Major Signal Transduction Pathways Research Articles

Regulation and activities of alpha-fetoprotein.

alpha-Fetoprotein (AFP) is one of the major serum proteins in the early life of mammals. The function of this protein is not yet fully understood. AFP is an oncodevelopmental gene product which is expressed at high levels in the embryonic yolk sac and fetal liver. The synthesis of AFP decreases dramatically after birth. Only trace amounts of AFP are synthesized in the adult liver. However, expression of the AFP gene is reactivated in the adult during liver regeneration and hepatocarcinogenesis. AFP is an excellent model system for studying the temporal and tissue-specific regulation of oncodevelopmental gene expression. Experiments with transgenic mice and DNA transfection studies have revealed several transcriptional control regions and cis-acting elements in the AFP gene. A large number of trans-acting protein factors interacting with these cis-acting elements have also been identified. Recent studies demonstrated that expression of AFP is regulated by at least two major signal transduction pathways in response to extracellular stimuli. The interactions between steroid hormone receptors and transcriptional factors which respond to separate signal transduction pathways result in transcriptional regulation of AFP gene expression. Trans-acting protein factors or steroid receptors complexed with given response elements can display different activities in different cell types due to cross-talk among both local protein-protein interactions within the DNA-binding domain, and distal protein-protein interactions. However, the detailed mechanisms of AFP gene expression are still not completely understood.

Exogenous human immunodeficiency virus type‐1 Tat protein selectively stimulates a phosphatidylinositol‐specific phospholipase C nuclear pathway in the Jurkat T cell line

We investigated the effect of extracellular Tat protein of human immunodeficiency virus-type 1 (HIV-1) on the phosphatidylinositol (PI) cycle, which represents a major signal transduction pathway in lymphoid cells. Recombinant Tat, recombinant HIV-1 p24 and cross-linked anti-CD3 monoclonal antibody (mAb) were added in culture for 1-60 min to Jurkat lymphoblastoid CD4+ T cells. The stimulation of T cell receptor by cross-linked anti-CD3 mAb resulted in a rapid increase of the phosphatidylinositol-specific phospholipase C (PI-PLC) activity in whole cell lysates. On the other hand, Tat protein, either alone or in combination with anti-CD3 mAb, showed little effect on the PI turnover of whole cell extracts. Tat, however, selectively stimulated a nuclear-specific PI-PLC with a peak of activity after 30 min from the addition in culture to Jurkat cells. Interestingly, this time corresponded to that required for the uptake and nuclear localization of recombinant Tat protein, as demonstrated by electron microscope immunocytochemistry experiments with anti-Tat mAb. Moreover, exogenous Tat reached the nucleus of Jurkat cells in a bioactive form, as shown in a HIV-1 long terminal repeat-chloramphenicol acetyl transferase transactivation assay. The specific increase of a nuclear PI-PLC activity was further demonstrated by the ability of Tat to stimulate PI turnover also when added directly to isolated nuclei. As a whole, these data demonstrate that Tat selectively stimulates a nuclear polyphosphoinositide hydrolysis, which appears to be independent of the cellular PI turnover. The relevance of these findings for a better understanding of the biological functions of extracellular Tat is discussed.

Endothelin Receptors, Second Messengers, and Actions in Bone

Endothelins are a class of peptides that are produced by and elicit responses in many tissues. A growing literature documents the presence and effects of endothelins in bone. Both endothelinA and endothelinB receptors have been demonstrated in osteoblastic cells by ligand binding. Major signal transduction pathways for endothelin in bone cells appear to be stimulation of phospholipid turnover, by activation of A, C and D phospholipases, stimulation of calcium flux from intracellular and extracellular stores and activation of tyrosine kinases. Endothelins also modulate calcium signaling elicited by other agents in osteoblastic cells. The parathyroid hormone-stimulated calcium transient in UMR-106 cells is enhanced by endothelins, acting through an endothelinB receptor, whereas the parathyroid hormone-stimulated increase in cyclic AMP is inhibited by endothelins. Phenotypic responses to endothelin-1 include changes in alkaline phosphatase activity, stimulation of osteocalcin and osteopontin message, stimulation of collagen and noncollagenous protein synthesis, inhibition of osteoclast motility and stimulation of prostaglandin-dependent resorption. Endothelin-1 also enhances the interleukin-1-induced increase in interleukin-6. Endothelins can also potentially affect calcium metabolism through their actions to inhibit the secretion of parathyroid hormone.

Agonist properties of a stable hexapeptide analog of neurotensin, NαMeArg-Lys-Pro-Trp-tLeu-Leu (NT1)

The major signal transduction pathway for neurotensin (NT) receptors is the G-protein-dependent stimulation of phospholipase C, leading to the mobilization of intracellular free Ca 2+ ([Ca 2+] i) and the stimulation of cyclic GMP. We investigated the functional actions of an analog of NT(8–13), N α MeArg-Lys-Pro-Trp-tLeu-Leu (NT1), and other NT related analogs by quantitative measurement of the cytosolic free Ca 2+ concentration in HT-29 (human colonic adenocarcinoma) cells using the Ca 2+-sensitive dye fura-2/AM and by effects on cyclic GMP levels in rat cerebellar slices. The NT receptor binding affinities for these analogs to HT-29 cell membranes and newborn (10-day-old) mouse brain membranes were also investigated. Data obtained from HT-29 cell and mouse brain membrane preparations showed saturable single high-affinity sites and binding densities ( B max) of 130.2 and 87.5 fmol/mg protein, respectively. The respective K D values were 0.47 and 0.39 nM, and the Hill coefficients were 0.99 and 0.92. The low-affinity levocabastine-sensitive site was not present ( K I > 10,000) in either membrane preparation. Although the correlation of binding betwen HT-29 cell membranes and mouse brain membranes was quite significant ( r = 0.92), some of the reference agents had lower binding affinities in the HT-29 cell membranes. The metabolically stable compound NT1 plus other NT analogs and related peptides [NT, NT(8–13), xenopsin, neuromedin N, NT(9–13), kinetensin and ( d-Trp 11)-NT] increased intracellular Ca 2+ levels in HT-29 cells, indicating NT receptor agonist properties. The effect of NT1 in mobilizing [Ca 2+] i blocked by SR 48692, a non-peptide NT antagonist. Receptor binding affinities of NT analogs to HT-29 cell membranes were positively correlated with potencies for mobilizing intracellular calcium in the same cells. In addition, NT1 increased cyclic GMP levels in rat cerebellar slices, confirming the latter findings of its NT agonist action. These results substantiate the in vitro NT agonist properties of the hexapeptide NT analog NT1.

Regulation of mRNA expression involved in Ras and PKA signal pathways during rat hypoglossal nerve regeneration.

Using in situ hybridization histochemistry and immunohistochemistry, the present study examines the cooperative regulation of transcription of molecules involved in the Ras-signal and the cAMP dependent protein kinase (PKA) pathways during peripheral nerve regeneration in rats. Injury to hypoglossal motor neurons resulted in an increase in extracellular regulated kinase (ERK, or MAP kinase) and ERK kinase (MEK, or MAP kinase kinase) mRNAs, but in a decrease in the expression of the catalytic subunits of PKA (C alpha and C beta) mRNAs. These results show the importance of the Ras-signal pathway in the nerve regeneration process and extend recent observation which suggested a cross-talk between the Ras and PKA pathways in vitro. The down-regulation of PKA may facilitate the activation of the Ras pathway which is located downstream of the growth factor receptor. The present study may suggest a possibility of regulatory talk between these two major signal transduction pathways.

Modulation of protein tyrosine phosphorylation in rat insular cortex after conditioned taste aversion training.

Protein tyrosine phosphorylation is a major signal transduction pathway involved in cellular metabolism, growth, and differentiation. Recent data indicate that tyrosine phosphorylation also plays a role in neuronal plasticity. We are using conditioned taste aversion, a fast and robust associative learning paradigm subserved among other brain areas by the insular cortex, to investigate molecular correlates of learning and memory in the rat cortex. In conditioned taste aversion, rats learn to associate a novel taste (e.g., saccharin) with delayed poisoning (e.g., by LiCl injection). Here we report that after conditioned taste aversion training, there is a rapid and marked increase in tyrosine phosphorylation of a set of proteins in the insular cortex but not in other brain areas. A major protein so modulated, of 180 kDa, is abundant in a membrane fraction and remains modulated for more than an hour after training. Exposure of the rats to the novel taste alone results in only a small modulation of the aforementioned proteins whereas administration of the malaise-inducing agent per se has no effect. To the best of our knowledge, this is the first demonstration of modulation of protein tyrosine phosphorylation in the brain after a behavioral experience.

Mutual antagonistic interactions between the thyrotropin (adenosine 3',5'-monophosphate) and protein kinase C/epidermal growth factor (tyrosine kinase) pathways in cell proliferation and differentiation of cultured human thyroid follicles.

Our aim has been to delineate the role of the major signal transduction pathways believed implicated in the control of thyroid function and growth: the cAMP-, epidermal growth factor (EGF) (tyrosine kinase)-, and protein kinase C (PKC)-mediated mechanisms. The experimental model used was our system of thyroid follicles of human origin cultured in suspension under serum-free conditions in which the follicular three-dimensional structure is retained. The phorbol ester 12-O tetradecanoylphorbol 13-acetate (TPA) time and dose dependently (10(-11)-10(-7) M) inhibited TSH-stimulated thyroid functions (cAMP formation, iodide uptake and organification, and T3 secretion). TPA also inhibited such forskolin- and 8-BrcAMP-stimulated effects, suggesting that the attenuation of the cAMP-dependent pathway occurs at steps both pre- and post-cAMP formation. The effects of TPA were mimicked by another PKC activator, phorbol 12,13-dibutyrate, but not by a phorbol ester that fails to activate PKC, 4 alpha-phorbol 12,13-didecanoate, and were reversed by staurosporine, a PKC inhibitor. The TPA actions seem, therefore, to be PKC-mediated. EGF exhibited a time- and dose-dependent (0.02-8 nM) restraining influence on the above TSH-stimulated differentiated functions, except for cAMP, which was enhanced. EGF also blunted such forskolin- and 8-BrcAMP-induced response parameters, suggesting inhibition at a post-cAMP locus. Regarding cell proliferation, during the initial stages of culture (day 2), TPA dose dependently (10(-11)-10(-7) M) attenuated cell proliferation, but subsequently (day 7 of culture) the same doses of TPA stimulated cell multiplication. The TPA-mitogenic and antimitogenic effects could not be mimicked by 4 alpha-phorbol-12,13-didecanoate and were reversed by staurosporine, thus indicating a PKC-mediated pathway for such TPA actions. EGF, on the other hand, only enhanced cell proliferation at a late stage (coincident with the TPA-mitogenic effect). TSH (0.5 U/liter) inhibited both the mitogenic and antimitogenic actions of TPA as well as the cell-proliferative influence of EGF. In conclusion, the data demonstrate mutual antagonistic interactions between the signal transduction pathways: the PKC and EGF (tyrosine kinase) pathways seem to inhibit TSH (cAMP)-mediated human thyroid cell differentiation, whereas TSH attenuates PKC-mediated thyroid cell mitogenesis and antimitogenesis as well as EGF-mediated cell proliferation.

Genetic regulation of leishmanial and mycobacterial infections: the Lsh / Ity / Bcg gene story continues

A common basis to genetic regulation of leishmanial and mycobacterial infections is provided by the action of the murine Lsh / Ity / Bcg gene in controlling the priming/activation of macrophages for antimicrobial activity. This relies on the TNF-α-dependent sustained expression of the inducible nitric oxide synthase (iNOS) gene responsible for the generation of large amounts of toxic nitric oxide (NO). The Lsh / Ity / Bcg gene has many pleiotropic effects, including differential expression of the early response gene KC following stimulation of macrophages with bacterial lipopolysaccharide (LPS) and mycobacterial lipoarabinomannan (LAM). The major signal transduction pathway involved in KC induction requires the generation of low levels of NO via constitutive nitric oxide synthase (cNOS) activity, leading to activation of guanylate cyclase and the cGMP-dependent kinase pathway. NO therefore appears to provide a common link between the early influence of Lsh in regulating the expression of genes which mediate many pleiotropic effects, and the later production of NO as the final effector mechanism for kill. The recently cloned candidate for Lsh / Ity / Bcg, designated Nramp for Natural resistance associated macrophage protein, encodes a polytopic integral membrane protein that has structural features common to prokaryotic and eukaryotic transporters and includes a conserved binding-protein-dependent transport motif which may be involved in interaction with peripheral ATP-binding subunits. The N-terminal sequence also carries a proline / serine rich putative SH3 binding domain, consistent with a role for tyrosine kinases in regulating Nramp function. This is also supported by the demonstration that ligation of β 1 integrins, which signal via tyrosine kinases, by plating of macrophages onto extracellular matrix proteins is sufficient to mediate differential TNF-α release by macrophages from congenic Lsh resistant and susceptible mice. Transfection studies with the resistant allele demonstrate that Nramp plays a role, either directly or as an additional pleiotropic effect, in interferon-γ/LPS upregulated L-arginine transport across the macrophage membrane, thus providing the substrate required to generate NO for both signal transduction and antimicrobial activity. Nramp also shows 55–58% sequence similarity with the yeast genes SMF1 and SMF2, which influence protein import into mitochondria. A high degree of conservation over the region of Nramp which contains the susceptible Nramp mutation indicates a possible common function at the level of protein translocation across membranes of intracellular compartments. Analysis of human NRAMP has identified a novel 3 × 9 nucleotide repeat in the putative SH3 binding domain, with a rare second allele bearing a 2 × 9 nucleotide repeat occurring at low frequency in the Brazilian population. Studies in progress will attempt to determine the function of human NRAMP, and hence to identify its role in parallel activation pathways in man. This is of particular interest in the light of studies demonstrating ( i) that NO generated by iNOS is not used for antimicrobial activity in human macrophages, and ( ii) that the iNOS gene itself and the interferon-γ-inducible JAK tyrosine kinases are candidates for two other major genes, Scl1 and Scl2, identified and mapped in mice for their role in controlling different leishmanial resistance phenotypes. Further analysis of genetic regulation of pathways leading to iNOS-mediated NO production may provide the key to understanding why human macrophages do not use this as an antimicrobial pathway, and may also provide the basis for development of novel immunotherapeutic strategies for disease control.

How receptors work: mechanisms of signal transduction.

In recent years major advances have been made regarding our understanding of the mechanisms underlying cell surface receptor activation. In particular, how information is transferred from these receptors to the interior of the cell. The aim of this article is to give a brief overview of the major signal transduction pathways employed by cells and to highlight where such information has allowed insight into the molecular mechanisms underlying certain disease states. In many instances this information has permitted the development of novel therapeutic strategies. While most cells seem to possess a bewildering array of agonist-specific cell surface receptors capable of recognizing incoming hormonal, neurotransmitter, cytokine and growth factor signals, a far more limited number ofpathways exist to relay this information to the cell interior. In light of our recent understanding of these signalling pathways, receptors can now be classified in a far more functional way based on the mode of signal transduction rather than purely on the basis of agonist specificity (Figure 1). Such transmembrane signalling mechanisms include receptor regulation of ion channel function, activation ofa membrane-associated phospholipase, generation of cyclic nucleotides or stimulation of a receptor-coupled tyrosine kinase. One important caveat to this scheme, however, is the recognition that a single agonist may activate a variey of receptor subtypes (for example, for acetylcholine the ml-m5 muscarinic cholinoceptors), each of which in turn may be coupled to one or more signal transduction pathways.

Multiple regulatory elements in the interleukin-6 gene mediate induction by prostaglandins, cyclic AMP, and lipopolysaccharide.

Induction of interleukin-6 (IL-6) gene expression is mediated by numerous agents involving all major signal transduction pathways. We have compared the effects of prostaglandins and their second messenger cyclic AMP (cAMP) with the effect of lipopolysaccharide (LPS) on IL-6 gene expression. We demonstrate that secretion of IL-6 is induced by cAMP in murine monocytic PU5-1.8 cells, even though to a lesser extent than by LPS. Nevertheless, cAMP and prostaglandins of the E series in the presence of theophylline induce transcription of the IL-6 promoter more strongly than LPS, suggesting distinctive effects of cAMP and LPS on posttranscriptional events. Mutations within four regulatory elements, namely, the multiple response element (MRE), AP-1, NF-IL6, and NF-kappa B sites, significantly reduce, but do not completely abrogate, inducibility by cAMP and prostaglandin E1, whereas alterations of four additional sites have no effects. LPS-induced promoter activity, however, is almost completely abolished by mutations in the NF-kappa B site, suggesting that a single regulatory element is crucial for inducibility by LPS. Stimulation by cAMP is correlated with the binding of inducible factors to the AP-1, NF-IL6, and NF-kappa B elements, whereas factors binding to the MRE are constitutively expressed. Recombinant cAMP response element-binding protein binds to the MRE, indicating a potential role for this factor in the cAMP response. Our results suggest that cAMP and prostaglandins act through multiple, partially redundant regulatory elements to induce IL-6 expression in monocytic cells. Nuclear events that overlap partially with the LPS response but also exhibit distinctive features are involved.

Guinea Pig Histamine H1 Receptor. II. Stable Expression in Chinese Hamster Ovary Cells Reveals the Interaction with Three Major Signal Transduction Pathways

A cDNA encoding a guinea pig histamine H1 receptor was stably expressed in Chinese hamster ovary (CHO) cells. In one resulting clone, named CHO(H1), the H1 receptor was found to be coupled to several major signal transduction pathways. In each case the involvement of a Gi/Go protein with pertussis toxin (PTX) was assessed, as well as the influence of extracellular Ca2+ and of protein kinase C activation by phorbol 12-myristate 13-acetate (PMA). Histamine induced, in a PTX- and PMA-insensitive manner, a biphasic increase in the intracellular Ca2+ level of which only the second sustained phase was dependent on the extracellular Ca2+ level. Histamine also caused a threefold elevation of inositol phosphate production, which was PTX-insensitive, but slightly inhibited by PMA and reduced by 75% in the absence of extracellular Ca2+. Histamine also caused a massive release of arachidonic acid, which occurred in a Ca(2+)- and PMA-sensitive manner, probably through the activation of a cytosolic phospholipase A2, which partly involves coupling to a PTX-sensitive G protein. In comparison, in HeLa cells endowed with a native H1 receptor, the histamine-induced arachidonic acid release was also Ca(2+)- and PMA-sensitive, but totally PTX-insensitive. Finally, in CHO(H1) cells, histamine in very low concentrations potentiated the cyclic AMP accumulation induced by forskolin. This response appeared to be insensitive to PTX, extracellular Ca2+, and PMA.(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations in gene expression and signal transductions in human melanocytes and melanoma cells.

The development of techniques to cultivate human primary melanocytes in vitro has provided the technical foundation for understanding the biology of this cell. Human melanocytes require various growth factors and agents for proliferation in vitro. These compounds activate two major signal transduction pathways: a calcium- and phospholipid-dependent (protein kinase C or PKC) pathway and a cyclic AMP (cAMP)-dependent (protein kinase A or PKA) pathway. Alterations in these signal transduction pathways coupled with changes in specific genes (protooncogenes, growth factors, and tumor suppressor genes) have been observed in human melanoma cells compared with normal melanocytes. Our own work indicates that loss in the expression of the PKC beta II isotype is a common, if not universal, alteration that occurs early in human melanocyte transformation. In this review, we concentrate on alterations in the signal transduction pathways in human melanocytes and melanoma cells and delineate how an understanding of these changes may allow us to understand the molecular mechanisms involved in human melanocyte transformation.

Cholinergic and serotonergic stimulation of phosphoinositide hydrolysis is decreased in Alzheimer's disease

Agonist-stimulated phosphoinositide (PPI) hydrolysis is a major signal transduction pathway in brain. These studies investigated neurotransmitter stimulated PPI hydrolysis in postmortem human brain. Preliminary studies using rat brain suggested that moderate postmortem delay has little effect on PPI hydrolysis and that human tissue might be reliably studied for differences in receptor-PLC coupling. Studies in human brain membranes (frontal cortex) indicated that the time course for GTP γS and carbachol/GTPγS-stimulated PPI hydrolysis was linear for at least 20 min. GTPγS-stimulated [ 3H] inositol phosphate (InsP) formation was enhanced by carbachol (232%) and 5-Hydroxytryptamine (5HT - 147%). SAX-HPLC seperation of [ 3H] inositol polyphosphates indicated that the major isomer of inositol trisphosphate (InsP 3) was Ins(1,4,5)P3, the expected product of PtdIns(4,5)P2 hydrolysis. Ca 2+ increased PPI hydrolysis progressively from 100 nM through 50 μM and synergistically enhanced carbachol/GTPγS stimulation. Comparisons of age-matched controls with Alzheimer's patients indicated that GTPγS, carbachol/GTPγS, and 5HT/GTPγS-stimulation of PPI hydrolysis is reduced approximately 50% in membranes prepared from Alzheimer's patients. Ca 2+ stimulation of PPI hydrolysis was not different between controls and Alzheimer's patients suggesting that muscarinic cholinergic and serotonergic receptors are uncoupled from PLC in Alzheimer's disease. These studies indicate that there are changes in cholinergic and serotonergic signal transduction in Alzheimer's disease. Further, this method can be used to study signal transduction events in postmortem human brain.

Activation of cAMP-dependent protein kinase alters the chromatin structure of the urokinase-type plasminogen activator gene promoter.

In LLC-PK1 cells, the urokinase-type plasminogen activator (uPA) gene is induced by two of the major signal transduction pathways, the protein kinase C (PKC) and the cAMP-dependent protein kinase (PKA) pathways. We have analyzed the chromatin structure of 26 kb of the uPA gene locus and have shown that PKA activation but not PKC activation induce major chromatin structural alterations in the uPA gene promoter. In uninduced cells, several DNase I hypersensitive (HS) sites were detected in the 5' and 3' flanking regions but not in the transcribed region. Two of the sites correspond to previously characterized regulatory sites: a cAMP responsive site at nucleotide position -3500 with respect to the initiation site, and the PEA3/AP1 site at -2100 that mediates PKC activation. After the activation of PKA but not PKC, a strong HS site was induced at -2600. Functional analysis of this region revealed cAMP responsive activity. Chromatin structural alterations again brought about specifically by PKA but not by PKC were were also detected in the upstream of the promoter by topoisomerase I cleavage site analysis, with two prominent sites appearing at -2800 and -3300. These results suggest that the strong cAMP induction of the uPA gene requires structural alterations that permit cooperative interactions between the multiple cAMP responsive sites.

Involvement of PKC-alpha in PDGF-mediated mitogenic signaling in human mesangial cells.

Platelet-derived growth factor (PDGF) is a potent mitogen for a variety of cells. The calcium/phospholipid-dependent protein kinase C (PKC) represents a major signal transduction pathway for many growth stimuli including PDGF. Various isoforms of PKC are differentially expressed in the same or in different cells and tissues, and diverse stimuli may selectively activate one or more PKC isoforms. We studied the effect of PDGF on DNA synthesis and on the activity of PKC in human mesangial cells and vascular pericytes in the glomerular microvascular bed. PKC activity was measured as the amount of phosphorylated myelin basic protein-derived peptide substrate in the absence and presence of an inhibitor, a peptide spanning the pseudosubstrate region of PKC. PDGF (15 ng/ml) stimulated PKC activity within 5 min, and the effect was sustained for 60 min. Pretreatment of mesangial cells with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), an inhibitor of PKC, abolished the stimulation of PKC and DNA synthesis in response to PDGF. This effect of H-7 was specific, because H-7 did not inhibit the tyrosine phosphorylation of the PDGF receptor in vivo when added to the cells or the in vitro kinase activity in the PDGF beta-receptor immunoprecipitates. Utilizing isotype-specific antibodies against PKC-alpha, -beta, or -gamma for immunoprecipitation of PDGF-treated mesangial cell extracts, followed by assay of PKC activity, we demonstrated the activation of PKC-alpha only. Northern blot analysis of mRNA prepared from mesangial cells also revealed two transcripts, 3.7 kb and 1.8 kb, that hybridized with cDNA specific for PKC-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of the phosphorelay and the initiation of sporulation in Bacillus subtilis.

The initiation of sporulation of bacteria is a complex cellular event controlled by an extensive network of regulatory proteins that serve to ensure that a cell embarks on this differentiation process only when appropriate conditions are met. The major signal-transduction pathway for the initiation of sporulation is the phosphorelay, which responds to environmental, cell cycle, and metabolic signals, and phosphorylates the Spo0A transcription factor activating its function. Signal input into the phosphorelay occurs through activation of kinases to phosphorylate a secondary-messenger protein, Spo0F. Spo0F-P serves as a substrate for phosphoprotein phosphotransferase, Spo0B, which phosphorylates Spo0A. The pathway is regulated by transcriptional control of its component proteins and by regulating phosphate flux through the pathway. This is accomplished by several regulatory proteins, and by activated Spo0A, which regulates transcription of genes for its own synthesis. Spo0A-P indirectly controls the transcription of numerous genes by regulating the level of other transcription regulators and directly activates the transcription of several regulatory proteins and sigma factors required for progression to the second stage of sporulation. Although the pathway and regulatory proteins have been identified, the signals and effectors for these regulators remain a mystery.

Identification of a G protein alpha subunit from Neurospora crassa that is a member of the Gi family

Heterotrimeric G proteins, consisting of alpha, beta, and gamma subunits, are implicated in major signal transduction pathways controlling a diversity of functions in eukaryotic organisms. In the filamentous fungus Neurospora crassa, G proteins are implicated in the regulation of several environmental responses. As a first step in studying the role of G proteins in these processes, we have cloned the genes for two alpha subunits, gna-1 and gna-2, from Neurospora. The genes are located on different chromosomes and are differentially regulated during asexual development. The encoded proteins (Gna-1 and Gna-2) are the same size as members of the Gi-alpha family (approximately 40 kDa). The Gna-1 protein sequence is 55% identical overall to members of the Gi family and contains the consensus sequences for ADP-ribosylation by pertussis toxin and incorporation of myristic acid, which are found in this group. These properties make Gna-1 the first identified microbial alpha subunit to be a member of any class. Furthermore, incubation of a N. crassa plasma membrane fraction with pertussis toxin results in ADP-ribosylation of a protein substrate which is the approximate size of Gna-1. The predicted Gna-2 protein sequence does not share a high degree of sequence identity with the Gi class. However, the coding region contains at least one intron in a position conserved in the Gi family. We propose that the Gi family of alpha subunits is ancient and during evolution may have first appeared in filamentous fungi.

Signal transduction pathways and cellular intoxication with Clostridium difficile toxins

In cultured cells the cytopathic effects (CPE) of Clostridium difficile toxins A and B are superficially similar. The irreversible CPEs involve a reorganization of the cytoskeleton, but the molecular details of the mechanism(s) of action are unknown. As part of the work to elucidate the events leading to the CPE, cultured cells were preincubated with agents known to either stimulate or inhibit some major signal transduction pathways, whereupon toxin was added and the development of the CPE was followed. Both toxin-induced CPEs were enhanced by phorbol esters and mezerein, which stimulate protein kinase C, while they were inhibited by the phospholipase A2 inhibitors quinacrine and 4-bromophenacylbromide. Agents affecting certain G-proteins, cGMP and cAMP levels, phosphatases, prostacyclin, lipoxygenase, and phospholipase C did not affect the development of the CPE of either toxin. Thus, the cytoskeletal effect induced by toxins A or B appears to require PLA2 activity and involves at least part of a protein kinase C-dependent pathway, but not pertussis toxin-sensitive G-proteins, cyclic nucleotides, eicosanoid metabolites, or phospholipase C activity. In addition, both toxins were shown to activate phospholipase A2.

Hormonal control of gene expression: multiplicity and versatility of cyclic adenosine 3',5'-monophosphate-responsive nuclear regulators.

A central goal of modern biology is to obtain insights into the molecular mechanisms by which extracellular stimuli modulate the expression of specific genes in a temporal and/or tissue-specific manner. These mechanisms are central to processes such as cellular growth, differentiation, and response to environmental changes. This review focuses on the mechanisms that underlie CAMP-induced changes in gene expression. The CAMP-signalling pathway is one of the major intracellular signal transduction pathways operating in eukaryotic cells (1, 2). The intracellular concentration of CAMP is increased upon stimulation of the adenylyl cyclases. This results in activation of the catalytic subunit of protein kinase-A (PKA), which, in turn, phosphorylates a number of substrates (2). Several genes have been found to be inducible upon an increase in intracellular CAMP concentrations (2, 4). In some cases, activation of specific G-protein-coupled receptors by the respective ligands has been shown to result in either stimulation or repression of CAMP-induced gene expression (4). The identification of CAMP response elements (CREs) in the promoters of several CAMP-inducible genes provided the first link between activation of PKA and CAMP-induced gene expression (5-7; see Ref. 4 for a review). Subsequently, it was shown that specific nuclear factors bind to the various CREs. These proteins were of heterogeneous size and characteristics, suggesting the presence of a family (8). The identification and cloning of the gene encoding for

TYR-MIF-1, but not MIF-1 or morphine, decreases cyclic AMP-dependent protein kinase activity extracted from HeLa cells

The biochemical effects of Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH 2), a naturally occurring opiate modulator, on cellular function are poorly understood. We looked for changes in cyclic AMP-dependent protein kinase (cAMP-kinase; protein kinase A) after treating HeLa cells with this peptide. Binding sites for Tyr-MIF-1 (IC 50 = 83 n M), but not for DAMGO, a mu opiate, were found on these cells. Brief incubation of HeLa cells with 10 −7 to 10 −5 M Tyr-MIF-1 significantly decreased, in a rapid and dose-dependent manner, cAMP-kinase activity in cell extracts assayed in reaction mixtures with added cAMP but not the much lower basal enzymatic activity that is dependent on endogenous cAMP. In contrast, incubation of the cells with the related peptide MIF-1 (Pro-Leu-Gly-NH 2) or with morphine did not significantly alter cAMP-kinase activity. This study shows that Tyr-MIF-1 can affect part of a major signal transduction pathway independently of binding to opiate receptors.