The dependence receptor DCC (deleted in colorectal cancer) defines an alternative mechanism for caspase activation

The expression of DCC (deleted in colorectal cancer) is often markedly reduced in colorectal and other cancers. However, the rarity of point mutations identified in DCC coding sequences and the lack of a tumor predisposition phenotype in DCC hemizygous mice have raised questions about its role as a tumor suppressor. DCC also mediates axon guidance and functions as a dependence receptor; such receptors create cellular states of dependence on their respective ligands by inducing apoptosis when unoccupied by ligand. We now show that DCC drives cell death independently of both the mitochondria-dependent pathway and the death receptor/caspase-8 pathway. Moreover, we demonstrate that DCC interacts with both caspase-3 and caspase-9 and drives the activation of caspase-3 through caspase-9 without a requirement for cytochrome c or Apaf-1. Hence, DCC defines an additional pathway for the apoptosome-independent caspase activation.

The ADP Ribosylation Factor-Nucleotide Exchange Factors Gea1p and Gea2p Have Overlapping, but Not Redundant Functions in Retrograde Transport from the Golgi to the Endoplasmic Reticulum

The activation of the small ras-like GTPase Arf1p requires the action of guanine nucleotide exchange factors. Four Arf1p guanine nucleotide exchange factors have been identified in yeast: Sec7p, Syt1p, Gea1p, and its homologue Gea2p. We identified GEA2 as a multicopy suppressor of a sec21-3 temperature-sensitive mutant. SEC21 encodes the γ-subunit of coatomer, a heptameric protein complex that together with Arf1p forms the COPI coat. GEA1 and GEA2 have at least partially overlapping functions, because deletion of either gene results in no obvious phenotype, whereas the double null mutant is inviable. Conditional mutants defective in both GEA1 and GEA2 accumulate endoplasmic reticulum and Golgi membranes under restrictive conditions. The two genes do not serve completely overlapping functions because a Δgea1 Δarf1 mutant is not more sickly than a Δarf1 strain, whereas Δgea2 Δarf1 is inviable. Biochemical experiments revealed similar distributions and activities for the two proteins. Gea1p and Gea2p exist both in membrane-bound and in soluble forms. The membrane-bound forms, at least one of which, Gea2p, can be visualized on Golgi structures, are both required for vesicle budding and protein transport from the Golgi to the endoplasmic reticulum. In contrast, Sec7p, which is required for protein transport within the Golgi, is not required for retrograde protein trafficking.

Corepressor-induced organization and assembly of the biotin repressor: A model for allosteric activation of a transcriptional regulator

The Escherichia coli biotin repressor binds to the biotin operator to repress transcription of the biotin biosynthetic operon. In this work, a structure determined by x-ray crystallography of a complex of the repressor bound to biotin, which also functions as an activator of DNA binding by the biotin repressor (BirA), is described. In contrast to the monomeric aporepressor, the complex is dimeric with an interface composed in part of an extended β-sheet. Model building, coupled with biochemical data, suggests that this is the dimeric form of BirA that binds DNA. Segments of three surface loops that are disordered in the aporepressor structure are located in the interface region of the dimer and exhibit greater order than was observed in the aporepressor structure. The results suggest that the corepressor of BirA causes a disorder-to-order transition that is a prerequisite to repressor dimerization and DNA binding.

Considerations of transcriptional control mechanisms: Do TFIID–core promoter complexes recapitulate nucleosome-like functions?

The general transcription initiation factor TFIID was originally identified, purified, and characterized with a biochemical assay in which accurate transcription initiation is reconstituted with multiple, chromatographically separable activities. Biochemical analyses have demonstrated that TFIID is a multiprotein complex that directs preinitiation complex assembly on both TATA box-containing and TATA-less promoters, and some TFIID subunits have been shown to be molecular targets for activation domains in DNA-binding regulatory proteins. These findings have most commonly been interpreted to support the view that transcriptional activation by upstream factors is the result of enhanced TFIID recruitment to the core promoter. Recent insights into the architecture and cell-cycle regulation of the multiprotein TFIID complex prompt both a reassessment of the functional role of TFIID in gene activation and a review of some of the less well-appreciated literature on TFIID. We present a speculative model for diverse functional roles of TFIID in the cell, explore the merits of the model in the context of published data, and suggest experimental approaches to resolve unanswered questions. Finally, we point out how the proposed functional roles of TFIID in eukaryotic class II transcription fit into a model for promoter recognition and activation that applies to both eubacteria and eukaryotes.

Effects of anesthesia on functional activation of cerebral blood flow and metabolism

Functional brain mapping based on changes in local cerebral blood flow (lCBF) or glucose utilization (lCMRglc) induced by functional activation is generally carried out in animals under anesthesia, usually α-chloralose because of its lesser effects on cardiovascular, respiratory, and reflex functions. Results of studies on the role of nitric oxide (NO) in the mechanism of functional activation of lCBF have differed in unanesthetized and anesthetized animals. NO synthase inhibition markedly attenuates or eliminates the lCBF responses in anesthetized animals but not in unanesthetized animals. The present study examines in conscious rats and rats anesthetized with α-chloralose the effects of vibrissal stimulation on lCMRglc and lCBF in the whisker-to-barrel cortex pathway and on the effects of NO synthase inhibition with NG-nitro-l-arginine methyl ester (l-NAME) on the magnitude of the responses. Anesthesia markedly reduced the lCBF and lCMRglc responses in the ventral posteromedial thalamic nucleus and barrel cortex but not in the spinal and principal trigeminal nuclei. l-NAME did not alter the lCBF responses in any of the structures of the pathway in the unanesthetized rats and also not in the trigeminal nuclei of the anesthetized rats. In the thalamus and sensory cortex of the anesthetized rats, where the lCBF responses to stimulation had already been drastically diminished by the anesthesia, l-NAME treatment resulted in loss of statistically significant activation of lCBF by vibrissal stimulation. These results indicate that NO does not mediate functional activation of lCBF under physiological conditions.

The extreme C terminus of progesterone receptor contains a transcriptional repressor domain that functions through a putative corepressor.

Binding of a hormone agonist to a steroid receptor leads to the dissociation of heat shock proteins, dimerization, specific DNA binding, and target gene activation. Although the progesterone antagonist RU486 can induce most of these events, it fails to activate human progesterone receptor (hPR)-dependent transcription. We have previously demonstrated that a conformational change is a key event leading to receptor activation. The major conformational distinction between hormone- and antihormone-bound receptors occurs within the C-terminal portion of the molecule. Furthermore, hPR mutants lacking the C terminus become transcriptionally active in the presence of RU486. These results suggest that the C terminus contains a repressor domain that inhibits the transcriptional activity of the RU486-bound hPR. In this study, we have defined a 12 amino acid (12AA) region in the C terminus of hPR that is necessary and sufficient for the repressor function when fused to the C-terminal truncated hPR or to the GAL4 DNA-binding domain. Mutations in the 12AA domain (aa 917-928) generate an hPR that is active in the presence of RU486. Furthermore, overexpression of the 12AA peptide activates the RU486-bound wild-type hPR without affecting progesterone-dependent activation. These results suggest that association of the 12AA repressor region with a corepressor might inactivate hPR activity when it is bound to RU486. We propose that binding of a hormone agonist to the receptor changes its conformation in the ligand-binding domain so that association with coactivator is promoted and activation of target gene occurs.

Prostaglandin E2 receptors of the EP2 and EP4 subtypes regulate activation and differentiation of mouse B lymphocytes to IgE-secreting cells.

Prostaglandin E2 (PGE2) is a potent lipid molecule with complex proinflammatory and immunoregulatory properties. PGE2 can shape the immune response by stimulating the production of IgE antibody by B lymphocytes and the synthesis of T-helper type 2 cytokines [e.g., interleukin (IL)-4, IL-10], while inhibiting production of Th1 cytokines (e.g., interferon-gamma, IL-12). It is unknown what type of receptor binds PGE2 and modulates these responses. Recent analyses in nonhematopoietic cells have identified six PGE2 receptors (EP1, EP2, EP3 alpha, EP3 beta, EP3 gamma, and EP4). This investigation examines quiescent B lymphocytes and reports that these cells express mRNA encoding EP1, EP2, EP3 beta, and EP4 receptors. The immunoregulatory functions of each receptor were investigated using small molecule agonists that preferentially bind EP receptor subtypes. Unlike agonists for EP1 and EP3, agonists that bound EP2 or EP2 and EP4 receptors strongly inhibited expression of class II major histocompatibility complex and CD23 and blocked enlargement of mouse B lymphocytes stimulated with IL-4 and/or lipopolysaccharide. PGE2 promotes differentiation and synergistically enhances IL-4 and lipopolysaccharide-driven B-cell immunoglobulin class switching to IgE. Agonists that bound EP2 or EP2 and EP4 receptors also strongly stimulated class switching to IgE. Experiments employing inhibitors of cAMP metabolism demonstrate that the mechanism by which EP2 and EP4 receptors regulate B lymphocyte activity requires elevation of cAMP. In conclusion, these data suggest that antagonists to EP2 and EP4 receptors will be important for diminishing allergic and IgE-mediated asthmatic responses.

Activation of the human homologue of the Drosophila sina gene in apoptosis and tumor suppression.

Developmentally regulated genes in Drosophila, which are conserved through evolution, are potential candidates for key functions in biological processes such as cell cycle, programmed cell death, and cancer. We report cloning and characterization of the human homologue of the Drosophila seven in absentia gene (HUMSIAH), which codes for a 282 amino acids putative zinc finger protein. HUMSIAH is localized on human chromosome 16q12-q13. This gene is activated during the physiological program of cell death in the intestinal epithelium. Moreover, human cancer-derived cells selected for suppression of their tumorigenic phenotype exhibit constitutively elevated levels of HUMSIAH mRNA. A similar pattern of expression is also displayed by the p21waf1. These results suggest that mammalian seven in absentia gene, which is a target for activation by p53, may play a role in apoptosis and tumor suppression.

Expression of a second receptor rescues self-specific T cells from thymic deletion and allows activation of autoreactive effector function.

Allelic exclusion at the T-cell receptor alpha chain locus is incomplete resulting in the generation of T cells that express two T-cell receptors. The potential involvement of such T cells in autoimmunity has been suggested [Padovan, E., Casorati, G., Dellabona, P., Meyer, S., Brockhaus, M. & Lanzavecchia, A. (1993) Science 262, 422-424; Heath, W. R. & Miller, J. F. A. P. (1993) J. Exp. Med. 178, 1807-1811]. Here we show that expression of a second T-cell receptor can rescue T cells with autospecific receptors from thymic deletion and allow their exit into the periphery. Dual receptor T cells, created by constitutive expression of two transgenic T-cell receptors on a Rag1-/- background, are tolerant to self by maintaining low levels of autospecific receptor, but selfreactive effector function (killing) can be induced through activation via the second receptor. This opens the possibility that T cells carrying two receptors in the periphery of normal individuals contain putatively autoreactive cells that could engage in autoimmune effector functions after recognition of an unrelated environmental antigen.

Antibody-induced engagement of beta 2 integrins on adherent human neutrophils triggers activation of p21ras through tyrosine phosphorylation of the protooncogene product Vav.

It is known that beta 2 integrins are crucial for leukocyte cell-cell and cell-matrix interactions, and accumulating evidence now suggests that integrins serve not only as a structural link but also as a signal-transducing unit that controls adhesion-induced changes in cell functions. In the present study, we plated human neutrophils on surface-bound anti-beta 2 (CD18) antibodies and found that the small GTP-binding protein p21ras is activated by beta 2 integrins. Pretreatment of the cells with genistein, a tyrosine kinase inhibitor, led to a complete block of p21ras activation, an effect that was not achieved with either U73122, which abolishes the beta 2 integrin-induced Ca2+ signal, or wortmannin, which totally inhibits the phosphatidylinositol 3-kinase activity. Western blot analysis revealed that antibody-induced engagement of beta 2 integrins causes tyrosine phosphorylation of several proteins in the cells. One of these tyrosine-phosphorylated proteins had an apparent molecular mass of 95 kDa and was identified as the protooncogene product Vav, a p21ras guanine nucleotide exchange factor that is specifically expressed in cells of hematopoietic lineage. A role for Vav in the activation of p21ras is supported by the observations that antibody-induced engagement of beta 2 integrins causes an association of Vav with p21ras and that the effect of genistein on p21ras activation coincided with its ability to inhibit both the tyrosine phosphorylation of Vav and the Vav-p21ras association. Taken together, these results indicate that antibody-induced engagement of beta 2 integrins on neutrophils triggers tyrosine phosphorylation of Vav and, possibly through its association, a downstream activation of p21ras.

Separation of the transcriptional coactivator and antirepression functions of transcription factor IIA.

Human transcription factor IIA (TFIIA) is composed of three subunits (alpha, beta, and gamma). TFIIA interacts with the TATA-box binding protein and can overcome repression of transcription. TFIIA was found to be necessary for VP16-mediated transcriptional activation through a coactivator function. We have separated the coactivator and antirepression activities of TFIIA. A TFIIA lacking the alpha subunit was isolated from HeLa cells. This "mini-TFIIA" interacts with the TATA-box binding protein and can overcome repression of transcription, but it is defective in transcriptional coactivator function.

Different functions for the interleukin 8 receptors (IL-8R) of human neutrophil leukocytes: NADPH oxidase and phospholipase D are activated through IL-8R1 but not IL-8R2.

Two monoclonal antibodies, anti-IL8R1 and anti-IL8R2, raised against both interleukin 8 receptors (IL-8R) of human neutrophils, IL-8R1 and IL-8R2, were used to study individual receptor functions after stimulation with IL-8, GRO alpha, or NAP-2. Efficacy and selectivity of the antibodies were tested in Jurkat cells transfected with cDNA coding for one or the other receptor. The binding of 125 I labeled IL-8 and IL-8-induced changes of the cytosolic free Ca2+ concentration were inhibited by anti-IL8RI in cells expressing IL-8R1 and by anti-IL8R2 in cells expressing IL-8R2. In human neutrophils, release of elastase was observed after stimulation with IL-8 or GRO alpha. The response to IL-8 was inhibited slightly by anti-IL8R1 and more substantially when both monoclonal antibodies were present, while the response to GRO alpha was inhibited by anti-IL8R2 but was not affected by anti-IL8R1. These results indicate that both IL-8 receptors can signal independently for granule enzyme release. Superoxide production, a measure of the respiratory burst, was obtained with increasing concentrations of IL-8 with maximum effects at 25 to 50 nM, but no response was observed upon challenge with GRO alpha or NAP-2 up to 1000 nM. The superoxide production induced by IL-8 was inhibited by anti-IL8R1, but was not affected by anti-IL8R2. Stimulation of neutrophils with IL-8, in contrast to GRO alpha or NAP-2, also elicited phospholipase D activity. The effect of IL-8 was again inhibited by anti-IL-8R1 but not by anti-IL8R2, indicating that this response, like the respiratory burst, was mediated by IL-8R1. Taken together, our results show that IL-8R1 and IL-8R2 are functionally different. Responses, such as cytosolic free Ca2+ changes and the release of granule enzymes, are mediated through both receptors, whereas the respiratory burst and the activation of phospholipase D depend exclusively on stimulation through IL-8R1.

Sp1 functions in a chromatin-dependent manner to augment human alpha-globin promoter activity.

The 5' flanking region of the human alpha-globin gene is highly G + C rich and contains multiple copies of the consensus sequence for the Sp1 binding site. We investigated the role of this G + C-rich region in augmenting alpha-globin promoter activity in the presence of the far-upstream alpha-globin enhancer, HS-40. We show that in transiently transfected erythroid cells, deletion of the alpha-globin G + C-rich 5' flanking region has no effect on alpha-globin promoter activity. However, upon stable integration into chromatin, deletion of this region causes a nearly 90% decrease in promoter activity compared with expression from an alpha-globin promoter retaining this region. These results suggest that the alpha-globin G + C-rich 5' flanking region augments alpha-globin promoter activity in a chromatin-dependent manner. We further show that this G + C-rich region is required for the activation of alpha-globin gene expression during erythroid differentiation. Finally, we show by both footprint analysis and functional assays that the ability of the G + C-rich region to increase alpha-globin promoter activity from a stably integrated alpha-globin gene is mediated by its multiple binding sites for the transcription factor Sp1.

Convergent and parallel activation of low-conductance potassium channels by calcium and cAMP-dependent protein kinase.

K+ channels, which have been linked to regulation of electrogenic solute transport as well as Ca2+ influx, represent a locus in hepatocytes for the concerted actions of hormones that employ Ca2+ and cAMP as intracellular messengers. Despite considerable study, the single-channel basis for synergistic effects of Ca2+ and cAMP on hepatocellular K+ conductance is not well understood. To address this question, patch-clamp recording techniques were applied to a model liver cell line, HTC hepatoma cells. Increasing the cytosolic Ca2+ concentration ([Ca2+]i) in HTC cells, either by activation of purinergic receptors with ATP or by inhibition of intracellular Ca2+ sequestration with thapsigargin, activated low-conductance (9-pS) K+ channels. Studies with excised membrane patches suggested that these channels were directly activated by Ca2+. Exposure of HTC cells to a permeant cAMP analog, 8-(4-chlorophenylthio)-cAMP, also activated 9-pS K+ channels but did not change [Ca2+]i. In excised membrane patches, cAMP-dependent protein kinase (the downstream effector of cAMP) activated K+ channels with conductance and selectivity identical to those of channels activated by Ca2+. In addition, cAMP-dependent protein kinase activated a distinct K+ channel type (5 pS). These data represent the differential regulation of low-conductance K+ channels by signaling pathways mediated by Ca2+ and cAMP. Moreover, since low-conductance Ca(2+)-activated K+ channels have been identified in a variety of cell types, these findings suggest that differential regulation of K+ channels by hormones with distinct signaling pathways may provide a mechanism for hormonal control of solute transport and Ca(2+)-dependent cellular functions in the liver as well as other nonexcitable tissues.

Modulation of c-Myb-induced transcription activation by a phosphorylation site near the negative regulatory domain.

The c-myb protooncogene encodes a highly conserved transcription factor that functions as both an activator and a repressor of transcription. The v-myb oncogenes of E26 leukemia virus and avian myeloblastosis virus encode proteins that are truncated at both the amino and the carboxyl terminus, deleting portions of the c-Myb DNA-binding and negative regulatory domains. This has led to speculation that the deleted regions contain important regulatory sequences. We previously reported that the 42-kDa mitogen-activated protein kinase (p42mapk) phosphorylates chicken and murine c-Myb at multiple sites in the negative regulatory domain in vitro, suggesting that phosphorylation might provide a mechanism to regulate c-Myb function. We now report that three tryptic phosphopeptides derived from in vitro phosphorylated c-Myb comigrate with three tryptic phosphopeptides derived from metabolically labeled c-Myb immunoprecipitated from murine erythroleukemia cells. At least two of these peptides are phosphorylated on serine-528. Replacement of serine-528 with alanine results in a 2- to 7-fold increase in the ability of c-Myb to transactivate a Myb-responsive promoter/reporter gene construct. These findings suggest that phosphorylation serves to regulate c-Myb activity and that loss of this phosphorylation site from the v-Myb proteins may contribute to their transforming potential.