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NRE1 is a DNA sequence element through which Ku antigen/DNA-dependent protein kinase (DNA-PK) catalytic subunit represses the induction of mouse mammary tumor virus transcription by glucocorticoids. Although Ku is an avid binder of DNA ends and has the ability to translocate along DNA, we report that direct sequence-specific Ku binding occurs with higher affinity (Kd = 0.84 +/- 0.24 nM) than DNA end binding. Comparison of Ku binding to several sequences over which Ku can accumulate revealed two classes of sequence. Sequences with similarity to NRE1 competed efficiently for NRE1 binding. Conversely, sequences lacking similarity to NRE1 competed poorly for Ku and were not recognized in the absence of DNA ends. Phosphorylation of glucocorticoid receptor (GR) fusion proteins by DNA-PK reflected Ku DNA-binding preferences and demonstrated that co-localization of GR with DNA-PK on DNA in cis was critical for efficient phosphorylation. Phosphorylation of the GR fusion protein by DNA-PK mapped to a single site, Ser-527. This site occurs adjacent the GR nuclear localization sequence between the DNA and ligand binding domains of GR, and thus its phosphorylation, if confirmed, has the potential to affect receptor function in vivo.

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Glucocorticoid receptor (GR) exchanges between an active nuclear form and a complexed inactive, steroid-sensitive cytoplasmic form. Using a semi-quantitative indirect immunofluorescence assay to measure the kinetics of subcellular redistribution of GR in response to challenge during G(o), we have found that the ability to bind DNA is an important determinant for localization and tight binding of GR to the nucleus. The transfer of GR DNA-binding mutants to the nucleus after treatment with hormone agonists and antagonists was markedly reduced. Further, mutant receptors localized to the nucleus were only weakly associated with the nuclear compartment as they were released into cytosol upon hypotonic lysis of the cell membrane. Moreover, after agonist withdrawal, GR redistributed to the cytoplasm more rapidly when unable to bind DNA. By contrast, withdrawal of the hormone antagonist RU486 was found to yield a form of wild type GR that was completely unable to redistribute to the cytoplasm. However, this did not appear to result from a block in nuclear export as selective inactivation of nuclear import with energy inhibitor released RU486-withdrawn GRs from the nucleus at the same rates as agonist-withdrawn receptors. In addition, GR mutants unable to bind DNA, which retained a significant presence in the cytoplasm both during and after antagonist treatment, also failed to redistribute. The effect of RU486 treatment did not appear to be mediated through a block in reassociation of GR into a steroid-responsive form as RU486-withdrawn wild type receptors retained full potential to activate transcription from a glucocorticoid-responsive promoter after a second challenge with hormone. Therefore, reassociation of GR into a steroid-responsive form appears to be independent of signals important for the retention of GR in the cytoplasm.

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We have identified in mammalian cells a novel cyclic AMP response element (CRE)-binding protein of molecular mass 47 kDa. This protein was not recognized by either the CREB-327/341 or c-Jun antisera, and its tissue distribution did not overlap with those of the CREB and Jun families. For example, hepatoma and placental tissue did not contain the 47-kDa DNA-binding protein, but did contain the CREB isoforms. On the other hand, S49 lymphoma cells contained a high level of the 47-kDa DNA-binding protein but did not contain a 47-kDa Jun-related protein which was found in normal liver and hepatoma. This new 47-kDa factor bound to the CRE in the dephosphorylated form, and phosphorylation of the protein by the catalytic subunit of protein kinase A completely abolished its DNA-binding activity. The isoforms of the CREB-327/341 family, on the other hand, bound to DNA in the phosphorylated form, and alkaline phosphatase treatment reduced significantly their interaction with CRE sequence. This reverse effect of phosphorylation/dephosphorylation on the DNA-binding property of this new 47-kDa protein in particular distinguishes it from other known CREB factors and suggests that the protein might play a unique role in the regulation of cAMP-mediated transcription.

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The mechanism of switching between the cell cycle and active cell death (apoptosis) was investigated in cytokine-dependent CTLL cells. These cells proliferate in the presence of interleukin 2 (IL2), but accumulate in early G1 and undergo apoptosis in its absence. In the absence of IL2 the cells also become sensitive to glucocorticoid-induced apoptosis. Using specific inhibitors of protein kinase C and tyrosine kinases we established that two signals are required to fully repress cell death and stimulate G1 progression. One of these signals activates protein kinase C (PKC) which represses cell death and the other activates a tyrosine kinase which confers glucocorticoid resistance and permits cell cycle progression. Thus, phorbol esters can activate PKC and maintain cell viability in the absence of IL2, but the cells cannot proliferate. Moreover, the cells remain sensitive to glucocorticoid-induced apoptosis unless the tyrosine kinase-mediated signal is also given. There is a correlation between the presence of AP1 DNA-binding activity and the repression of the cell death pathway. The c-jun gene is expressed constitutively and both IL2 and phorbol esters induce the expression of c-fos to generate a functional AP1 capable of repressing cell death. However, only interleukin 2 can initiate the tyrosine kinase-mediated modification that confers dexamethasone resistance and permits G1 progression. In the absence of IL2 glucocorticoids stimulate AP1 degradation and induce apoptosis.

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We analyzed the ability of cyclic AMP-response element binding proteins (CREBs) to interact with the CRE sequences derived from different genes and examined the role of sequences flanking the core CRE element in rendering cAMP-responsiveness to the enhancer. We were able to detect reproducibly, sing the Southwestern blotting technique, five major CREB factors of molecular weights 56, 47, 40, and 36-34 kDa which were present in various rat tissues and cultured cells. The 34-40 kDa proteins (CREB-327/341) were able to bind to the CRE of cAMP-inducible genes (somatostatin, c-fos, E2A), but not to genes whose expression is not controlled by cAMP (glucagon, parathyroid hormone). The novel 47 kDa CREB had a high specificity for the core octameric CRE sequence and it bound equally well to the consensus CRE of cAMP-inducible and noninducible genes. On the other hand, the 47 kDa CREB did not bind at all to the phorbol ester response element (TRE), whereas the 56 kDa protein, reminiscent of the CRE-BP1 protein, could bind to both elements. A computer aided sequence analysis of cAMP-inducible gene promoters revealed the presence of an additional conserved element starting 4-6 nucleotides 3' to the octomer with the consensus C/GAGA/C. We have shown this element to be essential for maximal cAMP-responsiveness of the enhancer in transient expression assays of CRE-CAT plasmid constructs indicating that the functional interaction of CREB proteins with the cAMP-inducible enhancer involves an additional 8-10 base pairs immediately downstream from the CRE core element.

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Using a combination of immunoblotting, double immunoprecipitation, immunoglobulin-affinity chromatography, and isoelectrofocusing, we have been able to identify a group of proteins that display CDP-reductase activity and contain antigenic epitopes recognized by anti-ribonucleotide reductase M1 subunit and anti-ubiquitin antibodies. In the cytoplasm of rat liver cells, we could detect a total of five proteins with molecular masses of 92, 89, 56, 45, and 37 kilodaltons which reacted with the anti-M1 subunit serum. All of them, except the 89-kilodalton protein (the nascent unmodified M1), were also recognized by the anti-ubiquitin antibody. In normal liver cells, all of the apparently ubiquitinated species of the M1 protein were found in the cytoplasm, but not in the nuclear envelope associated pool of the enzyme. However, we did not detect ubiquitinated M1 protein fragments in the cytoplasm of Morris hepatoma 5123tc. The level of the apparently ubiquitinated fragments of the M1 subunit increased in parallel to the DNA-synthetic activity of normal liver cells, suggesting that ubiquitination plays a key role in the regulation of the activity of the enzyme during the cell cycle.

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We applied Southwestern and Western blotting and gel retardation techniques to investigate the changes that occur in the cyclic adenosine monophosphate (cAMP)-responsive element (CRE) binding (CREB) proteins in rapidly growing, chemically induced 5123tc and 5123D Morris hepatomas. Using the CRE sequences from the c-fos, E2A, and somatostatin gene promoters, we identified in the nuclear proteins from normal unstimulated or proliferating rat liver cells six different protein factors of Mr 34,000, 36,000, 40,000, 47,000, 56,000, and 72,000 capable of binding to the element. The Mr 47,000 protein had the highest specificity for the core CRE, suggesting its importance in cAMP-mediated gene expression. We could not find the Mr 47,000 CREB protein in the 5123tc and 5123D hepatomas. Our efforts to detect this protein in the tumors by (a) using the CRE sequence from different gene promoters, (b) altering the protocol for extracting nuclear proteins, or (c) attempting to restore its DNA-binding property by phosphorylation [with endogenous protein kinase(s), a catalytic subunit of cAMP-dependent protein kinase, and protein kinase C/dephosphorylation (with alkaline phosphatase)] were unsuccessful. The loss of tje Mr 47,000 CREB protein from solid tumors of the Morris hepatoma is likely to be related to the neoplastic properties of the tumor cell rather than to cell growth because the level of this protein remained unchanged during a 6-day period of liver regeneration. The nuclear extract from the Morris hepatoma that did not have the Mr 47,000 CRE-binding factor contained proteins immunologically related to the CREB, c-Jun, and c-Fos proteins. We conclude that the Mr 47,000 factor represents a distinct member of the CRE-binding protein family and that its absence from the hepatomas may lead to aberrant expression of cAMP-inducible genes.

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Utilizing the gel electrophoresis/DNA binding assay and a new technique of direct binding of radioactive DNA to protein blots, we have investigated putative factors selective for the cAMP-responsive element (CRE) of the lactate dehydrogenase A subunit promoter in rat ovary nuclear extracts. Analysis of linker-scanning mutants of lactate dehydrogenase A subunit promoter fragments by DNA binding assay identified DNA binding activity selective for the 11-nucleotide sequence 5' TCTGACGTCAG 3' located between positions -51 and -41 relative to the transcription initiation site. This sequence contains the previously identified CRE 5' TGACGTCA 3'. Probing of protein blots with radioactive promoter fragments containing the CRE demonstrated that ovarian nuclear extracts contain a protein of relative molecular mass 47,000 (Mr 47,000) which exhibits selective binding affinity for the CRE. The 47-kDa CRE binding protein was found to be present in comparable levels in the ovaries of normal and hypophysectomized rats. Furthermore, our data suggest the presence of a 37,000-dalton (Mr 37,000) protein which possesses selective binding affinity for part of the CRE sequence. The binding activity/level of the 37-kDa CRE binding protein appeared to be modulated by short-term hypophysectomy/follicle-stimulating hormone administration. These results provide evidence for the presence of CRE binding factors in rat ovarian nuclei, which may be involved in the molecular events responsible for transcriptional regulation of ovarian cAMP-inducible genes.

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Biochemical as well as immunochemical studies were carried out to quantitatively and qualitatively evaluate the hormonal regulation of nuclear cAMP-dependent protein kinase subunits in ovaries from estrogen-treated hypophysectomized rats. Photoaffinity labeling of nuclear extracts with 8-azido-[32P]cAMP and electrophoretic analysis showed the existence of three variants of the regulatory subunit RI and of a 52,000-dalton RII variant (RII-52) in ovarian nuclei of estrogen-primed hypophysectomized rats. After follicle-stimulating hormone (FSH) stimulation, an additional variant of RII (RII-51, Mr = 51,000) was detected in nuclei. The cytosolic RII-54 variant (Mr = 54,000) could not be identified in nuclei by photoaffinity labeling. The FSH-mediated appearance of the nuclear RII-51 variant was accompanied by an approximate 2-fold increase of nuclear catalytic subunit activity. Using quantitation by enzyme-linked immunosorbent assay, we identified a marked FSH-mediated increase of nuclear RII variant(s) and confirmed the increase of nuclear catalytic subunit levels. Furthermore, morphometric analysis of nuclear and cytoplasmic antigen density by immunogold electron microscopy demonstrated a cell-specific modulation by FSH of RII and C subunit density. In granulosa cells, both nuclear as well as cytoplasmic RII density was increased by FSH, whereas catalytic subunit density was increased in the nuclear area only. In thecal cells, FSH increased only the nuclear catalytic subunit density. These results provide biochemical as well as immunochemical evidence for a cell-specific FSH regulation of nuclear RII and catalytic subunit levels which may be involved in the molecular events responsible for the FSH-mediated differentiation of the rat ovary.

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The studies described in this report suggest a rather complex, albeit incomplete, sequence of molecular events that we believe form part of the cascade of reactions through which a series of hormones, via cAMP, regulates the expression of specific gene products. The majority of our own studies relate to cAMP-mediated induction of LDH. Some, if not all, of the molecular steps discussed in this paper may ultimately be recognized as part of a universal mechanism by which cAMP controls gene expression in higher eukaryotes. The idea of a functional role for cAMP-dependent protein kinase subunits in cAMP-mediated gene control has already had experimental support, but our identification of the regulatory subunit RII as a topoisomerase now more firmly points to a complex function for the kinase in regulating gene function at the DNA level. We look forward to the elucidation of the function of those nuclear proteins that serve as substrate for the catalytic subunit of cAMP-dependent protein kinase. Further studies related to the molecular interaction of RII with chromosomal DNA should be a fruitful area for future research.

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An immunocolloidal gold electron microscopy method is described allowing the ultrastructural localization and quantitation of the regulatory subunits RI and RII and the catalytic subunit C of cAMP-dependent protein kinase. Using a postembedding indirect immunogold labeling procedure that employs specific antisera, the catalytic and regulatory subunits were localized in electron-dense regions of the nucleus and in cytoplasmic areas with a minimum of nonspecific staining. Antigenic domains were localized in regions of the heterochromatin, nucleolus, interchromatin granules, and in the endoplasmic reticulum of different cell types, such as rat hepatocytes, ovarian granulosa cells, and spermatogonia, as well as cultured H4IIE hepatoma cells. Morphometric quantitation of the relative staining density of nuclear antigens indicated a marked modulation of the number of subunits per unit area under various physiologic conditions. For instance, following partial hepatectomy in rats, the staining density of the nuclear RI and C subunits was markedly increased 16 h after surgery. Glucagon treatment of rats increased the staining density of only the nuclear catalytic subunit. Dibutyryl cAMP treatment of H4IIE hepatoma cells led to a marked increase in the nuclear staining density of all three subunits of cAMP-dependent protein kinase. These studies demonstrate that specific antisera against cAMP-dependent protein kinase subunits may be used in combination with immunogold electron microscopy to identify the ultrastructural location of the subunits and to provide a semi-quantitative estimate of their relative cellular density.

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