Biomedical subjects

P Schaap

Publications and source records attributed to P Schaap.

At least 19 recordsLinked to original sources

PubMed2001

Adenylyl cyclase A expression is tip-specific in Dictyostelium slugs and directs StatA nuclear translocation and CudA gene expression.

cAMP oscillations, generated by adenylyl cyclase A (ACA), coordinate cell aggregation in Dictyostelium and have also been implicated in organizer function during multicellular development. We used a gene fusion of the ACA promoter with a labile lacZ derivative to study the expression pattern of ACA. During aggregation, most cells expressed ACA, but thereafter expression was lost in all cells except those of the anterior tip. Before aggregation, ACA transcription was strongly upregulated by nanomolar cAMP pulses. Postaggregative transcription was sustained by nanomolar cAMP pulses, but downregulated by a continuous micromolar cAMP stimulus and by the stalk-cell-inducing factor DIF. Earlier work showed that the transcription factor StatA displays tip-specific nuclear translocation and directs tip-specific expression of the nuclear protein CudA, which is essential for culmination. Both StatA and CudA were present in nuclei throughout the entire slug in an aca null mutant that expresses ACA from the constitutive actin15 promoter. This suggests that the tip-specific expression of ACA directs tip-specific nuclear translocation of StatA and tip-specific expression of CudA.

Active Transport, Cell Nucleus↗

PubMed2000

Guanylyl cyclase activity associated with putative bifunctional integral membrane proteins in Plasmodium falciparum.

We report here that guanylyl cyclase activity is associated with two large integral membrane proteins (PfGCalpha and PfGCbeta) in the human malaria parasite Plasmodium falciparum. Unusually, the proteins appear to be bifunctional; their amino-terminal regions have strong similarity with P-type ATPases, and the sequence and structure of the carboxyl-terminal regions conform to that of G protein-dependent adenylyl cyclases, with two sets of six transmembrane sequences, each followed by a catalytic domain (C1 and C2). However, amino acids that are enzymatically important and present in the C2 domain of mammalian adenylyl cyclases are located in the C1 domain of the P. falciparum proteins and vice versa. In addition, certain key residues in these domains are more characteristic of guanylyl cyclases. Consistent with this, guanylyl cyclase activity was obtained following expression of the catalytic domains of PfGCbeta in Escherichia coli. In P. falciparum, expression of both genes was detectable in the sexual but not the asexual blood stages of the life cycle, and PfGCalpha was localized to the parasite/parasitophorous vacuole membrane region of gametocytes. The profound structural differences identified between mammalian and parasite guanylyl cyclases suggest that aspects of this signaling pathway may be mechanistically distinct.

Amino Acid Sequence↗

PubMed1999

Fingerprinting of adenylyl cyclase activities during Dictyostelium development indicates a dominant role for adenylyl cyclase B in terminal differentiation.

Activation of cAMP-dependent protein kinase (PKA) triggers terminal differentiation in Dictyostelium, without an obvious requirement for the G-protein-coupled adenylyl cyclase, ACA, or the osmosensory adenylyl cyclase, ACG. A third adenylyl cyclase, ACB, was recently detected in rapidly developing mutants. The specific characteristics of ACA, ACG, and ACB were used to determine their respective activities during development of wild-type cells. ACA was highly active during aggregation, with negligible activity in the slug stage. ACG activity was not present at significant levels until mature spores had formed. ACB activity increased strongly after slugs had formed with optimal activity at early fruiting body formation. The same high activity was observed in slugs of ACG null mutants and ACA null mutants that overexpress PKA (acaA/PKA), indicating that it was not due to either ACA or ACG. The detection of high adenylyl cyclase activity in acaA/PKA null mutants contradicts earlier conclusions (B. Wang and A. Kuspa, Science 277, 251-254, 1997) that these mutants can develop into fruiting bodies in the complete absence of cAMP. In contrast to slugs of null mutants for the intracellular cAMP-phosphodiesterase REGA, where both intact cells and lysates show ACB activity, wild-type slugs only show activity in lysates. This indicates that cAMP accumulation by ACB in living cells is controlled by REGA. Both REGA inhibition and PKA overexpression cause precocious terminal differentiation. The developmental regulation of ACB and its relationship to REGA suggest that ACB activates PKA and induces terminal differentiation.

Adenylyl Cyclases↗

PubMed1999

Dictyostelium development-socializing through cAMP.

Starving Dictyostelium amoebae use cAMP as a chemoattractant to gather into aggregates, as a hormone-like signal to induce cell differentiation, and as an intracellular messenger to control stalk- and spore cell maturation and germination of spores. In this chapter we describe the respective roles of the three adenylyl cyclases ACA, ACB and ACG in controlling cAMP signaling during development and we discuss how cAMP signals are processed by the cells to trigger the large repertoire of gene regulatory events that is under control of this signal molecule.

Animals↗

PubMed1999

Evidence that the Dictyostelium Dd-STATa protein is a repressor that regulates commitment to stalk cell differentiation and is also required for efficient chemotaxis.

Dd-STATa is a structural and functional homologue of the metazoan STAT (Signal Transducer and Activator of Transcription) proteins. We show that Dd-STATa null cells exhibit several distinct developmental phenotypes. The aggregation of Dd-STATa null cells is delayed and they chemotax slowly to a cyclic AMP source, suggesting a role for Dd-STATa in these early processes. In Dd-STATa null strains, slug-like structures are formed but they have an aberrant pattern of gene expression. In such slugs, ecmB/lacZ, a marker that is normally specific for cells on the stalk cell differentiation pathway, is expressed throughout the prestalk region. Stalk cell differentiation in Dictyostelium has been proposed to be under negative control, mediated by repressor elements present in the promoters of stalk cell-specific genes. Dd-STATa binds these repressor elements in vitro and the ectopic expression of ecmB/lacZ in the null strain provides in vivo evidence that Dd-STATa is the repressor protein that regulates commitment to stalk cell differentiation. Dd-STATa null cells display aberrant behavior in a monolayer assay wherein stalk cell differentiation is induced using the stalk cell morphogen DIF. The ecmB gene, a general marker for stalk cell differentiation, is greatly overinduced by DIF in Dd-STATa null cells. Also, Dd-STATa null cells are hypersensitive to DIF for expression of ST/lacZ, a marker for the earliest stages in the differentiation of one of the stalk cell sub-types. We suggest that both these manifestations of DIF hypersensitivity in the null strain result from the balance between activation and repression of the promoter elements being tipped in favor of activation when the repressor is absent. Paradoxically, although Dd-STATa null cells are hypersensitive to the inducing effects of DIF and readily form stalk cells in monolayer assay, the Dd-STATa null cells show little or no terminal stalk cell differentiation within the slug. Dd-STATa null slugs remain developmentally arrested for several days before forming very small spore masses supported by a column of apparently undifferentiated cells. Thus, complete stalk cell differentiation appears to require at least two events: a commitment step, whereby the repression exerted by Dd-STATa is lifted, and a second step that is blocked in a Dd-STATa null organism. This latter step may involve extracellular cAMP, a known repressor of stalk cell differentiation, because Dd-STATa null cells are abnormally sensitive to the inhibitory effects of extracellular cyclic AMP.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1998

Two distinct signaling pathways mediate DIF induction of prestalk gene expression in Dictyostelium.

During Dictyostelium development, the differentiation inducing factor (DIF) triggers expression of the prestalk gene ecmB and induces stalk cell differentiation, a form of programmed cell death. The effects of DIF are mediated by a sustained increase in cytosolic Ca2+ levels. The Ca2+ ATPase inhibitor BHQ causes a similar rise in Ca2+ levels and also induces prestalk gene expression. We show here that Ca2+ is a specific intermediate for prestalk gene induction, since BHQ represses transcription of the cAMP-inducible aggregative gene PDE, the postaggregative gene CP2, and the prespore gene D19. The prestalk gene ecmA is also induced by DIF, but induction appears to occur in two steps, which occur within 1 h and after 2 h, respectively. The slow step shows the same kinetics as ecmB induction and similar to ecmB induction, this step is BHQ inducible and requires an initial round of protein synthesis. The fast step does not require protein synthesis and cannot be induced by BHQ. This indicates that in addition to the slow Ca2+-mediated pathway, there is probably a second fast Ca2+-independent signal transduction pathway for DIF.

Animals↗

PubMed1998

A novel adenylyl cyclase detected in rapidly developing mutants of Dictyostelium.

Disruption of either the RDEA or REGA genes leads to rapid development in Dictyostelium. The RDEA gene product displays homology to certain H2-type phosphotransferases, while REGA encodes a cAMP phosphodiesterase with an associated response regulator. It has been proposed that RDEA activates REGA in a multistep phosphorelay. To test this proposal, we examined cAMP accumulation in rdeA and regA null mutants and found that these mutants show a pronounced accumulation of cAMP at the vegetative stage that is not observed in wild-type cells. This accumulation was due to a novel adenylyl cyclase and not to the known Dictyostelium adenylyl cyclases, aggregation stage adenylyl cyclase (ACA) or germination stage adenylyl cyclase (ACG), since it occurred in an acaA/rdeA double mutant and, unlike ACG, was inhibited by high osmolarity. The novel adenylyl cyclase was not regulated by G-proteins and was relatively insensitive to stimulation by Mn2+ ions. Addition of the cAMP phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX) permitted detection of the novel adenylyl cyclase activity in lysates of an acaA/acgA double mutant. The fact that disruption of the RDEA gene as well as inhibition of the REGA-phosphodiesterase by IBMX permitted detection of the novel AC activity supports the hypothesis that RDEA activates REGA.

1-Methyl-3-isobutylxanthine↗

PubMed1998

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.

Adenylyl Cyclases↗

PubMed1998

Functional promiscuity of gene regulation by serpentine receptors in Dictyostelium discoideum.

Serpentine receptors such as smoothened and frizzled play important roles in cell fate determination during animal development. In Dictyostelium discoideum, four serpentine cyclic AMP (cAMP) receptors (cARs) regulate expression of multiple classes of developmental genes. To understand their function, it is essential to know whether each cAR is coupled to a specific gene regulatory pathway or whether specificity results from the different developmental regulation of individual cARs. To distinguish between these possibilities, we measured gene induction in car1 car3 double mutant cell lines that express equal levels of either cAR1, cAR2, or cAR3 under a constitutive promoter. We found that all cARs efficiently mediate both aggregative gene induction by cAMP pulses and induction of postaggregative and prespore genes by persistent cAMP stimulation. Two exceptions to this functional promiscuity were observed. (i) Only cAR1 can mediate adenosine inhibition of cAMP-induced prespore gene expression, a phenomenon that was found earlier in wild-type cells. cAR1's mediation of adenosine inhibition suggests that cAR1 normally mediates prespore gene induction. (ii) Only cAR2 allows entry into the prestalk pathway. Prestalk gene expression is induced by differentiation-inducing factor (DIF) but only after cells have been prestimulated with cAMP. We found that DIF-induced prestalk gene expression is 10 times higher in constitutive cAR2 expressors than in constitutive cAR1 or cAR3 expressors (which still have endogenous cAR2), suggesting that cAR2 mediates induction of DIF competence. Since in wild-type slugs cAR2 is expressed only in anterior cells, this could explain the so far puzzling observations that prestalk cells differentiate at the anterior region but that DIF levels are actually higher at the posterior region. After the initial induction of DIF competence, cAMP becomes a repressor of prestalk gene expression. This function can again be mediated by cAR1, cAR2, and cAR3.

Adenosine↗

PubMed1997

Phosphorylation of chemoattractant receptors is not essential for chemotaxis or termination of G-protein-mediated responses.

In several G-protein-coupled signaling systems, ligand-induced receptor phosphorylation by specific kinases is suggested to lead to desensitization via mechanisms including receptor/G-protein uncoupling, receptor internalization, and receptor down-regulation. We report here that elimination of phosphorylation of a chemoattractant receptor of Dictyostelium, either by site-directed substitution of the serines or by truncation of the C-terminal cytoplasmic domain, completely prevented agonist-induced loss of ligand binding but did not impair the adaptation of several receptor-mediated responses including the activation of adenylyl and guanylyl cyclases and actin polymerization. In addition, the phosphorylation-deficient receptors were capable of mediating chemotaxis, aggregation, and differentiation. We propose that for chemoattractant receptors agonist-induced phosphorylation regulates surface binding activity but other phosphorylation-independent mechanisms mediate response adaptation.

Actins↗

PubMed1997

Two ras genes in Dictyostelium minutum show high sequence homology, but different developmental regulation from Dictyostelium discoideum rasD and rasG genes.

The social amoeba Dictyostelium discoideum expresses five ras genes at different stages of development. One of them, DdrasD is expressed during postaggregative development and transcription is induced by extracellular cAMP. A homologue of DdrasD, the DdrasG gene, is expressed exclusively during vegetative growth. We cloned two ras homologues Dmras1 and Dmras2 from the primitive species D. minutum, which show high homology to DdrasD and DdrasG and less homology to the other Ddras genes. In contrast to the DdrasD and DdrasG genes, both the Dmras1 and Dmras2 genes are expressed during the entire course of development. The expression levels are low during growth, increase at the onset of starvation and do not decrease until fruiting bodies have formed. Expression of neither Dmras1 or Dmras2 is regulated by cAMP. So even though the high degree of homology between the ras genes of different species suggests conservation of function, this function is apparently not associated with a specific developmental stage.

Amino Acid Sequence↗

PubMed1996

A slow sustained increase in cytosolic Ca2+ levels mediates stalk gene induction by differentiation inducing factor in Dictyostelium.

During Dictyostelium stalk cell differentiation, cells vacuolate, synthesize a cellulose cell wall and die. This process of programmed cell death is accompanied by expression of the prestalk gene ecmB and induced by the differentiation inducing factor DIF. Using cell lines expressing the recombinant Ca2+-sensitive photoprotein apoaequorin, we found that 100 nM DIF increases cytosolic Ca2+ ([Ca2+]i) levels from approximately 50 to 150 nM over a period of 8 h. The Ca2+-ATPase inhibitor 2,5-di(tert-butyl)-1,4-hydroquinone (BHQ) induced a similar increase in [Ca2+]i levels and induced expression of the prestalk gene ecmB to the same level as DIF. The [Ca2+]i increases induced by DIF and BHQ showed similar kinetics and preceded ecmB gene expression by approximately 1-2 h. The Ca2+ chelator 1,2-bis(o-aminophenoxy)-ethane-N,N,N'N'-tetra-acetic acid (BAPTA) efficiently inhibited the BHQ-induced [Ca2+]i increase and blocked DIF-induced expression of the ecmB gene. These data indicate that the effects of DIF on stalk gene expression are mediated by a sustained increase in [Ca2-]i. Sustained [Ca2+]i elevation mediates many forms of programmed cell death in vertebrates. The Dictyostelium system may be the earliest example of how this mechanism developed during early eukaryote evolution.

Animals↗

PubMed1996

Adenylyl cyclase G, an osmosensor controlling germination of Dictyostelium spores.

Dictyostelium cells express a G-protein-coupled adenylyl cyclase, ACA, during aggregation and an atypical adenylyl cyclase, ACG, in mature spores. The ACG gene was disrupted by homologous recombination. acg- cells developed into normal fruiting bodies with viable spores, but spore germination was no longer inhibited by high osmolarity, a fairly universal constraint for spore and seed germination. ACG activity, measured in aca-/ACG cells, was strongly stimulated by high osmolarity with optimal stimulation occurring at 200 milliosmolar. RdeC mutants, which display unrestrained protein kinase A (PKA) activity and a cell line, which overexpresses PKA under a prespore specific promoter, germinate very poorly, both at high and low osmolarity. These data indicate that ACG is an osmosensor controlling spore germination through activation of protein kinase A.

Adenylyl Cyclases↗

PubMed1996

Extracellular cAMP depletion triggers stalk gene expression in Dictyostelium: disparities in developmental timing and dose dependency indicate that prespore induction and stalk repression by cAMP are mediated by separate signaling pathways.

During Dictyostelium development, amoebae differentiate into spores and stalk cells. Earlier studies showed that extracellular cAMP is essential for induction of prespore differentiation and that cAMP represses stalk gene expression in vitro. We show that the repressive pathway is operative in vivo, because activation of the stalk-specific promoter region of the ecmB gene is strongly enhanced by overexpression of a phosphodiesterase that depletes extracellular cAMP. To test whether a single cAMP transduction pathway controls the choice between prespore or stalk cell differentiation, we compared the timing and dose dependency of the effects of cAMP on both responses. Cells acquire competence for cAMP repression of ecmB promoter activity 4 hr later than for prespore gene induction. Half-maximal prespore induction requires 30 microM stable cAMP analog Sp-cAMPs, while ecmB induction is half-maximally repressed by 200 nM Sp-cAMPs, which is equivalent to about 3 to 13 nM cAMP. At concentrations exceeding 10 microM, Sp-cAMPs stimulates ecmB expression from the intact promoter, but not from the stalk-specific subregion. These data suggest that distinct signaling pathways operating at different developmental stages control induction of prespore genes on one hand and repression of stalk genes on the other. Both stalk gene repression and prespore gene induction by Sp-cAMPs are antagonized by millimolar adenosine concentrations. However, an adenosine analog that is resistant to extracellular metabolism is active at 10 microM. Since adenosine inhibits cAMP binding to cAMP receptors, it may facilitate stalk gene expression by reducing the perceived cAMP concentration.

Adenosine↗

PubMed1995

Dual role of cAMP during Dictyostelium development.

cAMP plays an essential role during Dictyostelium development both outside and inside the cell. Membrane-bound receptors and adenylyl cyclase are responsible for sensing and producing extracellular cAMP, whereas a phosphodiesterase is responsible for maintaining a low basal level. The molecular events underlying this type of hormone like signalling, which are now beginning to be deciphered, will be presented, in the light of cAMP analogue studies. The importance of intracellular cAMP for cell differentiation has been demonstrated by the central role of the cAMP dependent protein kinase. Mutants as well as strains obtained by reverse genetics will be reviewed which lead to our current understanding of the role of intracelluar cAMP in the differentiation of both stalk and spore cells.

Adenylyl Cyclases↗

PubMed1995

cAMP-dependent protein kinase activity is essential for preaggregative gene expression in Dictyostelium.

Constitutive inhibition of cAMP-dependent protein kinase (PKA) in Dictyostelium cells blocks cell aggregation and development. We investigated the cause of the aggregation defect in transformants overexpressing dominant-negative PKA regulatory subunits (PKA-RM) under an actin 15 promoter. These mutants could not relay pulses of the chemoattractant cAMP, due to a defect in expression of the aggregative adenylyl cyclase (ACA) gene. Unstimulated and cAMP pulse-induced expression of other aggregative genes encoding the cAMP receptor cAR1, adhesive contact sites A and cAMP-phosphodiesterase were also strongly reduced in the mutants. Additionally, the expression of the discoidin I gene, that is expressed early in development in response to cell density sensing factors, was almost completely absent. These data are in interesting contrast with observations that cAMP relay and aggregative gene expression are normal in null mutants for the PKA catalytic (C) subunit and suggest the presence of multiple C subunit genes in Dictyostelium and an almost universal requirement for PKA activity in developmental gene expression.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1995

Regulation of Dictyostelium adenylylcyclases by morphogen-induced modulation of cytosolic pH or Ca2+ levels.

Inhibition of cAMP production and consequent inactivation of protein kinase A (PKA) by the putative morphogen ammonia has been suggested to block culmination and stalk cell differentiation in Dictyostelium. Since other weak bases mimic and weak acids act oppositely to ammonia, its effects were attributed to cytosolic or vesicular alkalinization; the latter resulting in impaired Ca2+ sequestration. We investigated whether weak bases and acids modulate the activity of the two Dictyostelium adenylylcyclases ACA and ACG in a manner consistent with their effects on development. It appeared that ammonia inhibits both ACG activity and ACA activation only transiently and does not significantly affect cAMP levels in slugs. Surprisingly, weak acids inhibit both ACA and ACG permanently, but do not affect secretion of cAMP as was suggested earlier. The effects of weak acids, which reduce cytosolic pH, are consistent with the pH dependence of ACA and ACG. In lysates, basal and GTP gamma S-stimulated ACA activity as well as ACG activity are optimal at pH 8 and are virtually absent below pH 7. ACG activity in cell lysates is completely insensitive to Ca2+, while GTP gamma S-stimulated ACA activity is maximally 50% reduced by supraphysiological Ca2+ concentrations. The observation that weak acids strongly inhibit ACA and ACG while promoting a PKA-dependent process such as stalk cell differentiation suggests that in Dictyostelium PKA can be activated in the absence of cAMP production.

Adenylyl Cyclases↗

PubMed1995

Transformation with vectors harboring the NEOR selection marker induces germination-specific adenylylcyclase activity in Dictyostelium cells.

Dictyostelium cells express an aggregative adenylylcyclase (ACA), responsible for oscillatory cAMP signaling, and a spore germination-specific adenylylcyclase (ACG). Overexpression of PKA regulatory (R) subunits blocks oscillatory cAMP signaling but increases basal cAMP levels, while neither ACA nor ACG mRNA could be detected. To test whether a novel type of adenylylcyclase (AC) was responsible for cAMP synthesis, dominant-negative PKA-R subunits (PKA-RM) and control R-subunits (PKA-RC) were overexpressed in ACA null mutants. Both transformations as well as transformation with an unrelated vector, carrying the same NEOR selection marker, induced an AC activity in growing cells with the biochemical characteristics of ACG. Similar vectors with a different URA selection marker did not increase AC activity. We conclude that the amino-glycoside phosphotransferase encoded by the very commonly used NEOR selection marker induces ectopic ACG activity in Dictyostelium cells.

Adenylyl Cyclases↗