Herman Polet

The effects of concanavalin A and other agents on protein degradation and migration of non-histone proteins (NHP) to the nucleus in lymphocytes

Concanavalin A (conA) inhibits the degradation of [3H]leucine-labeled cellular proteins of human lymphocytes. The lectin also stimulates the migration of non-histone proteins (NHP) from the cytoplasm to the nucleus. The increased nuclear level of NHP is associated with increased cellular binding of [3H]actinomycin D [(3H]AD). Decreased protein breakdown and increased migration of NHP are parallel events, i.e. both changes occur as a function of the lectin concentration and display a similar time course, suggesting that these events could be related. Similar effects are observed with fluoride, chloroquine and iodoacetate: these agents simultaneously decrease proteolysis and increase the nuclear level of NHP, associated with increased cellular [3H]AD binding. Fractionation of the acidic NHP according to pH 2.5-6.5 shows that proteins with a high degree of degradation in unstimulated cells correspond to proteins with a high degree of migration in conA-stimulated cells. A similar correlation was observed in fluoride-treated lymphocytes. conA, fluoride and iodoacetate decrease cellular [3H]chloroquine [(3H]CQ) accumulation, indicating a lysosomotropic effect. These and previously reported data suggest, but do not prove that conA inhibits degradation of cellular proteins via the lysosomal pathway. Ammonium chloride, methylamine and sodium azide also inhibit proteolysis and increase cellular [3H]AD binding; however, their effects are weak. On the basis of these observations it appears that lysosomal degradation and migration of NHP to the nucleus are linked; however, the mechanism of the linkage is unknown.

Role of nuclear proteins on [3H]actinomycin D binding during lymphocyte mitogenesis.

Abstract The uptake of [3H]actinomycin D ([3H]AD) by ConA-stimulated lymphocytes was followed during 96 h of incubation and correlated with the level of nuclear proteins in the nucleus, DNA synthesis and the degree of AD-induced inhibition of RNA and DNA synthesis. During the first 48 h there is a parallel increase of drug binding to cells and a rising level of non-histone proteins (NHP) in the nucleus. During the next 48 h, DNA synthesis occurs, drug uptake decreases and the nuclear level of NHP continues to rise. The level of histones remains constant during 96 h. The variations in cellular [3H]AD uptake during 96 h are not due to changes in cell membrane permeability, since similar variations in drug binding are observed in isolated cell nuclei. NHP, obtained as 0.25 M NaCl extracts of cell nuclei, increase binding of [3H]AD to nuclei isolated from non-stimulated lymphocytes, while histones have no such effect. NHP extracted with phenol, after washing the nuclei with salt and acid solutions, or extracted with 0.25 M NaCl from non-stimulated and stimulated lymphocytes and Chang liver cells are equally active to bind [3H]AD to nuclei of non-stimulated lymphocytes. NHP from Chang cells, purified by DNA-cellulose chromatography using calf thymus DNA, stimulated [3H]AD binding to lymphocyte nuclei, indicating that the drug-binding activity is due to proteins binding to DNA. NHP increase binding of [3H]AD to pure DNA in the absence of histones. The degree of [3H]AD binding to ConA-stimulated lymphocytes during 96 h correlated with the degree of inhibition of RNA and DNA synthesis by AD.

Effects of fibroblastic growth factor on protein degradation, the migration of non-histone proteins to the nucleus and DNA synthesis in diploid fibroblasts

Stimulation of resting WI38 cells, prelabeled with [3H]leucine, with fibroblastic growth factor (FGF) or serum, caused increased nuclear translocation of [3H]non-histone proteins [( 3H]NHP) and DNA synthesis, and a parallel decrease of proteolysis. [3H]NHP migration was independent of protein synthesis. Fractionation of the nuclear proteins in a pH gradient of 2.5-6.5 showed that [3H]NHP fractions with high degradation rates in resting cells corresponded to the [3H]NHP fractions with high migration rates in stimulated cells, suggesting that degradation and migration of [3H]NHP are linked. FGF inhibited cellular uptake of [3H]chloroquine, suggesting that FGF inhibits NHP degradation via lysosomes. The lysosomotropic amine eserine had similar effects as FGF. It is proposed that FGF induces NHP migration to the nucleus by inhibiting their lysosomal degradation. FGF also caused migration of [3H]histones, however, the mechanism is not clear.

The effects of amino acids on protein degradation and translocation of non-histone proteins to the nucleus in lymphocytes

Tryptophan, phenylalanine and leucine have two parallel effects in cultured lymphocytes, they inhibit cellular proteolysis and increase the translocation of non-histone proteins to the nucleus. The latter is associated with an increased cellular binding of [3H]actinomycin D, indicating an altered structure of chromatin. The amino acids also inhibit the cellular uptake of [3H]chloroquine, suggesting that inhibited protein degradation is lysosomal. Several amine catabolites of tryptophan and phenylalanine, some of which are known to play a role as biogenic amines, have similar actions, and can explain, at least in part, the effects of their parent amino acids. Fractionation of the nuclear 3H-labeled non-histone proteins according to pH 2.5-6.5 shows that such proteins with a high rate of degradation in untreated cells correspond to the 3H-labeled non-histone proteins with a high rate of translocation in tryptophan treated cells. These data suggest that the degradation and the translocation of the non-histone proteins are linked and that the increased translocation of the non-histone proteins to the nucleus may be the consequence of inhibited lysosomal degradation of these proteins by the amino acids.

Epidermal growth factor stimulates DNA synthesis while inhibiting cell multiplication of A-431 carcinoma cells☆

Epidermal growth factor (EGF) has been shown to inhibit the multiplication of the human epidermoid carcinoma cell line A-431. In the present report it is shown that, despite growth inhibition, EGF caused a marked synthesis of DNA and nonhistone proteins, without progression into mitosis. This event was associated with a retraction of the monolayer into colonies of cells. This suggests that the cell cycle of A-431 cells is controlled by two surface membrane signals: one generated by EGF stimulating the synthetic events of the G1 and S phases; a second signal, leading to progression into mitosis appears either not to be generated or to be inhibited by EGF.

Effects of platelet-derived growth factor on degradation, nuclear translocation of nonhistone proteins, and DNA synthesis.

Abstract Stimulation of resting normal rat kidney fibroblasts, prelabeled with [3H]leucine, by platelet-derived growth factor (PDGF) caused inhibition of cellular protein degradation and a parallel increased nuclear translocation of 3H-labeled nonhistone proteins (3H-NHP) and DNA synthesis. Nuclear translocation of these proteins was independent of protein synthesis. Fractionation of the nuclear 3H-NHP in a pH gradient of 2.5–6.5 showed that the protein fractions with a high degree of proteolysis in resting cells corresponded to the protein fractions with a high extent of translocation in stimulated cells, suggesting that degradation and translocation of these proteins may be related. PDGF inhibited cellular uptake of [3H]chloroquine, suggesting that PDGF inhibits NHP degradation via the lysosomal pathway. These observations support the hypothesis that PDGF induces NHP translocation to the nucleus by inhibiting lysosomal degradation of these proteins.

Cytochalasin D inhibits nuclear translocation of nonhistone proteins induced by lectin and other agents in lymphocytes

Abstract Cytochalasin D (CD), known to interfere with microfilament activity, inhibits RNA and protein synthesis and the cellular uptake of [3H]actinomycin D in concanavalin A-stimulated lymphocytes. It also inhibits the nuclear translocation of nonhistone proteins (NHP) induced by the lectin. Since NHP mediate RNA and protein synthesis and [3H]actinomycin D binding, inhibited nuclear translocation of NHP can explain the inhibition of the former events. CD also inhibits nuclear translocation of NHP caused by NaF or chloroquine. The mechanism of how CD inhibits nuclear translocation of the NHP is obscure. Since NaF and chloroquine enter cells by diffusion, it would appear that CD acts on an intracellular target, rather than abrogating a cell surface-mediated signal generated by binding of the lectin to a cell surface receptor.

Effects of epidermal growth factor on protein degradation, the translocation of non-histone proteins to the nucleus and DNA synthesis

Stimulation of resting Chang liver or monkey kidney cells, prelabeled with [3H]leucine, by epidermal growth factor (EGF), caused inhibition of cellular protein degradation and a parallel increase nuclear translocation of 3H-labeled non-histone proteins and DNA synthesis. Nuclear translocation of these proteins was independent of protein synthesis. Fractionation of the nuclear 3H-labeled non-histone proteins in a pH gradient of 2.5-6.5 showed that the protein fractions with a high degree of proteolysis in resting cells corresponded to the protein fractions with a high extent of translocation in stimulated cells, suggesting that degradation and translocation of these proteins may be related. EGF inhibited cellular uptake of [3H]chloroquine, suggesting that EGF inhibits non-histone protein degradation via the lysosomal pathway. These observations support the hypothesis that EGF induces non-histone protein translocation to the nucleus by inhibiting lysosomal degradation of these proteins.

Endocytosed non-histone proteins translocate in part to the nucleus in lymphocytes

[3H]Non-histone proteins ([3H]NHP), dissolved in the culture medium, are endocytosed by lymphocytes and equilibrate rapidly between the cytoplasm and the nucleus. During incubation, the proteins are gradually degraded in the lysosomes. The lysosomotropic agents conA, NaF, eserine and atropine have two parallel effects on resting lymphocytes, after they have endocytosed [3H]NHP: inhibition of degradation and increased translocation of [3H]NHP from the cytoplasm to the nucleus. This indicates that lysosomal degradation and translocation of [3H]NHP to the nucleus are linked and suggests that this translocation may be the result of inhibited lysosomal degradation of the [3H]NHP. The behaviour of endocytosed [3H]NHP appears similar to that of endogenous [3H]NHP in cells prelabeled with [3H]leucine, when subjected to the same lysosomotropic agents, reported previously (Polet, H, Exp cell res 148 (1983) 345). This observation may provide a model to study the mechanism(s) controlling nucleo-cytoplasmic traffic of NHP.