Temperature-sensitive Gbeta mutants discriminate between G protein-dependent and -independent signaling mediated by serpentine receptors.

Deletion of the single gene for the Dictyostelium G protein beta-subunit blocks development at an early stage. We have now isolated temperature-sensitive alleles of Gbeta to investigate its role in later development. We show that Gbeta is directly required for adenylyl cyclase A activation and for morphogenetic signaling during the entire developmental program. Gbeta was also essential for induction of aggregative gene expression by cAMP pulses, a process that is mediated by serpentine cAMP receptors (cARs). However, Gbeta was not required for cAR-mediated induction of prespore genes and repression of stalk genes, and neither was Gbeta needed for induction of prestalk genes by the differentiation inducing factor (DIF). cAMP induction of prespore genes and repression of stalk genes is mediated by the protein kinase GSK-3. GSK-3 also determines cell-type specification in insects and vertebrates and is regulated by the wingless/wnt morphogens that are detected by serpentine fz receptors. The G protein-dependent and -independent modes of cAR-mediated signaling reported here may also exist for the wingless/wnt signaling pathways in higher organisms.     

Cyclic AMP-elevating agents induce the expression of MAP kinase phosphatase-1 in PC12 cells

Stimulation of pheochromocytoma PC12 cells by cAMP-elevating agents caused the induction of the immediate early gene 3CH134, which encodes MAP kinase phosphatase-1 (MKP-1). Forskolin was as potent as serum in stimulating MKP-1 gene expression, whereas dibutyryl-cAMP and neuropeptide PACAP were less effective. Induction of the MKP-1 gene was accompanied by neo-synthesis of MKP-1 protein. MAP kinase activation was not involved in the cAMP-induced MKP-1 gene expression. The MAP kinase inactivation, that would result from MKP-1 induction in response to increased intracellular cAMP level, contributes to explain how hormones or neurotransmitters signaling through cAMP influence cell growth and differentiation.     

Evidence that phosphatidylcholine-specific phospholipase C is a key molecule mediating insulin-induced enhancement of gene expression from human cytomegalovirus promoter in CHO cells

The signal transduction from insulin to its receptors and Ras has been extensively studied, while little has been reported beyond these steps. We found that the expression of human interleukin 6 gene under the control of immediate early gene promoter of human cytomegalovirus was enhanced by insulin sitmulation in Chinese hamster ovary cells. The induction effect of insulin was not significantly affected by inhibitors or activators of conventional protein kinase C, cAMP dependent protein kinase and phosphoinositide -3 kinase, however, pre-incubation of the cells with D609, a specific inhibitors of phosphatidylcholine-specific phospholipase C completely abolished the induction effect. These results clearly demonstrate that phosphatidylcholine-specific phospholipase C is a key molecule mediating insulin-induced enhancement of hIL-6 expression from the human cytomegalovirus promoter in Chinese hamster ovary cells and strongly suggest that it plays an important role in the insulin signaling pathways.Abbreviations CHO – Chinese hamster ovary; hCMV promoter – immediate early gene promoter of human cytomegalovirus; hIL-6 – human interleukin 6; PC-PLC-phosphatidylcholine-specific phospholipase C; PI-3 kinase – phosphoinositide 3 kinase; PKA – cAMP dependent protein kinase; PKC – protein kinase C.     

DC6, a novel type of Dictyostelium discoideum gene regulated by secreted factors but not by cAMP

We have isolated a gene, DC6, which is induced in the early aggregative stages of development in Dictyostelium discoideum. The increase in DC6 expression is dependent on high cell density, indicating that cellular interactions are required for DC6 induction. In low-cell-density cultures, the induction of DC6 occurs if supplied with conditioned medium of developing cells, suggesting that secreted factors are involved in DC6 induction. The expression of DC6 is not affected (1) in the presence of caffeine or adenosine, which block the production or the action of cAMP pulses, (2) in the presence of high concentrations of cAMP, or (3) in mutant strains (Synag7 and FrigidA), which are defective in transduction pathways of cAMP pulse signals. These results indicate that the induction of DC6 does not require extracellular cAMP pulse signals, which are known to regulate the expression of many genes in the early development. Independence of cAMP signals and dependence on other unknown cellular interactions are prominent characteristics of DC6.     

Synergistic induction of ornithine decarboxylase by diacylglycerol, A23187, and cholera toxin in guinea pig lymphocytes

When guinea pig lymphocytes were cultured with 1-oleoyl-2-acetyl-glycerol (OAG), A23187, and cholera toxin, ornithine decarboxylase activity was induced synergistically, peaking at 6 h. Addition of 12-O-tetradecanoyl-phorbol 13-acetate (TPA), A23187, and dibutyryl cAMP caused the same kind of induction. Cholera toxin potentiated the ability of A23187 to induce ornithine decarboxylase, but not that of OAG. Dibutyryl cAMP augmented the induction caused by A23187 but not by TPA. These results suggest that both the activation of Ca++-sensitive, phospholipid-dependent protein kinase (protein kinase C) and the increase in intracellular levels of Ca++ and cAMP are necessary for this induction. cAMP may potentiate the induction by modulating a Ca++ messenger system other than that for protein kinase C activation.     

cAMP regulation of early gene expression in signal transduction mutants of Dictyostelium

We have examined the regulation of three early developmentally regulated genes in Dictyostelium. Two of these genes (D2 and M3) are induced by pulses of cAMP and the other (K5) is repressed. Expression of these genes has been examined in a number of developmental mutants that are specifically blocked in various aspects of the signal transduction/cAMP relay system involved in aggregation and control of early development. The mutant strains include Synag mutants, which are blocked in receptor-mediated activation of adenylate cyclase and do not relay cAMP pulses; FrigidA mutants, which are blocked in receptor-mediated activation of both adenylate cyclase and the putative phosphoinositol bisphosphate (PIP2) turnover pathway and appear to be mutations in the gene encoding one of the G alpha protein subunits; and a StreamerF allele, which lacks cGMP-specific cGMP phosphodiesterase. From the analysis of the developmental expression of these genes under a variety of conditions in these mutant strains, we have drawn a number of conclusions concerning the modes of regulation of these genes. Full induction of D2 and M3 genes requires cAMP interaction with the cell surface receptor and an "oscillation" of the receptor between active and adapted forms. Induction of these genes does not require activation of the signal transduction pathway that leads to adenylate cyclase activation and cAMP relay, but does require activation of other receptor-mediated intracellular signal transduction pathways, possibly that involving PIP2 turnover. Likewise, repression of the K5 gene requires pulses of cAMP. Expression of this gene is insensitive to cAMP pulses in FrigidA mutants, suggesting that a signal transduction pathway is necessary for its repression. Results using the StreamerF mutant suggest that the rise in cGMP in response to cAMP/receptor interactions may not be directly related to control of the pulse-induced genes. In addition, we have examined the effect of caffeine, which M. Brenner and S.D. Thomas (1984, Dev. Biol., 101, 136-146) showed preferentially blocks the cAMP relay system by blocking receptor-mediated activation of adenylate cyclase. We show that in many of the mutants and in an axenic wild-type strain, caffeine causes the induction of pulse-induced gene expression to almost wild-type levels or in some cases to higher than wild-type levels. Our data suggest that caffeine works by activating some step in the signal transduction pathway that must lie downstream from both the receptor and at least one of the G proteins and thus has effects other than simply blocking the receptor-mediated cAMP relay system.     

Cholera toxin induces expression of the immediate-early response gene JE via a cyclic AMP-independent signaling pathway.

Cholera toxin (CT) activates expression of two immediate-early response genes (JE and TIS10) in quiescent BALB/c 3T3 cells. Increases in cyclic AMP (cAMP) levels in response to CT are likely responsible for the induction of TIS10 gene expression, since treatment with 8-Br-cAMP and increasing the intracellular levels of cAMP by treatment with forskolin induce TIS10 gene expression. In contrast, neither forskolin nor 8-Br-cAMP induces JE gene expression. 3-Isobutyl-1-methylxanthine, which stabilizes intracellular cAMP, potentiates CT-induced TIS10 gene expression but has no effect on CT-induced JE gene expression. Thus, induction of JE by CT is independent of the cAMP produced in response to CT. Induction of JE by CT does not require protein kinase C (PKC), since depleting cells of PKC activity has no effect on the induction of JE by CT. CT-induced expression of JE can be distinguished from CT-induced TIS10 gene expression by using protein kinase inhibitors and inhibitors of arachidonic acid metabolism, further suggesting distinct signaling pathways for CT-induced JE and TIS10 gene expression. Thus, induction of JE gene expression by CT results from the activation of an intracellular signaling pathway that is independent of cAMP production. This pathway is independent of PKC activity and uniquely sensitive to inhibitors of protein kinases and arachidonic acid metabolism.     

Genome-wide expression analyses of gene regulation during early development of Dictyostelium discoideum

Using genome-wide microarrays, we recognized 172 genes that are highly expressed at one stage or another during multicellular development of Dictyostelium discoideum. When developed in shaken suspension, 125 of these genes were expressed if the cells were treated with cyclic AMP (cAMP) pulses at 6-min intervals between 2 and 6 h of development followed by high levels of exogenous cAMP. In the absence of cAMP treatment, only three genes, carA, gbaB, and pdsA, were consistently expressed. Surprisingly, 14 other genes were induced by cAMP treatment of mutant cells lacking the activatable adenylyl cyclase, ACA. However, these genes were not cAMP induced if both of the developmental adenylyl cyclases, ACA and ACR, were disrupted, showing that they depend on an internal source of cAMP. Constitutive activity of the cAMP-dependent protein kinase PKA was found to bypass the requirement of these genes for adenylyl cyclase and cAMP pulses, demonstrating the critical role of PKA in transducing the cAMP signal to early gene expression. In the absence of constitutive PKA activity, expression of later genes was strictly dependent on ACA in pulsed cells.     

Arachidonic acid and cyclic adenosine monophosphate stimulation of c-fos expression by a pathway independent of phorbol ester-sensitive protein kinase C

The stimulation of cell proliferation by platelet-derived and other growth factors is associated with a rapid increase in the expression of the c-fos protooncogene. We and others have shown that phosphosphoinositide turnover and protein kinase C play a role in the activation of this gene by growth factors, but that a second, kinase C-independent pathway(s) exist. Because cAMP potentiates the actions of a number of growth factors and is elevated in platelet-derived growth factor-stimulated Swiss 3T3 cells, we examined the ability of cAMP to stimulate c-fos expression in this cell type. Forskolin, a direct activator of adenylate cyclase, elicited marked increases in c-fos mRNA levels. Receptor-mediated activation of adenylate cyclase by prostaglandin E1 and stimulation with the cAMP analog 8-bromo-cAMP also enhanced c-fos expression. In cells made protein-kinase C deficient, c-fos induction by phorbol ester was abolished; by contrast, c-fos was still induced by cAMP-elevating agents in protein kinase C-depleted cells. Platelet-derived growth factor causes cAMP accumulation by stimulating arachidonic acid release and the formation of prostaglandins capable of activating adenylate cyclase. The addition of arachidonic acid and the arachidonate metabolite prostaglandin E2 to Swiss 3T3 cultures stimulated c-fos expression. These data suggest the existence of a pathway from growth factor receptor to gene induction that is mediated by cAMP and does not depend on a phorbol ester-sensitive protein kinase C.     

A developmentally regulated, putative serine/threonine protein kinase is essential for development in Dictyostelium.

Using PCR technology, we have cloned parts of three developmentally regulated putative serine/threonine kinases from Dictyostelium. All show significant homology to members of the cAMP-dependent protein kinase A/protein kinase C subfamilies. A genomic clone encoding one of these, DdPK3, has been isolated and sequenced. The open reading frame encodes a protein of 648 amino acids with the conserved kinase domain in the C-terminal half. The protein encoded by this gene is unusual in that it contains long homopolymer runs in the N-terminal half of the protein, including a long run of 88 amino acids in which 73 are glutamine residues. To examine the function of DdPK3, a gene disruption was created via homologous recombination. Ddpk3- cells do not aggregate by themselves but will co-aggregate with wild-type cells. However, after aggregation these cells are 'sloughed off' and do not proceed further through development, but are found as a discrete mass alongside the fruiting body formed by the wild-type cells. Analysis of signal transduction pathways indicates that cAMP pulse-induced expression of aggregation stage-specific genes is normal in Ddpk3- cells, as is induction of the prestalk gene Ddras in single cell assays. However, cAMP induction of the late promoters of cAMP receptor cAR1 and of two prespore-specific genes is absent under similar conditions. These cells show normal activation of adenylate cyclase and normal phosphorylation of the G alpha protein G alpha 2 in response to cAMP. The possible role of DdPK3 in Dictyostelium development is discussed.     

Control of cAMP-induced gene expression by divergent signal transduction pathways.

A compilation of literature data and recent experiments led to the following conclusions regarding cyclic adenosine 3':5' monophosphate (cAMP) regulation of gene expression. Several classes of cAMP-induced gene expression can be discriminated by sensitivity to stimulation kinetics. The aggregation-related genes respond only to nanomolar cAMP pulses. The prestalk-related genes respond both to nanomolar pulses and persistent micromolar stimulation. The prespore specific genes respond only to persistent micromolar stimulation. The induction of the aggregation- and prestalk-related genes by nanomolar cAMP pulses may share a common transduction pathway, which does not involve cAMP, while involvement of the inositol 1,4,5-trisphosphate (IP3)/Ca2+ pathway is unlikely. Induction of the expression of prespore and prestalk-related genes by micromolar cAMP stimuli utilizes divergent signal processing mechanisms. cAMP-induced prespore gene expression does not involve cAMP and probably also not cyclic guanosine 3'.5' monophosphate (cGMP) as intracellular intermediate. Involvement of cAMP-induced phospholipase C (PLC) activation in this pathway is suggested by the observation that IP3 and 1,2-diacylglycerol (DAG) can induce prespore gene expression, albeit in a somewhat indirect manner and by the observation that Li+ and Ca2+ antagonists inhibit prespore gene expression. Cyclic AMP induction of prestalk-related gene expression is inhibited by IP3 and DAG and promoted by Li+, and is relatively insensitive to Ca2+ antagonists, which indicates that PLC activation does not mediate prestalk-related gene expression. Neither prespore nor prestalk-related gene expression utilizes the sustained cAMP-induced pHi increase as intracellular intermediate.     

A functional role for interleukin (IL)-4-driven cyclic amp accumulation in human b lymphocytes

Interleukin-4 (IL-4) regulates the expression of the 55-kDa alpha-subunit (CD25) of the IL-2 receptor complex in human B lymphocytes. This report suggests that the cAMP/protein kinase A (PKA) component of the IL-4 receptor signalling programme in human tonsillar B cells has a functionally important role in regulating expression of the CD25 gene by attenuating activity of a protein binding to a potent negative regulatory element (NRE) in the CD25 promoter; this effect can be mimicked by agents that elevate cAMP and blocked by inhibitors of PKA but not protein kinase C (PKC). In a B-cell line that fails to elevate cAMP, attenuate NRE-binding protein (NRE-BP) activity or express CD25 following IL-4 treatment, stimulation of cAMP accumulation by forskolin facilitates IL-4-mediated induction of both the endogenous gene and an exogenous reporter gene under the control of a minimal promoter/enhancer fragment of the CD25 gene.     

Examination of the involvement of protein kinase A in D2 dopamine receptor antagonist-induced immediate early gene expression

Immediate early genes (IEGs) are induced by different signaling pathways. It has been proposed that D2 dopamine receptor blockade induces IEG expression through activation of protein kinase A (PKA), although few studies have examined this issue in vivo. We infused the PKA inhibitor H-89 into the striatum of male rats, followed 30 min later by systemic administration of eticlopride. Eticlopride-induced c-fos and zif268 mRNA expression in striatum was not blocked by H-89. In addition, eticlopride did not produce measurable levels of PKA activity in striatum, whereas the cAMP activator Sp-8-Br-cAMPs increased levels of activated PKA. Neither the adenosine A2a receptor agonist CGS 21680 nor the phosphodiesterase-4 inhibitor rolipram, each of which should increase PKA activation, potentiated eticlopride-induced IEG expression. To test whether other signaling pathways are involved in eticlopride-mediated gene induction, we also infused inhibitors of the mitogen-activated and calcium/calmodulin-dependent protein kinases into animals and then treated them with eticlopride. The data suggest that eticlopride-induced IEG expression is not solely dependent on these kinases either. These data suggest that PKA activation may not be necessary for induction of IEGs by D2 dopamine receptor antagonists and that other intracellular signaling pathways may be involved.