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.     

cAMP-controlled gene expression in Dictyostelium mutants exhibiting abnormal patterns of developmental cAMP metabolism

We have previously described the developmental regulation of the M4-1 gene in Dictyostelium. M4-1 is expressed in undifferentiated, vegetative cells but is repressed after the establishment of the cAMP signal and relay system. We have suggested that regulation is dependent upon the establishment of intracellular cAMP signaling. We have now extended these studies of M4-1 developmentally regulated gene expression to a series of mutants that exhibit abnormal patterns of cAMP metabolism. These include cAMP signal deficient mutants and mutants which accumulate abnormally high or low levels of intracellular and/or extracellular cAMP. Mutant cells able to establish aggregates in the absence of normal cAMP signaling are unable to repress M4-1 expression. Rather these cells continue to express M4-1 at vegetative levels. In another mutant which displays precocious cAMP signaling, repression of M4-1 occurs more rapidly than is normal. A third mutant, with decreased signaling activity, exhibits delayed repression of M4-1 expression. Taken in conjunction with our previous results, these data suggest an intimate association between M4-1 gene regulation and changes in intracellular cAMP levels and that certain genes in Dictyostelium may be regulated by a cAMP-dependent mechanism which is common to other eukaryotes.     

Different molecular mechanisms for cAMP regulation of gene expression during Dictyostelium development

Alterations in cAMP concentrations have been implicated in developmentally regulated gene expression in Dictyostelium. Using a variety of culture conditions to control the metabolism of cAMP during cytodifferentiation, I have examined the role of the cyclic nucleotide in development. Conditions which allow intracellular synthesis of cAMP promote the normal developmental repression of gene M4-1 by a mechanism which is completely independent of the formation of multicellular aggregates. If, however, cells are inhibited in their ability to activate adenylate cyclase and, thus, intracellular cAMP signaling, they prove unable to repress M4-1, even in the presence of exogenous cAMP. In contrast, expression of genes which exhibit maximal activity after aggregate formation depends upon accumulation of extracellular cAMP. Inhibition of intracellular cAMP signaling does not prevent the expression of these genes if cultures are simultaneously exposed to high levels of exogenously added extracellular cAMP. These results indicate that there are at least two independent mechanisms involved in the developmental regulation of gene expression by cAMP in Dictyostelium. I discuss plausible molecular mechanisms through which cAMP might alter gene expression.     

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.     

Regulation of gene expression by the intracellular second messengers IP3 and diacylglycerol

In Dictyostelium, extracellular cAMP interacts specifically with cell-surface receptors to promote the accumulation of a variety of intracellular second messengers, such as 3'-5' cyclic adenosine monophosphate (cAMP) and 1,4,5 inositol trisphosphate (IP3). We and others have shown that activation of the cell-surface cAMP receptor can also modulate the expression of the Dictyostelium genome during development. In at least one instance, synthesis of intracellular cAMP is required for appropriate gene regulation. However, the induction of most cAMP-dependent gene expression can occur in the absence of receptor-mediated activation of adenylate cyclase and a consequent accumulation of intracellular cAMP. These results suggest that other intracellular second messengers produced in response to receptor activation may potentially act as signal transducers to modulate gene expression during development. In vertebrate cells, IP3 and diacylglycerol (DAG) are intracellular activators of specific protein kinases; they are produced in equimolar amounts by cleavage of phosphoinositol bisphosphate after a receptor-mediated activation of a membrane-bound phosphodiesterase. IP3 and, thus, by inference, diacyl-glycerol are synthesized in Dictyostelium as a response to cAMP interacting with its cell-surface receptor. Using defined conditions to inhibit the accumulation of extracellular cAMP, we have examined the effects of these compounds on the expression of genes that require cAMP for their maximal expression. Our results suggest that intracellular IP3 and DAG may in part mediate the action of extracellular cAMP on the expression of the Dictyostelium genome.     

Catabolite repression mutants of yeast

The mechaṅism of catabolite repression in yeast is not well understood, although it has been established that cAMP does not play a role similar to that found in Escherichia coli. To identify the elements implicated in catabolite repression in yeast, a variety of mutants affected in this process have been isolated by different research groups. A systematic review of the results reported in the literature is presented. The conclusion that can be drawn is that the mechanism of catabolite repression is a complex one, with no single gene controlling all the genes subject to repression. The expression of a given gene or set of genes is controlled by several regulatory genes, but it is not yet known whether these genes act cooperatively or sequentially.     

Regulation of galactokinase gene expression in Tetrahymena thermophila. I. Intracellular catecholamine control of galactokinase expression

The addition of glucose to the medium of Tetrahymena thermophila results in a 7-fold repression of galactokinase (EC 2.7.1.6; ATP:D-galactose-1-phosphotransferase). The presence of millimolar amounts of the catecholamines dopa, dopamine, norepinephrine, and epinephrine or the hormone glucagon also results in the repression of galactokinase in the absence of glucose. The addition of millimolar amounts of adrenergic agonists (isoproterenol, tyramine, 2-amino-6,7-dihydroxytetrahydronaphthalene) results in significant repression of galactokinase in the absence of glucose; concentrations of 2-amino-6,7-dihydroxytetrahydronaphthalene less than or equal to 10(-4) M result in a derepression of galactokinase specific activity. Addition of adrenergic antagonists (propranolol, dichloroisoproterenol) have no effect on galactokinase activity at concentrations less than 10(-4) M but do arrest cell growth at greater concentrations. The addition of the cAMP analogs caffeine or theophylline in millimolar amounts results in repression of galactokinase activity; however, cell growth is greatly slowed or completely arrested at these concentrations. Analysis of the repression response of several mutants demonstrates that mutants deficient in catecholamine biosynthesis are altered in their regulation of galactokinase. Measurements of intracellular cAMP levels for 0-24 h following the addition of several of the above compounds to exponentially growing cells did not demonstrate any change over this period. Measurement of intracellular cAMP levels for 24 h following the addition of glucose or galactose to exponentially growing wild-type and mutant cell strains did not demonstrate any difference in cAMP concentrations over this period although a wide range of galactokinase activity was exhibited. Starvation of wild-type cells prior to the addition of glucose in minimal medium without added carbohydrate resulted in a significant increase in cAMP following the addition of glucose. This increase is demonstrated to be dependent upon the ability of the cells to resume division after the arrest of growth and is not correlated with galactokinase regulation. These results support the conclusion that cAMP is not involved in the repression of galactokinase gene expression initiated by glucose or hormone-like effectors and demonstrate the participation of an adrenergic control system in galactokinase regulation which is subordinate to the regulation by glucose. A possible model is discussed.     

Conditions that elevate intracellular cyclic AMP levels promote spore formation in Dictyostelium

We have been using sporogenous mutants of Dictyostelium discoideum strain V12M2 to study regulation of cell fate during terminal differentiation of spores and stalk cells. Analyses of intracellular cAMP accumulation, cAMP secretion, cAMP binding to cell surface receptors, and chemotactic sensitivity to exogenous cAMP during aggregation showed that all of these functions were identical in V12M2 and HB200, a sporogenous mutant. We used several methods of altering intracellular cAMP levels in HB200 cells to test the hypothesis that intracellular cAMP levels affect cell fate. First, HB200 amoebae were treated with 5 mM caffeine for 4 h during growth, washed, and allowed to develop in the absence of caffeine. Treated cells had normal levels of intracellular cAMP and adenylate cyclase activities at the beginning of differentiation; by 6 h development, they contained two to three times more intracellular cAMP and two times more GTP-dependent adenylate cyclase activity than untreated cells. However, their level of basal Mn++-dependent adenylate cyclase activity was the same as untreated controls. Thus, treatment of growing HB200 amoebae with caffeine for only 4 h leads to hyperinduction of a GTP-dependent regulator (or inhibition of a negative regulator) of adenylate cyclase during subsequent differentiation, without induction of basal activity. The fraction of amoebae forming spores increased twofold when HB200 amoebae were treated with caffeine during growth. Spore (but not stalk cell) differentiation by such treated cells was blocked by inhibitors of cAMP accumulation. Second, cells grown on nutrient agar accumulated higher levels of intracellular cAMP and formed more spores in vitro than cells grown in shaken suspension.(ABSTRACT TRUNCATED AT 250 WORDS)     

A pleiotropic defect in cAMP-regulated gene expression in the Dictyostelium agg- mutant synag 7

During differentiation of Dictyostelium discoideum, cAMP functions as a diffusible, extracellular signal to direct chemotaxis and regulate developmental gene expression. The availability of signal-transduction mutants of Dictyostelium now makes it feasible to pursue a genetic analysis of cAMP signaling. The synag 7 mutant is defective in receptor-mediated adenylate cyclase stimulation and cannot relay a cAMP signal. To further characterize this mutant, mRNA levels of several cAMP-regulated genes were measured during development. cAMP-regulated gene expression was found to be dramatically altered in synag 7:several different genes which require cAMP for expression in wild-type cells were induced in synag 7 in the absence of cAMP. In addition, the gene-encoding discoidin I, which is normally expressed in starved cells and repressed by cAMP, is expressed at very low levels in starved synag 7 cells, possibly due to precocious repression. These results suggest that a pleiotropic regulator of cAMP-regulated gene expression is uncoupled from its normal controls during development in synag 7.