. The results show that queuine as a free base influences protein tyrosine phosphorylation in intact cells, suggesting that it might interfere with protein phosphorylation involved in signal transduction pathways.

Topics: Adenosine Triphosphate; Epidermal Growth Factor; Guanine; HeLa Cells; Humans; Membrane Proteins; Phosphoproteins; Phosphorylation; Phosphotyrosine; Signal Transduction; Tyrosine

Mitogenic stimulation of quiescent mammalian cells triggers an array of early events crucial for cell cycle progression. Here we show that the activity of the anoxic stress protein, lactate dehydrogenase 6/k, transiently increased after mitogenic stimulation of serum-starved HeLa cells. Regulation of lactate dehydrogenase 6/k activity in early G1 depended on the activity of a receptor tyrosine kinase and on protein and mRNA synthesis, but did not involve protein kinase C. The guanine analog, queuine, an ubiquitously occurring tRNA base of bacterial origin, suppressed the mitogen-induced protein synthesis and also the transient increase in lactate dehydrogenase 6/k activity. The results suggest that queuine relieves hypoxic stress resulting from mitogenic stimulation by suppressing protein synthesis during G0/G1 transition.

Topics: Amanitins; Cell Cycle; Cell Division; Cell Hypoxia; Cycloheximide; Cytosol; Epidermal Growth Factor; G1 Phase; Guanine; Heat-Shock Proteins; HeLa Cells; Humans; Isoenzymes; Kinetics; L-Lactate Dehydrogenase; Protein Kinase C; Receptor Protein-Tyrosine Kinases; RNA, Messenger; RNA, Transfer; Tetradecanoylphorbol Acetate

Epidermal growth factor (EGF) induces autophosphorylation of its cognate receptor at tyrosine residues. Here we show that queuine (q), a widely distributed modified guanine analogue occurring free or as a tRNA wobble base, modulates this EGF receptor activity in vitro and in intact cells. Autophosphorylation of the immunopurified receptor from human A431 epidermoid carcinoma cells was enhanced three to fourfold in the presence of physiological concentrations of q. Using a membrane fraction of A431 cells, a twofold increase in autophosphorylation activity in the presence of q was observed, however, only when the receptor was activated by the ligand. In intact A431 cells, q enhanced the initial ligand-induced autophosphorylation of the EGF receptor three to fourfold. However, upon longer treatment of the cells with EGF in the presence of q, significantly less autophosphorylated receptor was detectable compared with stimulation of cells in the absence of q. A similar q-dependent modulation of EGF receptor autophosphorylation was observed also in human cervical carcinoma cells HeLa-S3. Treatment of q-deficient HeLa cells with EGF induced the c-fos gene expression, transiently increased the activity of the anoxic stress protein LDH k, and stimulated proliferation. Treatment of HeLa cells with EGF in the presence of q resulted in a delayed c-fos gene expression and an accelerated increase and decrease of LDH k activity. The stimulatory effect of low doses of EGF on HeLa cell proliferation was completely antagonized in the presence of q. The results suggest that the mitogenic signalling initiated by the EGF receptor is modulated by q.

Topics: Cell Line; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation; Genes, fos; Guanine; HeLa Cells; Humans; L-Lactate Dehydrogenase; Phosphorylation

Cell cultures derived from human neonatal foreskins (HF cells) are susceptible to phorbol-12,13-didecanoate- (PDD) induced inhibition of queuine uptake, but this inhibition is pronounced only in early passage HF cells. The present analysis of five different primary cultures demonstrated that, between 10 and 30 population doublings beyond the primary cultures, HF cells gradually became refractile to PDD-induced inhibition of queuine uptake, after which PDD begins to stimulate queuine uptake. Treating late passage HF cells with conditioned medium from early passage HF cells partially restored the PDD-induced inhibition of queuine uptake. This indicates the existence of a factor produced by early passage HF cells that permits PDD to inhibit queuine uptake. The tumor promoter, teleocidin, mimics the effects of PDD on queuine uptake. Both PDD and teleocidin are known to activate protein kinase C; therefore, this kinase may be an intermediary in tumor promoter-induced effects on queuine uptake. Epidermal growth factor, platelet-derived growth factor, and transforming growth factor beta stimulated queuine uptake in both early and late passage HF cells. Growth factor stimulation of uptake was enhanced by PDD in late passage cells but inhibited by PDD in early passage cells. Polyinosinic polycytidylic acid treatment of late passage HF cells partially restored PDD-induced inhibition of queuine uptake. Human recombinant beta-interferon, plus or minus PDD, had no effect on queuine uptake. PDD did not inhibit queuine uptake in the immortal human and non-human cell lines examined.

Topics: Cells, Cultured; Epidermal Growth Factor; Fibroblasts; Guanine; Humans; Kinetics; Lyngbya Toxins; Peptides; Phorbol Esters; Platelet-Derived Growth Factor; Poly I-C; Transforming Growth Factors