Janice Saxton

Ternary complex factors: prime nuclear targets for mitogen-activated protein kinases

Ternary complex factors (TCFs), a subgroup of the ETS protein family, were first described in the context of c-fos gene regulation. Subsequently, their early identification as nuclear targets for mitogen-activated protein kinases served to exemplify the fundamental links in eukaryotic cells between growth factor-mediated signalling pathways and gene control. This article provides an overview of recent work on ternary complex factors, addressing their expression and molecular structure, as well as how selective interactions with members of other protein families serve to up-1 regulate or restrict their activity. Although only one genetic study on ternary complex factors has been published to date, unravelling of the underlying molecular events provides a basis for tentative predictions about their biological roles in mammalian organisms.

Mitogen-induced recruitment of ERK and MSK to SRE promoter complexes by ternary complex factor Elk-1.

Many eukaryotic genes are acutely regulated by extra-cellular signals. The c-fos serum response element (SRE) mediates transcriptional activation in response to mitogens through serum response factor (SRF)-dependent recruitment of Elk-1, a mitogen-activated protein kinase (MAPK)-responsive transcription factor. How subsequent events at SRE promoters stimulate initiation of transcription has yet to be fully resolved. Here we show that extra-cellular signal-regulated kinase (ERK) and mitogen and stress-activated kinase (MSK) are recruited to SRE promoter complexes in vitro and in vivo. Their recruitment in vitro correlates with Elk-1 binding and for ERK the D domain/KIM of Elk-1 is specifically involved. In vivo, recruitment of ERK and MSK is stimulated by mitogens, correlates with histone H3 phosphorylation and is impaired by Elk-1 knockdown. Immunocytochemistry and confocal microscopy reveal that ERK appears to associate to some extent with initiating rather than elongating RNA polymerase II. Taken together, our data add to the body of evidence implying that ERK and related MAPKs may fulfil a generic role at the promoters of acutely regulated genes.

Purification and partial characterization of the high and low molecular weight form (S- and F-form) of invertase secreted by Aspergillus nidulans.

Two forms of secreted invertase have been purified from Aspergillus nidulans by ion-exchange and gel-filtration chromatography. S-invertase gave a single, broad, glycoprotein band on PAGE and SDS-PAGE corresponding in size to 185 and 78 kDa, respectively, compared with 94 and 110 kDa for F-invertase. The carbohydrate of S-invertase contained mainly mannose (14%) and less galactose (5%) whereas the F-form yielded mainly galactose (29%) and less mannose (12%). Three sharp bands of enzymically active glycoprotein for both the S-form (pI 4.9-5.2) and the F-form (pI 3-4.2) were observed after isoelectric focusing. Deglycosylation with Endo H simplified this pattern to one enzymically active protein band (pI 5.2). The aglycoenzymes gave narrow bands on PAGE and SDS-PAGE corresponding to 115 kDa and 60 kDa respectively for both S- and F-forms. The specific activity of S-invertase was three-fold higher than that of F-invertase both before and after deglycosylation. The Km values of the two forms of invertase were very similar. Significant homology existed between the N-terminal amino-acid sequences of S-invertase (and of internal peptides derived from it) and sequences of invertase from other species. It is suggested that the higher carbohydrate content in F-invertase results in the native enzyme existing as a monomer and having a greater negative charge and lower specific enzyme activity compared with the dimeric S-enzyme.

Possible relationships between changes in body weight set-point and stress metabolism after treating rats chronically with D-fenfluramine: effects of feeding rats acutely with fructose on the metabolism of corticosterone, glucose, fatty acids, glycerol and triacylglycerol

Rats were maintained on a corn oil diet and treated with D-fenfluramine at doses of 2.5 mg/kg twice a day for 11 days or with 10 mg or 25 mg/kg once a day for 12 days. The lower dose of D-fenfluramine produced no marked changes in body weight and after 11 days of treatment the weights of the rats on average were only 2% lower than the controls. The food intake of these rats was only decreased on the first day. The two higher doses of D-fenfluramine decreased the food consumption for about 3 days but thereafter it was similar to that of the control rats. The body weight of these rats fell on the first day, but after about four days the gain in body weight paralleled rather than approached that of the control rats. Increasing the dose of D-fenfluramine progressively decreased the relative size of the epididymal fat pad. At the end of the treatment period the rats were fed acutely with fructose to increase the circulating concentrations of corticosterone and to stimulate triacylglycerol synthesis. All three doses of D-fenfluramine decreased the concentration of circulating triacylglycerol after fructose feeding. The 10 mg/kg dose also decreased the basal concentration of triacylglycerol. The two higher doses of fenfluramine decreased the rises in the circulating concentrations of corticosterone, glycerol and fatty acids that are produced by fructose feeding. The basal concentrations of these compounds in the absence of fructose feeding were not significantly affected by the 10 mg/kg dose of D-fenfluramine. The possible relationship between the effect of chronic treatment with D-fenfluramine in decreasing a metabolic stress response and lipolysis is discussed relative to its hypotriglyceridaemic action and its effect on body weight-set point. The results demonstrate that D-fenfluramine produced persistent changes in metabolism at a time when the treated rats were growing at the same rate as the control rats and when they were eating similar quantities of food.

Effects of chronic administration of benfluorex to rats on the metabolism of corticosterone, glucose, triacylglycerols, glycerol and fatty acid

(1) Rats were fed on diets enriched with sucrose, beef tallow or corn oil and treated for 11-16 days with 50 mg of benfluorex per kg of body weight. By these times the growth rate and food intake were not significantly different from those of control rats. (2) Benfluorex approximately halved the concentration of circulating triacylglycerol in rats fed the beef tallow or sucrose diets. (3) It did not significantly alter the total lipoprotein lipase activity in diaphragm, heart and adipose tissue. (4) The clearance of triacylglycerols from chylomicrons exhibited two t 1/2 values of about 0.6 and 6.9 min in rats fed the beef tallow diet. Benfluorex did not significantly alter these values. (5) Benfluorex did not significantly alter the rate of appearance of triacylglycerol in the blood of rats injected with Triton WR 1339 to block triacylglycerol uptake. It did, however, decrease the rise in circulating glucose which presumably resulted from the stress of the procedure. (6) Benfluorex decreased the extent and duration of the rise in serum corticosterone when rats maintained on the corn oil diet were fed acutely with fructose. It also decreased the circulating concentrations of glycerol, triacylglycerol and glucose after fructose feeding. (7) Rats fed on the corn oil diet and then treated with benfluorex had lower concentrations of circulating glucose, triacylglycerol, glycerol and fatty acids after being injected with 2-deoxyglucose. (8) It is proposed that some of the long-term hypoglycaemic and hypotriglyceridaemic effects of benfluorex could be mediated indirectly through changes in endocrine balance, perhaps via the serotonergic system and in particular, by decreasing the effects of stress hormones relative to insulin. The implications of these findings are discussed in relation to controlling metabolism in stress conditions and for the management of obesity, diabetes and atherosclerosis.

Dimer formation and conformational flexibility ensure cytoplasmic stability and nuclear accumulation of Elk-1

The ETS (E26) protein Elk-1 serves as a paradigm for mitogen-responsive transcription factors. It is multiply phosphorylated by mitogen-activated protein kinases (MAPKs), which it recruits into pre-initiation complexes on target gene promoters. However, events preparatory to Elk-1 phosphorylation are less well understood. Here, we identify two novel, functional elements in Elk-1 that determine its stability and nuclear accumulation. One element corresponds to a dimerization interface in the ETS domain and the second is a cryptic degron adjacent to the serum response factor (SRF)-interaction domain that marks dimerization-defective Elk-1 for rapid degradation by the ubiquitin–proteasome system. Dimerization appears to be crucial for Elk-1 stability only in the cytoplasm, as latent Elk-1 accumulates in the nucleus and interacts dynamically with DNA as a monomer. These findings define a novel role for the ETS domain of Elk-1 and demonstrate that nuclear accumulation of Elk-1 involves conformational flexibility prior to its phosphorylation by MAPKs.

Effects of ulapualide A and synthetic macrolide analogues on actin dynamics and gene regulation.

Abstract.Several marine macrolide toxins act as potent and specific actin-severing molecules. Recent elucidation of their stereochemistries and modes of interaction with actin has allowed the syntheses of bioactive analogues. Here we used synthetic analogues in a structure-function analysis of ulapualide A, a trisoxazole-based macrolide. Ulapualide A harboured potent actin-depolymerising activity both in cells and in vitro. Its synthetic diastereoisomer was three orders of magnitude less active than the natural toxin and synthetic macrolide fragments lacked actin-capping/ severing activity altogether. Modulation of serum response factor (SRF)-dependent gene expression, as described for other actin-binding toxins, was also examined. Specific changes in response to ulapualide A were not observed, primarily due to its profound effects on cytoskeletal integrity and cell adhesion. Several synthetic fragments of ulapualide A also had no effect on SRF-dependent gene expression. However, inhibition was observed with a molecule corresponding to the extended aliphatic side chain of halichondramide, a structurally related macrolide. These findings indicate that side-chain derivatives of trisoxazole-based macrolides may serve to uncouple gene-regulatory events from actin dynamics.

Comparison of theN-glycoloylneuraminic andN-acetylneuraminic acid content of platelets and their precursors using high performance anion exchange chromatography

N-Acetylneuraminic acid (Neu5Ac) andN-glycoloylneuraminic acid (Neu5Gc) are distributed widely in nature. Using a Carbopac PA-1 anion exchange column, we have determined the ratios of Neu5Ac and Neu5Gc in hydrolysates of platelets and their precursors: a rat promegakaryoblastic (RPM) cell line and a human megakaryoblastic leukemia cell line (MEG-01). The ratio of Neu5Gc:Neu5Ac in cultured RPM cells is 16:1, whereas in platelet rich plasma and cultured MEG-01 cells it is 1:38 and 1:28, respectively. The nature of these sialic acids from RPM cells was verified using thin layer chromatography and liquid secondary ion mass spectrometry. The relevance of increased Neu5Gc levels in early stages of development is discussed.

Changes in the secretion and glycosylation of fibronectin by human skin fibroblasts associated with tuberous sclerosis

Fibroblasts from skin and skin lesions of patients with tuberous sclerosis (TS) and from skin of normal individuals were grown in culture. ELISA showed that the spent medium of those derived from TS skin lesions contained significantly more fibronectin (FN) than spent medium from the other cells. Amino acid compositional analysis of the FN from TS and normal sources revealed no substantial differences. However the FN of fibroblasts from TS-skin lesions was shown by HPAEC to contain a two- to three-fold increased content of carbohydrate. The changed monosaccharide composition was consistent with an increased content of N- and O-linked glycans and with the former containing polylactosamine chains. Fibroblasts from a normal individual were shown to proliferate more slowly and to produce larger cells when grown on FN from a TS skin lesion compared to growth on FN from normal skin.

Protein degradation in mammary gland explants evidence for limited heterogeneity of protein degradation rates

Mammary gland explants in organ culture were subjected to hormonal manipulation, and rates of protein degradation during 1 and 2 day periods were measured by a double-isotope method. Isotope ratios for protein subunits in subcellular fractions were measured after resolution by two-dimensional polyacrylamide gel electrophoresis. Frequency distribution analysis shows that isotope ratios for each fraction are grouped predominantly in exponential distributions corresponding to populations of protein subunits with different mean degradation rates. The results also suggest that heterogeneity of protein degradation rates within each population is limited. There is no consistent correlation of degradation rate with protein isoelectric point or subunit molecular weight either overall or within any population of degradation rates. Therefore, the similarity of protein degradation rates within each population is clearly related to these molecular properties of the proteins.