Glucocorticoids antagonize AP-1 by inhibiting the Activation/phosphorylation of JNK without affecting its subcellular distribution.

The immunosuppressive and antiinflammatory actions of glucocorticoid hormones are mediated by their transrepression of activating protein-1 (AP-1) and nuclear factor-kappa B (NFkappaB) transcription factors. Inhibition of the c-Jun NH(2)-terminal kinase (JNK) signaling pathway, the main mediator of AP-1 activation, has been described in extracts of hormone-treated cells. Here, we show by confocal laser microscopy, enzymatic assays, and immunoblotting that the synthetic glucocorticoid dexamethasone inhibited tumor necrosis factor alpha (TNF-alpha)-induced phosphorylation and activation of JNK in the cytoplasm and nucleus of intact HeLa cells. As a result, c-Jun NH(2)-terminal domain phosphorylation and induction were impaired. Dexamethasone did not block the TNF-alpha-induced JNK nuclear translocation, but rather induced, per se, nuclear accumulation of the enzyme. Consistently with previous findings, a glucocorticoid receptor mutant (GRdim), which is deficient in dimerization, DNA binding, and transactivation, but retains AP-1 transrepressing activity, was as efficient as wild-type GR in mediating the same effects of dexamethasone on JNK in transfected Cos-7 cells. Our results show that glucocorticoids antagonize the TNF-alpha-induced activation of AP-1 by causing the accumulation of inactive JNK without affecting its subcellular distribution.

M-phase-specific phosphorylation of the POU transcription factor GHF-1 by a cell cycle-regulated protein kinase inhibits DNA binding.

GHF-1 is a member of the POU family of homeodomain proteins. It is a cell-type-specific transcription factor responsible for determination and expansion of growth hormone (GH)- and prolactin-expressing cells in the anterior pituitary. It was previously suggested that cyclic AMP (cAMP)-responsive protein kinase A (PKA) phosphorylates GHF-1 at a site within the N-terminal arm of its homeodomain, thereby inhibiting its binding to the GH promoter. These results, however, are inconsistent with the physiological stimulation of GH production by the cAMP pathway. As reported here, cAMP agonists and PKA do not inhibit GHF-1 activity in living cells and although they stimulate the phosphorylation of GHF-1, the inhibitory phosphoacceptor site within the homeodomain is not affected. Instead, this site, Thr-220, is subject to M-phase-specific phosphorylation. As a result, GHF-1 DNA binding activity is transiently inhibited during the M phase. This activity is regained once cells enter G1, a phase during which GHF-1 phosphorylation is minimal. Thr-220 of GHF-1 is the homolog of the mitotic phosphoacceptor site responsible for the M-phase-specific inhibition of Oct-1 DNA binding Ser-382. As this site is conserved in all POU proteins, it appears that all members of this group are similarly regulated. A specific kinase activity distinct in its substrate specificity and susceptibility to inhibitors from the Cdc2 mitotic kinase or PKA was identified in extracts of mitotic cells. This novel activity could be involved in regulating the DNA binding activity of all POU proteins in a cell cycle-dependent manner.

UV irradiation induces the murine urokinase-type plasminogen activator gene via the c-Jun N-terminal kinase signaling pathway: requirement of an AP1 enhancer element.

UV irradiation leads to severe damage, such as cutaneous inflammation, immunosuppression, and cancer, but it also results in a gene induction protective response termed the UV response. The signal triggering the UV response was thought to originate from DNA damage; recent findings, however, have shown that it is initiated at or near the cell membrane and transmitted via cytoplasmic kinase cascades to induce gene transcription. Urokinase-type plasminogen activator (uPA) was the first protein shown to be UV inducible in xeroderma pigmentosum DNA repair-deficient human cells. However, the underlying molecular mechanisms responsible for the induction were not elucidated. We have found that the endogenous murine uPA gene product is transcriptionally upregulated by UV in NIH 3T3 fibroblast and F9 teratocarcinoma cells. This induction required an activator protein 1 (AP1) enhancer element located at -2.4 kb, since deletion of this site abrogated the induction. We analyzed the contribution of the three different types of UV-inducible mitogen-activated protein (MAP) kinases (ERK, JNK/SAPK, and p38) to the activation of the murine uPA promoter by UV. MEKK1, a specific JNK activator, induced transcription from the uPA promoter in the absence of UV treatment, whereas coexpression of catalytically inactive MEKK1(K432M) and of cytoplasmic JNK inhibitor JIP-1 inhibited UV-induced uPA transcriptional activity. In contrast, neither dominant negative MKK6 (or SB203580) nor PD98059, which specifically inhibit p38 and ERK MAP kinase pathways, respectively, could abrogate the UV-induced effect. Moreover, our results indicated that wild-type N-terminal c-Jun, but not mutated c-Jun (Ala-63/73), was able to mediate UV-induced uPA transcriptional activity. Taken together, we show for the first time that kinases of the JNK family can activate the uPA promoter. This activation links external UV stimulation and AP1-dependent uPA transcription, providing a transcription-coupled signal transduction pathway for the induction of the murine uPA gene by UV.

Structure and characterization of the gene encoding the ferredoxin-NADP reductase-binding protein from Zea mays L.

A genomic clone encoding ferredoxin-NADP reductase binding protein (BP) from Zea mays L. was sequenced and characterized. The promoter region (692 bp) shows several motives resembling those involved in enhancement, tissue-specific expression and light regulation in plants, besides the typical TATA and CAAT boxes. The coding sequence is interrupted by two introns. The deduced amino acid (aa) sequence corresponds to 22.85 kDa for the precursor polypeptide, including a transit peptide of 64 aa and a mature protein of 148 aa.

A pair of genes coding for lipid-transfer proteins in Sorghum vulgare.

Approximately five genes coding for lipid-transfer proteins (LTP) can be detected in Sorghum vulgare by DNA blots using a specific genomic probe. Two of these genes have been identified and sequenced. The two genes (ltp1 and ltp2) code for very similar (91.8% identity) proteins, they are separated by approx. 4 kb of DNA and their open reading frames may be read in the same direction. The gene (ltp1) located upstream has an intron placed in the same position already described for other ltp in maize and rice. Gene ltp2 has no intron. cDNAs corresponding to ltp1 have been identified in a 6-day-old plantlet library, but not for ltp2. The results of the comparison between the two sequences indicate the presence of a gap between the two genes in their promoter region. LTP seem to be coded for in plants by a small family of genes. At least in sorghum, two of its components are tightly clustered in the same genomic region.

Hormone-activated nuclear receptors inhibit the stimulation of the JNK and ERK signalling pathways in endothelial cells.

Glucocorticoid hormones, retinoids, and vitamin D3 display anti-angiogenic activity in tumor-bearing animals. However, despite their in vivo effect on the tumor vasculature little is known about their mechanism of action. Here we show that the synthetic glucocorticoid dexamethasone (Dex) and retinoic acid (RA) inhibit the activation of c-Jun N-terminal kinase (JNK) and extracellular-regulated kinase (ERK) signalling pathways by the pro-angiogenic agents tumor necrosis factor and vascular endothelial growth factor in endothelial cells. In contrast, Dex and RA failed to inhibit the activation of the p38 mitogen-activated protein kinase cascade. As a number of pro-angiogenic factors activate AP-1 transcription factor via the JNK and ERK pathways, our results suggest that the antagonism with AP-1 may underlie at least partially the anti-angiogenic effect of glucocorticoids and retinoids.

Aspects related to mevalonate biosynthesis in plants.

We purified and characterized a membrane-associated enzyme system from radish (Raphanus sativus L.) that is capable of converting acetyl-CoA into 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA). The enzyme system apparently comprises acetoacetyl-CoA thiolase (EC 2.3.1.9) and HMG-CoA synthase (EC 4.1.3.5). Its activity in vitro can be strongly stimulated by FeII. When ferrous ions are applied chelated with ethylenediaminetetraacetate, citrate or adenosine 5-triphosphate (ATP), the stimulation is further increased. Stimulation is due to a higher catalytic efficiency as indicated by an increase in Vmax, whereas the affinity of the enzyme towards acetyl-CoA remains constant (Km = 6 micro M). A considerable portion of HMG-CoA lyase activity is associated with the same membranes. HMG-CoA lyase (EC 4.1.3.4) is also solubilized and partially co-purified. Its activity requires comparatively high concentrations of Mg2+. The conversion of HMG-CoA to mevalonic acid is catalyzed by HMG-CoA reductase (EC 1.1.1.34) that is associated with the same membranes. By cDNA encoding the Arabidopsis HMG-CoA reductase, we isolated a corresponding gene from a cDNA library newly established from etiolated radish seedlings. This full-length cDNA, referred to as lambda cRS3, encodes a polypeptide 583 amino acids with a molecular mass of about 63 kDa. The hydropathy profile suggests the presence of two hydrophobic membrane-spanning domains within the N-terminal 165 amino acids. The carboxy-terminal part, where the catalytic site resides, is highly conserved in all eukaryotic HMG-CoA reductase genes sequenced so far.

p53-dependent apoptosis in the absence of transcriptional activation of p53-target genes.

The tumour suppressor p53 is required to induce programmed cell death (apoptosis) by DNA-damaging agents. As p53 is a transcriptional activator that mediates gene induction after DNA damage, it has been proposed to be a genetic switch that activates apoptosis-mediator genes. Here we evaluate the role of p53 in DNA-damage-induced apoptosis by establishing derivatives of GHFT1 cells, that are somatotropic progenitors immortalized by expression of SV40 T-antigen, which express a temperature-sensitive p53 mutant. In these cells induction of apoptosis by DNA damage depends strictly on p53 function. A shift to the permissive temperature triggers apoptosis following DNA damage, but this is independent of new RNA or protein synthesis. The extent of apoptotic DNA cleavage is directly proportional to the period during which p53 is functional. These results do not support the proposal that p53 is an activator of apoptosis-mediator genes but rather indicate that p53 either represses genes necessary for cell survival or is a component of the enzymatic machinery for apoptotic cleavage or repair of DNA.

Reaction of 5'-p-fluorosulfonylbenzoyladenosine with the catalytic and AMP allosteric sites of microsomal HMG-CoA reductase kinase.

The nucleotide analogue 5-p-fluorosulfonylbenzoyladenosine reacts with rat liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase kinase, causing a rapid loss of the AMP activation capacity and a slower inactivation of the catalytic activity. The rate constant for loss of AMP activation is eleven times higher (K1 = 0.107 min-1) than the rate constant for inactivation (K2 = 0.0094 min-1). Mg-ATP protects preferentially against inactivation, while Mg-AMP at a low concentration (7.5/0.05 mM) protects preferentially against loss of the AMP activation capacity. Oppositely, Mg-ADP at a low concentration (7.5/0.05 mM) hardly protects against loss of AMP activation capacity. We conclude that microsomal reductase kinase has distinct sites for activation and catalysis.

Allosteric activation of rat liver microsomal [hydroxymethylglutaryl-CoA reductase (NADPH)]kinase by nucleoside phosphates.

Microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase kinase activity is enhanced about 5 fold by 2 mM of either AMP or ADP. Activation constants, Ka, for AMP and ADP are 17 microM and 430 microM respectively, showing that AMP is a more potent activator than ADP. This property is expressed by increasing not only the rate of reductase inactivation but also the rate of reductase phosphorylation from [gamma-32P]ATP. GTP can replace ATP as substrate of reductase kinase but GMP and GDP cannot replace AMP as activators. Kinetic studies show that ATP can only act as a substrate. Nucleoside mono or diphosphates and nucleoside triphosphates, thus, appear to bind to different sites on microsomal HMG-CoA reductase kinase. Nucleoside mono or diphosphates act as allosteric activators of reductase kinase. The adenosyl moiety and the unaltered phosphate ester at the 5' position are two essential features of the activator molecule. Phosphorylation of reductase either by microsomal or cytosolic AMP-activated reductase kinase produces an 80% inactivation, with a concomitant incorporation of 0.8 mol of 32P per mol of reductase (Mr 55,000). In both cases exhaustive tryptic digestion of 32P-labeled HMG-CoA reductase, which had been denatured in 2M urea, yields two major phosphopeptides, the phosphoryl group being bound to serine residues.

Activation of rat liver cytosolic 3-hydroxy-3-methylglutaryl coenzyme A reductase kinase by adenosine 5'-monophosphate.

Inactivation of 3-hydroxy-3-methylglutaryl Coenzyme A reductase by reductase kinase and ATP-Mg needs either ADP or 5'-AMP as cofactors. 5'-AMP is a more potent activator of cytosolic reductase kinase than ADP. This capacity is expressed by increasing not only the rate of reductase inactivation, but also the rate of reductase phosphorylation from [gamma-32P]ATP. Activation constants, Ka, for 5'-AMP and ADP are 20 microM and 420 microM respectively. Neither 3'-AMP nor 2'-AMP activate reductase kinase. Other nucleoside monophosphates like UMP, CMP and GMP cannot replace 5'-AMP as activators of reductase kinase.

Affinity labeling of the catalytic and AMP allosteric sites of 3-hydroxy-3-methylglutaryl-coenzyme A reductase kinase by 5'-p-fluorosulfonylbenzoyladenosine.

The nucleotide analogue 5'-p-fluorosulfonylbenzoyladenosine (FSBA) reacts irreversibly with rat liver cytosolic 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase kinase, causing a rapid loss of the AMP activation capacity and a slower inactivation of the catalytic activity. The rate constant for loss of AMP activation is about 10 times higher (kappa 1 = 0.112 min-1) than the rate constant of inactivation (kappa 2 = 0.0106 min-1). There is a good correspondence between the time-dependent inactivation of reductase kinase and the time-dependent incorporation of 5'-p-sulfonylbenzoyl[14C]adenosine ([14C]SBA). An average of 1.65 mol of reagent/mol of enzyme subunit is bound when reductase kinase is completely inactivated. The time-dependent incorporation is consistent with the postulate that covalent reaction of 1 mol of SBA/mol of subunit causes complete loss of AMP activation, whereas reaction of another mole of SBA/mol of subunit would lead to total inactivation. Protection against inactivation by the reagent is provided by the addition of Mg2+, AMP, Mg-ATP, or Mg-AMP to the incubation mixtures. In contrast, addition of ATP, 2'-AMP, or 3'-AMP has no effect on the rate constants. Mg-ATP protects preferentially the catalytic site against inactivation, whereas Mg-AMP at low concentration protects preferentially the allosteric site. Mg-ADP affords less protection than Mg-AMP to the allosteric site when both nucleotides are present at a concentration of 50 microM with 7.5 mM Mg2+. Experiments done with [14C]FSBA in the presence of some protectants have shown that a close correlation exists between the pattern of protection observed and the binding of [14C]SBA. The postulate is that there exists a catalytic site and an allosteric site in the reductase kinase subunit and that Mg-AMP is the main allosteric activator of the enzyme.

Nuclear hormone receptor antagonism with AP-1 by inhibition of the JNK pathway.

The activity of c-Jun, the major component of the transcription factor AP-1, is potentiated by amino-terminal phosphorylation on serines 63 and 73 (Ser-63/73). This phosphorylation is mediated by the Jun amino-terminal kinase (JNK) and required to recruit the transcriptional coactivator CREB-binding protein (CBP). AP-1 function is antagonized by activated members of the steroid/thyroid hormone receptor superfamily. Recently, a competition for CBP has been proposed as a mechanism for this antagonism. Here we present evidence that hormone-activated nuclear receptors prevent c-Jun phosphorylation on Ser-63/73 and, consequently, AP-1 activation, by blocking the induction of the JNK signaling cascade. Consistently, nuclear receptors also antagonize other JNK-activated transcription factors such as Elk-1 and ATF-2. Interference with the JNK signaling pathway represents a novel mechanism by which nuclear hormone receptors antagonize AP-1. This mechanism is based on the blockade of the AP-1 activation step, which is a requisite to interact with CBP. In addition to acting directly on gene transcription, regulation of the JNK cascade activity constitutes an alternative mode whereby steroids and retinoids may control cell fate and conduct their pharmacological actions as immunosupressive, anti-inflammatory, and antineoplastic agents.

Glucocorticoid-induced apoptosis of human leukemic cells is caused by the repressive function of the glucocorticoid receptor.

Induction of apoptosis in lymphocytes, which may account for the therapeutic effects of glucocorticoids in various diseases including leukemia, depends on the glucocorticoid receptor. However, the events leading from the activated receptor to cell lysis are not understood. A prevailing hypothesis postulates induction of so-called 'lysis genes' by the activated receptor. In this study, we show that an activation-deficient glucocorticoid receptor mutant is as effective as the wild-type receptor in repression of AP-1 activity, inhibition of interleukin-2 production, inhibition of c-myc expression and induction of apoptosis. Furthermore, we show that retinoic acid can also induce apoptosis in these cells through the retinoic acid receptor, whose repressive functions but not target site specificity, are similar to those of the glucocorticoid receptor. Therefore, the primary effect of the receptor in glucocorticoid-mediated apoptosis correlates with transcriptional repression rather than activation and could be mediated by interference with other transcription factors required for cell survival.

Transactivation by NF-IL6/LAP is enhanced by phosphorylation of its activation domain.

One of the members of the bZIP family of transcriptional activators is NF-IL6/LAP (IL-6 DBP, C/EBP beta, CRP2). NF-IL6/LAP protein is highly expressed in liver nuclei, where it has been implicated as a master regulator of the acute-phase response, induced by interleukin-6 (IL-6) and other inflammatory mediators. Also, NF-IL6/LAP is involved in the activation of the IL-6 promoter in response to IL-1 and bacterial lipopolysaccharide. The control of NF-IL6/LAP expression and activity is complex and poorly understood. Under some conditions the NF-IL6/LAP gene is transcriptionally activated by IL-1 and lipopolysaccharide, whereas in other instances, its binding to cognate DNA sequences is enhanced by cytokines. Additionally, the ability of constitutively expressed NF-IL6/LAP to activate transcription is strongly augmented by IL-6, through an unknown signalling pathway. We now show that stimulation of the protein kinase C pathway increases the phosphorylation of Ser 105 within the activation domain of NF-IL6/LAP, and enhances its transcriptional efficacy.

Arabidopsis thaliana contains two differentially expressed 3-hydroxy-3-methylglutaryl-CoA reductase genes, which encode microsomal forms of the enzyme.

The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR; EC 1.1.1.34) catalyzes the first rate-limiting step in plant isoprenoid biosynthesis. Arabidopsis thaliana contains two genes, HMG1 and HMG2, that encode HMGR. We have cloned these two genes and analyzed their structure and expression. HMG1 and HMG2 consist of four exons and three small introns that interrupt the coding sequence at equivalent positions. The two genes share sequence similarity in the coding regions but not in the 5'- or 3'-flanking regions. HMG1 mRNA is detected in all tissues, whereas the presence of HMG2 mRNA is restricted to young seedlings, roots, and inflorescences. The similarity between the two encoded proteins (HMGR1 and HMGR2) is restricted to the regions corresponding to the membrane and the catalytic domains. Arabidopsis HMGR2 represents a divergent form of the enzyme that has no counterpart among plant HMGRs characterized so far. By using a coupled in vitro transcription-translation assay, we show that both HMGR1 and HMGR2 are cotranslationally inserted into endoplasmic reticulum-derived microsomal membranes. Our results suggest that the endoplasmic reticulum is the only cell compartment for the targeting of HMGR in Arabidopsis and support the hypothesis that in higher plants the formation of mevalonate occurs solely in the cytosol.

Isolation and structural characterization of a cDNA encoding Arabidopsis thaliana 3-hydroxy-3-methylglutaryl coenzyme A reductase.

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) catalyses the synthesis of mevalonate, the specific precursor of all isoprenoid compounds present in plants. We have characterized two overlapping cDNA clones that encompass the entire transcription unit of an HMG-CoA reductase gene from Arabidopsis thaliana. The transcription product has an upstream non-coding sequence of 70 nucleotides preceding an open reading frame of 1776 bases and a 3' untranslated region in which two alternative polyadenylation sites have been found. The analysis of the nucleotide sequence reveals that the cDNA encodes a polypeptide of 592 residues with a molecular mass of 63,605 Da. The hydropathy profile of the protein indicates the presence of two highly hydrophobic domains near the N-terminus. A sequence of 407 amino acids corresponding to the C-terminal part of the protein (residues 172-579), which presumably contains the catalytic site, shows a high level of similarity to the region containing the catalytic site of the hamster, human, yeast and Drosophila enzymes. The N-terminal domain contains two putative membrane-spanning regions, in contrast to the enzyme from other organisms which has seven trans-membrane regions. A. thaliana contains two different HMG-CoA reductase genes (HMG1 and HMG2), as estimated by gene cloning and Southern blot analysis. Northern blot analysis reveals a single transcript of 2.4 kb in leaves and seedlings, which presumably corresponds to the expression of the HMG1 gene.

v-erbA oncogene induces invasiveness and anchorage-independent growth in cultured glial cells by mechanisms involving platelet-derived growth factor.

The v-erbA oncogene coding for a mutated form of the thyroid hormone (T3) receptor (TR alpha 1) increased the invasion capacity of the mouse B3.1 glial cell line. This effect was mediated by the induction of platelet-derived growth factor (c-sis/PDGF B), as shown by its inhibition using an anti-PDGF BB antibody. Also, the low invasion capacity of parental B3.1 and c-erbA-expressing cells (B3.1 + TR alpha 1) was enhanced by exogenously added PDGF BB. This effect was independent of the growth-promoting activity of PDGF and unrelated to the secretion of metalloproteinases. All three cell types (parental B3.1, B3.1 + v-erbA, and B3.1 + TR alpha 1) secreted similar high levels of the M(r) 72,000 collagenase IV (A) independently of PDGF. Anchorage-independent cell growth was also enhanced by v-erbA; B3.1 + v-erbA cells but neither parental B3.1 nor B3.1 + TR alpha 1 cells formed foci in soft agar. The effect of v-erbA only happened in the presence of serum, suggesting that some serum factor(s) cooperate with PDGF to overcome the anchorage dependence of B3.1 + v-erbA cells. Supporting this, high doses of exogenous PDGF were much less efficient than serum, and the addition of an anti-PDGF BB antibody blocked only partially the effect of serum. Basic fibroblast growth factor was found to cooperate with PDGF to abolish anchorage dependence. Moreover, B3.1 + v-erbA cells detached and grew in suspension when cultured on plastic dishes. Interestingly, the transformation-competent c-jun and fra-1 oncogenes were induced by v-erbA in serum-free medium and are candidates to mediate v-erbA effects. In summary, our results show that v-erbA induces transformation parameters in the glial B3.1 cell line via an increase in c-sis/PDGF B and probably other mechanisms, suggesting a role for (autocrine) PDGF stimulation in glial cell transformation.

Phosphorylation of Ser2078 modulates the Notch2 function in 32D cell differentiation.

Notch signaling is involved in the regulation of many cell fate determination events in both embryonic development and adult tissue homeostasis. We previously demonstrated that Notch1 and Notch2 molecules inhibit myeloid differentiation in a cytokine-specific manner and that the Notch cytokine response domain is necessary for this functional specificity. We have now investigated the putative role of phosphorylation in the activity of Notch in response to cytokine signals. Our results show that the granulocyte colony-stimulating factor (G-CSF) stimulation of 32D cells expressing the intracellular Notch2 protein induces phosphorylation at specific sites of this molecule, rendering the molecule inactive and permitting differentiation of these cells. In contrast, when cells are stimulated with granulocyte macrophage colony-stimulating factor (GM-CSF), intracellular notch2 is not phosphorylated at these residues and differentiation is inhibited. We also show that deletion of the Ser/Thr-rich region between amino acids 2067 and 2099 abrogates G-CSF-induced phosphorylation and results in a molecule that inhibits differentiation in response to either G-CSF or GM-CSF. Our results further indicate that Ser(2078) is a critical residue for phosphorylation and modulation of Notch2 activity in the context of G-CSF-induced differentiation of 32D cells.