Sterol regulatory element-binding protein (SREBP)-1a is a transcription factor sensing cellular cholesterol levels and integrating gene regulatory signals mediated by MAP kinase cascades. Here we report the identification of serine 117 in SREBP-1a as the major phosphorylation site of the MAP kinases Erk1/2. This site was identified by nanoelectrospray mass spectrometry and peptide sequencing of recombinant fusion proteins phosphorylated by Erk1/2 in vitro. Serine 117 was verified as the major phosphorylation site by in vitro mutagenesis. Mutation of serine 117 to alanine abolished Erk2-mediated phosphorylation in vitro and the MAP kinase-related transcriptional activation of SREBP-1a by insulin and platelet-derived growth factor in vivo. Our data indicate that the MAP kinase-mediated effects on SREBP-1a-regulated target genes are linked to this phosphorylation site.Protein phosphorylation at serine and threonine residues is a key regulatory mechanism controlling proteins regulating metabolism, growth, differentiation, apoptosis, and gene expression of cells. One major class of serine/threonine kinases mediating signal transduction of various extracellular stimuli, including insulin and growth factors, are mitogen-activated protein kinases (MAPK) 1 (for review, see Ref. 1). Pathways involving MAPK consist of three kinases, i.e. MAPK kinase kinase (MKKK), MAPK kinase (MKK), and MAPK, which are sequentially activated. The activity of MAPK is stimulated by MKK-mediated dual phosphorylation (Thr-X-Tyr) in the activation loop. MKK is regulated by the serine/threonine kinase MKKK, which is linked by protein-protein interaction, phosphorylation, or subcellular relocalization to extracellular stimuli at the cell surface, similar to the receptor-associated tyrosine kinases of insulin and growth factors. Different MAPK cascades have been identified, but the best characterized in mammalian cells is the extracellular signal-regulated kinase (Erk) pathway leading to the activation of the MAPK isoforms Erk1 and Erk2. The majority of identified MAPK substrates are transcription factors regulating the expression of many genes (2). Transcription factors phosphorylated by activated Erk1/Erk2 are involved in hormone action (e.g. estrogen and glucocorticoid receptors), cell growth, and differentiation (e.g. Elk-1, Ets1, Sap-1a, c-Myc, STATs).Sterol-regulatory element binding proteins (called SREBP1a, SREBP-1c, and SREBP-2) are transcription factors that appear to transmit the signal of membrane-embedded cholesterol levels to the nucleus regulating the expression rate of multiple genes (3, 4). Recently, evidence is accumulating that SREBPs are not only involved in cholesterol-regulated events but are also gene regulatory targets of intracellular signaling pathways, e.g. MAP kinase cascades. In accordance with this hypothesis, we have previously shown that the effects of insulin and PDGF on LDL receptor promoter activity are abolished by a MAP kinase cascade inhibitor and are mediated via the SREBP-binding cis-element sre-1 (5, 6). Ov...

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The transcription factor sterol regulatory element binding protein (SREBP)-2 plays a pivotal role in lipid metabolism. Previously, we have shown that the mature form of SREBP-2 is a substrate of Erk-mitogen-activated protein kinases (MAPK). The aim of the present study was to identify Erk-specific phosphorylation sites. Using a protein chemistry approach, we could identify Ser-432 and Ser-455 as major phosphorylation sites. Further characterization by electrophoretic mobility shift assay and promoter reporter gene analyses revealed that phosphorylation does not influence protein/DNA interaction, but enhances trans-activity. In intact cells, SREBP-2 is phosphorylated by insulin, which seems to be related to their bio-responses on low density lipoprotein receptor activity. These results suggest that activation of Erk-MAPK pathways by hormones such as insulin might be related to a novel regulatory principle of SREBP-2.Sterol regulatory element binding proteins (SREBPs) 1 are a family of basic helix-loop-helix transcription factors that are embedded as precursor proteins in the endoplasmic reticulum and nuclear envelope. To date, three SREBP isoforms have been detected: SREBP-1a, SREBP-1c (shorter splicing variant of SREBP-1a), as well as SREBP-2 (1-5). SREBP-2 is a major regulator of cholesterol homeostasis, whereas SREBP-1c regulates predominantly de novo synthesis of fatty acids. In contrast, SREBP-1a seems to influence both lipogenic and cholesterogenic enzymes (6).Activation of SREBPs initiated by cellular cholesterol depletion is mediated by sequential cleavage (7). As a result, the amino-terminal domain of the protein translocates into the nucleus and activates transcription of target genes. Beside this mechanism, which controls the abundance of activated SREBPs in the cell, we have demonstrated that trans-activity of the N-terminal domain of SREBPs is regulated directly by extra cellular stimuli, e.g. by hormones such as insulin (8, 9). Moreover, in these studies, we have shown that the N-terminal domains of SREBP-1a, SREBP-1c, and SREBP-2 are substrates of the extracellular signal-regulated kinase (Erk) subfamily of mitogen-activated protein kinases (MAPK). In this study, we have identified Ser-432 and Ser-455 as the major phosphorylation sites of Erk-MAPK in SREBP-2 using protein chemistry methodology. This phosphorylation has no influence on DNA interaction but affects trans-activity of SREBP-2. Accordingly, in cells, activation of low density lipoprotein (LDL) receptor gene by insulin is coupled to the identified Erk-specific phosphorylation sites in SREBP-2.

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The germ line limited DNA of Ascaris suum was isolated from sperm and testis as a satellite DNA component in Hoechst 33 258 -- CsCl gradients. Employing restriction enzyme analysis, we show that the germ line limited DNA is composed entirely of two families of tandemly repeated sequences, one repeat unit is 125 bp, and the other 131 bp long. The total appr. 5 x 10(5) copies of the two families are physically separated from each other (segmental arrangement). Several repeat unit variants within both families could be detected. The copies of sequence variants are arranged in tandem (subsegmental arrangement). Reassociation and hybridization experiments revealed similar sequences of the two repeat units. The archaeotypic core sequence of both repeat units is probably a tetranucleotide which shows a 'theme and variation' pattern. During chromatin diminution in the presoma cells the satellite DNA is eliminated from the chromosomes. However, a limited number of tandemly repeated copies of both kinds of repeat units could be detected in the soma genome using radioactive probes of both repeat units in Southern blots of muscle and intestine of adult animals. The tandem arrangement and the hierarchical pattern of restriction sites throughout different subfamilies supports the model of successive segmental amplification events during the evolution of the germ line limited DNA. Since the germ line limited satellite DNA is exclusively located at the ends of the chromosomes, a fold back structure for the telomeric DNA sequences is proposed which might have generated this DNA.

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During the early cleavage divisions in some Ascarids, parts of the chromosomes are eliminated from the somatic blastomeres ("chromatin diminution", Boveri, 1887) while the chromosomes in the germ line cells maintain their integrity. To characterize the germ line and soma genome, DNA was isolated from gametes and embryonic somatic cells of two Ascarid species, Parascaris equorum var. univalens and Ascaris suum. It was shown that the germ line limited DNAs of these species have the same density and almost identical reassociation kinetics: in CsCl the predominant component of the germ line limited DNA of P. equorum and A. suum has the buoyant density of 1.697 g/cm3, while soma DNA of both species bands at 1.700 g/cm3. In P. equorum there is a small additional germ line limited satellite DNA component with the density of 1.690 g/cm3, identical to that of mitochondrial DNA of both organisms. Comparison of the reassociation kinetics of germ line and soma DNA demonstrates for both species that the eliminated DNA sequences are highly repetitive. In contrast to these similarities between the germ line limited DNAs of P. equorum and A. suum the analysis of their base composition revealed differences (40% guanine plus cytosine in P. equorum and 36% in A. suum). The only very fast reassociating DNA sequences which we could isolate from soma DNA was demonstrated to be foldback DNA. The reassociation kinetics of total A. suum soma DNA was investigated by hydroxylapatite chromatography. Least squares analysis of the data revealed about 10% of intermediate repetitive DNA sequences. Their interspersion between single copy DNA sequences was analyzed by comparing the reassociation kinetics of DNA fragments 0.35 and 7.2 kilobases long. Thus the DNA sequence arrangement of Ascaris does not follow the short period interspersion pattern observed in most organism.

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