The RdeA-RegA system, a eukaryotic phospho-relay controlling cAMP breakdown.

The regA and rdeA gene products of Dictyostelium are involved in the regulation of cAMP signaling. The response regulator, RegA, is composed of an N-terminal receiver domain linked to a C-terminal cAMP-phosphodiesterase domain. RdeA may be a phospho-transfer protein that supplies phosphates to RegA. We show genetically that phospho-RegA is the activated form of the enzyme in vivo, in that the predicted site of aspartate phosphorylation is required for full activity. We show biochemically that RdeA and RegA communicate, as evidenced by phospho-transfer between the two proteins in vitro. Phospho-transfer is dependent on the presumed phospho-accepting amino acids, histidine 65 of RdeA and aspartate 212 of RegA, and occurs in both directions. Phosphorylation of RegA by a heterologous phospho-donor protein activates RegA phosphodiesterase activity at least 20-fold. Our results suggest that the histidine phosphotransfer protein, RdeA, and the response regulator, RegA, constitute two essential elements in a eukaryotic His-Asp phospho-relay network that regulates Dictyostelium development and fruiting body maturation.     

The internal phosphodiesterase RegA is essential for the suppression of lateral pseudopods during Dictyostelium chemotaxis

Dictyostelium strains in which the gene encoding the cytoplasmic cAMP phosphodiesterase RegA is inactivated form small aggregates. This defect was corrected by introducing copies of the wild-type regA gene, indicating that the defect was solely the consequence of the loss of the phosphodiesterase. Using a computer-assisted motion analysis system, regA(-) mutant cells were found to show little sense of direction during aggregation. When labeled wild-type cells were followed in a field of aggregating regA(-) cells, they also failed to move in an orderly direction, indicating that signaling was impaired in mutant cell cultures. However, when labeled regA(-) cells were followed in a field of aggregating wild-type cells, they again failed to move in an orderly manner, primarily in the deduced fronts of waves, indicating that the chemotactic response was also impaired. Since wild-type cells must assess both the increasing spatial gradient and the increasing temporal gradient of cAMP in the front of a natural wave, the behavior of regA(-) cells was motion analyzed first in simulated temporal waves in the absence of spatial gradients and then was analyzed in spatial gradients in the absence of temporal waves. Our results demonstrate that RegA is involved neither in assessing the direction of a spatial gradient of cAMP nor in distinguishing between increasing and decreasing temporal gradients of cAMP. However, RegA is essential for specifically suppressing lateral pseudopod formation during the response to an increasing temporal gradient of cAMP, a necessary component of natural chemotaxis. We discuss the possibility that RegA functions in a network that regulates myosin phosphorylation by controlling internal cAMP levels, and, in support of that hypothesis, we demonstrate that myosin II does not localize in a normal manner to the cortex of regA(-) cells in an increasing temporal gradient of cAMP.     

Phosphodiesterases catalyze hydrolysis of cAMP-bound to regulatory subunit of protein kinase A and mediate signal termination

Although extensive structural and biochemical studies have provided molecular insights into the mechanism of cAMP-dependent activation of protein kinase A (PKA), little is known about signal termination and the role of phosphodiesterases (PDEs) in regulatory feedback. In this study we describe a novel mode of protein kinase A-anchoring protein (AKAP)-independent feedback regulation between a specific PDE, RegA and the PKA regulatory (RIα) subunit, where RIα functions as an activator of PDE catalysis. Our results indicate that RegA, in addition to its well-known role as a PDE for bulk cAMP in solution, is also capable of hydrolyzing cAMP-bound to RIα. Furthermore our results indicate that binding of RIα activates PDE catalysis several fold demonstrating a dual function of RIα, both as an inhibitor of the PKA catalytic (C) subunit and as an activator for PDEs. Deletion mutagenesis has localized the sites of interaction to one of the cAMP-binding domains of RIα and the catalytic PDE domain of RegA whereas amide hydrogen/deuterium exchange mass spectrometry has revealed that the cAMP-binding site (phosphate binding cassette) along with proximal regions important for relaying allosteric changes mediated by cAMP, are important for interactions with the PDE catalytic domain of RegA. These sites of interactions together with measurements of cAMP dissociation rates demonstrate that binding of RegA facilitates dissociation of cAMP followed by hydrolysis of the released cAMP to 5'AMP. cAMP-free RIα generated as an end product remains bound to RegA. The PKA C-subunit then displaces RegA and reassociates with cAMP-free RIα to regenerate the inactive PKA holoenzyme thereby completing the termination step of cAMP signaling. These results reveal a novel mode of regulatory feedback between PDEs and RIα that has important consequences for PKA regulation and cAMP signal termination.     

The cyclic AMP phosphodiesterase RegA critically regulates encystation in social and pathogenic amoebas

Amoebas survive environmental stress by differentiating into encapsulated cysts. As cysts, pathogenic amoebas resist antibiotics, which particularly counteracts treatment of vision-destroying Acanthamoeba keratitis. Limited genetic tractability of amoeba pathogens has left their encystation mechanisms unexplored. The social amoeba Dictyostelium discoideum forms spores in multicellular fruiting bodies to survive starvation, while other dictyostelids, such as Polysphondylium pallidum can additionally encyst as single cells. Sporulation is induced by cAMP acting on PKA, with the cAMP phosphodiesterase RegA critically regulating cAMP levels. We show here that RegA is deeply conserved in social and pathogenic amoebas and that deletion of the RegA gene in P. pallidum causes precocious encystation and prevents cyst germination. We heterologously expressed and characterized Acanthamoeba RegA and performed a compound screen to identify RegA inhibitors. Two effective inhibitors increased cAMP levels and triggered Acanthamoeba encystation. Our results show that RegA critically regulates Amoebozoan encystation and that components of the cAMP signalling pathway could be effective targets for therapeutic intervention with encystation.     

Loss of cAMP-specific phosphodiesterase rescues spore development in G protein mutant in Dictyostelium

Cyclic AMP (cAMP) is an important intracellular signaling molecule for many G protein-mediated signaling pathways but the specificity of cAMP signaling in cells with multiple signaling pathways is not well-understood. In Dictyostelium, at least two different G protein signaling pathways, mediated by the Gα2 and Gα4 subunits, are involved with cAMP accumulation, spore production, and chemotaxis and the stimulation of these pathways results in the activation of ERK2, a mitogen-activated protein kinase that can down regulate the cAMP-specific phosphodiesterase RegA. The regA gene was disrupted in gα2⁻ and gα4⁻ cells to determine if the absence of this phosphodiesterase rescues the development of these G protein mutants as it does for erk2⁻ mutants. The regA⁻ mutation had no major effects on developmental morphology but enriched the distribution of the Gα mutant cells to the prespore/prestalk border in chimeric aggregates. The loss of RegA function had no effect on Gα4-mediated folate chemotaxis. However, the regA gene disruption in gα4⁻ cells, but not in gα2⁻ cells, resulted in a substantial rescue and acceleration of spore production. This rescue in sporulation required cell autonomous signaling because the precocious sporulation could not be induced through intercellular signaling in chimeric aggregates. However, intercellular signals from regA⁻ strains increased the expression of the prestalk gene ecmB and accelerated the vacuolization of stalk cells. Intercellular signaling from the gα4⁻regA⁻ strain did not induce ecmA gene expression indicating cell-type specificity in the promotion of prestalk cell development. regA gene disruption in a Gα4^HC (Gα4 overexpression) strain did not result in precocious sporulation or stalk cell development indicating that elevated Gα4 subunit expression can mask regA⁻ associated phenotypes even when provided with wild-type intercellular signaling. These findings indicate that the Gα2 and Gα4-mediated pathways provide different contributions to the development of spores and stalk cells and that the absence of RegA function can bypass some but not all defects in G protein regulated spore development.     

Intracellular role of adenylyl cyclase in regulation of lateral pseudopod formation during Dictyostelium chemotaxis

Cyclic AMP (cAMP) functions as the extracellular chemoattractant in the aggregation phase of Dictyostelium development. There is some question, however, concerning what role, if any, it plays intracellularly in motility and chemotaxis. To test for such a role, the behavior of null mutants of acaA, the adenylyl cyclase gene that encodes the enzyme responsible for cAMP synthesis during aggregation, was analyzed in buffer and in response to experimentally generated spatial and temporal gradients of extracellular cAMP. acaA- cells were defective in suppressing lateral pseudopods in response to a spatial gradient of cAMP and to an increasing temporal gradient of cAMP. acaA- cells were incapable of chemotaxis in natural waves of cAMP generated by majority control cells in mixed cultures. These results indicate that intracellular cAMP and, hence, adenylyl cyclase play an intracellular role in the chemotactic response. The behavioral defects of acaA- cells were surprisingly similar to those of cells of null mutants of regA, which encodes the intracellular phosphodiesterase that hydrolyzes cAMP and, hence, functions opposite adenylyl cyclase A (ACA). This result is consistent with the hypothesis that ACA and RegA are components of a receptor-regulated intracellular circuit that controls protein kinase A activity. In this model, the suppression of lateral pseudopods in the front of a natural wave depends on a complete circuit. Hence, deletion of any component of the circuit (i.e., RegA or ACA) would result in the same chemotactic defect.     

Multiple phosphorylation sites on the RegA phosphodiesterase regulate Dictyostelium development

In Dictyostelium, the intracellular cAMP-specific phosphodiesterase RegA is a negative regulator of cAMP-dependent protein kinase (PKA), a key determinant in the timing of developmental morphogenesis and spore formation. To assess the role of protein kinases in the regulation of RegA function, this study identified phosphorylation sites on RegA and characterized the role of these modifications through the analysis of phospho-mimetic and phospho-ablative mutations. Mutations affecting residue T676 of RegA, a presumed target of the atypical MAP kinase Erk2, altered the rate of development and impacted cell distribution in chimeric organisms suggesting that phosphorylation of this residue reduces RegA function and regulates cell localization during multicellular development. Mutations affecting the residue S142 of RegA also impacted the rate developmental morphogenesis but in a manner opposite of changes at T676 suggesting the phosphorylation of the S142 residue increases RegA function. Mutations affecting residue S413 residue altered aggregate sizes and delayed developmental progression suggesting that PKA operates in a negative feedback mechanism to increase RegA function. These results suggest that the phosphorylation of different residues on RegA can lead to increased or decreased RegA function and therefore in turn regulate developmental processes such as aggregate formation, cell distribution, and the kinetics of developmental morphogenesis.     

SDF-2 Induction of Terminal Differentiation in Dictyostelium discoideum Is Mediated by the Membrane-Spanning Sensor Kinase DhkA

SDF-2 is a peptide released by prestalk cells during culmination that stimulates prespore cells to encapsulate. Genetic evidence indicates that the response is dependent on the dhkA gene. This gene encodes a member of the histidine kinase family of genes that functions in two-component signal transduction pathways. The sequence of the N-terminal half of DhkA predicts two hydrophobic domains separated by a 310-amino-acid loop that could bind a ligand. By inserting MYC6 epitopes into DhkA, we were able to show that the loop is extracellular while the catalytic domain is cytoplasmic. Cells expressing the MYC epitope in the extracellular domain of DhkA were found to respond only if induced with 100-fold-higher levels of SDF-2 than required to induce dhkA+ cells; however, they could be induced to sporulate by addition of antibodies specific to the MYC epitope. To examine the enzymatic activity of DhkA, we purified the catalytic domain following expression in bacteria and observed incorporation of labelled phosphate from ATP consistent with histidine autophosphorylation. Site-directed mutagenesis of histidine1395 to glutamine in the catalytic domain blocked autophosphorylation. Furthermore, genetic analyses showed that histidine1395 and the relay aspartate2075 of DhkA are both critical to its function but that another histidine kinase, DhkB, can partially compensate for the lack of DhkA activity. Sporulation is drastically reduced in double mutants lacking both DhkA and DhkB. Suppressor studies indicate that the cyclic AMP (cAMP) phosphodiesterase RegA and the cAMP-dependent protein kinase PKA act downstream of DhkA.     

Dynamics of phosphodiesterase-induced cAMP dissociation from protein kinase A: Capturing transient ternary complexes by HDXMS

cAMP signaling is a fundamental cellular process necessary for mediating responses to hormonal stimuli. In contrast to cAMP-dependent activation of protein kinase A (PKA), an important cellular target, far less is known on termination in cAMP signaling, specifically how phosphodiesterases (PDEs) facilitate dissociation and hydrolysis of bound cAMP. In this study, we have probed the dynamics of a ternary complex of PKA and a PDE–RegA with an excess of a PDE-nonhydrolyzable cAMP analog, Sp-cAMPS by amide hydrogen/deuterium exchange mass spectrometry (HDXMS). Our results highlight how HDXMS can be used to monitor reactions together with mapping conformational dynamics of transient signaling complexes. Our results confirm a two-state model for active RegA-mediated dissociation of bound cAMP. Further, our results reveal that Sp-cAMPS and RegA mediate mutually exclusive interactions with the same region of PKA and at specific concentrations of Sp-cAMPS, RegA is capable of blocking Sp-cAMPS reassociation to PKA. This provides a molecular basis for how PDEs modulate levels of intracellular cAMP so that PKA is better suited to responding to fluxes rather than constant levels of cAMP. This study underscores how HDXMS can be a powerful tool for monitoring reactions together with mapping conformational dynamics in signaling proteins. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.     

Autogenous Regulation of the Rega Gene of Bacteriophage T4: Derepression of Translation

The regA gene of phage T4 encodes a translational repressor that inhibits utilization of its own mRNA as well as the translation of a number of other phage-induced mRNAs. In recombinant plasmids, autogenous translational repression limits production of the RegA protein when the cloned structural gene is expressed under control of a strong, plasmid-borne promoter (lambda PL). We have found that a genetic fusion which places the regA ribosome binding domain in proximity to active translation leads to partial derepression of wild-type RegA protein synthesis. The derepression is not due to increased synthesis of regA RNA, suggesting that it occurs at the translational level. Derepressed clones of the wild-type regA gene were used to overproduce and purify the repressor. In an in vitro assay the wild-type target was sensitive and a mutant target was resistant to inhibition by the added protein. The results suggest that the sensitivity of a regA-regulated cistron to translational repression may depend on the competition between ribosomes and RegA protein for overlapping recognition sequences in the translation initiation domain of the mRNA.     

Multistate allostery in response regulators: phosphorylation and mutagenesis activate RegA via alternate modes

Response regulators (RRs) belong to two-component signaling pathways, widely prevalent in bacteria and lower eukaryotes, for sensing and mediating responses to diverse environmental stress stimuli. RRs are modular proteins, and in most instances, a receiver domain is found connected to diverse effector domain(s). All receiver domains contain a conserved aspartate, which is the site of phosphorylation by an associated histidine kinase. RRs function as phosphorylatable signaling switches whereby histidine-kinase-mediated phosphorylation of RRs alters its output function. It is largely unknown how phosphorylation of the receiver domain triggers activation of distally positioned effector domain(s). Although crystal structures have highlighted differences in conformations from comparisons of snapshots of the unphosphorylated and phosphorylated receiver domains, how this is translated into altered activity of a distal effector domain has remained a mystery. While allosteric relays have been identified within receiver domains by NMR and X-ray crystallography, phosphorylated states of larger multidomain RRs have not yet been characterized. In this study, we have used amide hydrogen/deuterium exchange mass spectrometry to probe the conformational dynamics of a multidomain RR, RegA from Dictyostelium discoideum, by comparisons of the unphosphorylated and phosphorylated states and an activating mutant. Our results reveal allosteric coupling between the site of phosphorylation and the activating mutation. Interestingly, however, the conformations of the effector domains in both instances are distinct. Hydrogen/deuterium exchange mass spectrometry indicates that the 'inactive' and 'active' conformations exist as ensembles of multiple conformations. This is consistent with the 'conformational selection' model for describing phosphorylation-dependent regulation of multidomain RRs.     

The guanine nucleotide exchange factor CNrasGEF activates ras in response to cAMP and cGMP

Small GTPase proteins such as Ras are key regulators of cellular proliferation and are activated by guanine nucleotide exchange/releasing factors (GEFs/GRFs). Three classes of Ras GRFs have been identified to date, represented by Sos1/2, Ras-GRF1/2 and Ras-GRP. Here, we describe a novel candidate Ras activator, cyclic nucleotide rasGEF (CNrasGEF), which contains CDC25, Ras exchange motif (REM), Ras-association (RA), PDZ and cNMP (cAMP/cGMP) binding (cNMP-BD) domains, two PY motifs and a carboxy-terminal SxV sequence. CNrasGEF can activate Ras in vitro, and it binds cAMP directly via its cNMP-BD. In cells, CNrasGEF activates Ras in response to elevation of intracellular cAMP or cGMP, or treatment with their analogues 8-Br-cAMP or 8-Br-cGMP, independently of protein kinases A and G (PKA and PKG). This activation is prevented in CNrasGEF lacking its CDC25 domain or cNMP-BD. CNrasGEF can also activate the small GTPase Rap1 in cells, but this activation is constitutive and independent of cAMP. CNrasGEF is expressed mainly in the brain and is localized at the plasma membrane, a localization dependent on the presence of intact PDZ domain but not the SxV sequence. These results suggest that CNrasGEF may directly connect cAMP-generating pathways or cGMP-generating pathways to Ras.     

Novel mechanisms in nutrient activation of the yeast protein kinase A pathway

In yeast the Protein Kinase A (PKA) pathway can be activated by a variety of nutrients. Fermentable sugars, like glucose and sucrose, trigger a spike in the cAMP level, followed by activation of PKA and phosphorylation of target proteins causing a.o. mobilization of reserve carbohydrates, repression of stress-related genes and induction of growth-related genes. Glucose and sucrose are sensed by a G-protein coupled receptor system that activates adenylate cyclase and also activates a bypass pathway causing direct activation of PKA. Addition of other essential nutrients, like nitrogen sources or phosphate, to glucose-repressed nitrogen- or phosphate-starved cells, also triggers rapid activation of the PKA pathway. In these cases cAMP is not involved as a second messenger. Amino acids are sensed by the Gap1 transceptor, previously considered only as an amino acid transporter. Recent results indicate that the amino acid ligand has to induce a specific conformational change for signaling. The same amino acid binding site is involved in transport and signaling. Similar results have been obtained for Pho84 which acts as a transceptor for phosphate activation of the PKA pathway. Ammonium activation of the PKA pathway in nitrogen-starved cells is mediated mainly by the Mep2 transceptor, which belongs to a different class of transporter proteins. Hence, different types of sensing systems are involved in control of the yeast PKA pathway by nutrients.     

Cell type specific inhibition of cAMP phosphodiesterase activity during terminal differential in Dictyostelium discoideum

Cyclic AMP phosphodiesterase (PDE) activity reaches a peak during the aggregation stage of development where it functions to regulate extracellular levels of cAMP. During the subsequent differentiation of the two cell types at the culmination stage, the activity reappears but only in stalk cells. We found that extracts from the culmination stage contained PDE which could be activated by preincubation with Mg2+ and dithiothreitol (DTT), a treatment which is known to release an endogenous inhibitor from the aggregation stage enzyme. When the culmination stage extracts were subjected to chromatography on Biogel P300, two peaks of activity were eluted, PDE-I (Mr greater than 260,000) and PDE-II (Mr 100,000). Treatment of the fractions with Mg-DTT did not affect the low-molecular-weight enzyme but caused activation of the high-molecular-weight enzyme and the appearance of a third, intermediate form. Kinetic analysis of the two peaks revealed Km values for cAMP of 2 mM and 10 microM for PDE-I and PDE-II, respectively. We tested the possibility that these forms of the enzyme might be distributed differently in the two cell types by measuring the Km for cAMP and the effect of Mg-DTT treatment on isolated sections of stalk and spore cells. The spore sections contained a high Km form of the enzyme (0.3 mM) which was activated by preincubation with Mg . DTT whereas stalk sections contained a low Km form (3 microM) which was not affected by the activation treatment. We conclude that both cell types contain enzyme protein and that the apparent localization of PDE activity in stalk cells is due to the inhibition of activity in spore cells.     

Osmotic stress response in Dictyostelium is mediated by cAMP

DokA, a homolog of bacterial hybrid histidine kinases, is essential for hyperosmotic stress resistance in Dictyostelium: We show that a transient intracellular cAMP signal, dependent on the presence of DokA, is generated in response to an osmotic shock. This variation of cAMP levels contributes to survival under hypertonic conditions. In contrast to the low cAMP levels observed in dokA(-) strains, overexpression of the receiver domain of DokA causes an increase in cAMP levels, resulting in a rapidly developing phenotype. We present biochemical and cell biological data indicating that the DokA receiver domain is a dominant-negative regulator of a phosphorelay, which controls the intracellular cAMP phosphodiesterase RegA. The activity of the DokA receiver domain depends on a conserved aspartate, mutation of which reverses the developmental phenotype, as well as the deregulation of cAMP metabolism.     

Epac activation converts cAMP from a proliferative into a differentiation signal in PC12 cells

Elevation of the intracellular cAMP concentration ([cAMP]i) regulates metabolism, cell proliferation, and differentiation and plays roles in memory formation and neoplastic growth. cAMP mediates its effects mainly through activation of protein kinase A (PKA) as well as Epac1 and Epac2, exchange factors activating the small GTPases Rap1 and Rap2. However, how cAMP utilizes these effectors to induce distinct biological responses is unknown. We here studied the specific roles of PKA and Epac in neuroendocrine PC12 cells. In these cells, elevation of [cAMP]i activates extracellular signal-regulated kinase (ERK) 1/2 and induces low-degree neurite outgrowth. The present study showed that specific stimulation of PKA triggered ERK1/2 activation that was considerably more transient than that observed upon simultaneous activation of both PKA and Epac. Unexpectedly, the PKA-specific cAMP analog induced cell proliferation rather than neurite outgrowth. The proliferative signaling pathway activated by the PKA-specific cAMP analog involved activation of the epidermal growth factor receptor and ERK1/2. Activation of Epac appeared to extend the duration of PKA-dependent ERK1/2 activation and converted cAMP from a proliferative into an anti-proliferative, neurite outgrowth-promoting signal. Thus, the present study showed that the outcome of cAMP signaling can depend heavily on the set of cAMP effectors activated.     

A new class of rapidly developing mutants in Dictyostelium discoideum: implications for cyclic AMP metabolism and cell differentiation

Rapidly developing (rde) mutants of Dictyostelium discoideum, in which cells precociously differentiated into stalk and spore cells without normal morphogenesis, were investigated genetically and biochemically. Genetic complementation tests demonstrated that the 16 rde mutants isolated could be classified into at least two groups (groups A and C) and that the first described rde mutant FR17 (D. R. Sonneborn, G. J. White, and M. Sussman, 1963, Dev. Biol. 7, 79-93) belongs to group A. Morphological studies revealed several differences in development and final morphology between group A and group C mutants. In group A mutants, the time required for cell differentiation from vegetative cells to aggregation competent cells is reduced, whereas the time required for spore and stalk cell differentiation following the completion of aggregation is shortened in group C mutants. This suggests that group C mutants represent a new class of rde mutants and that there exist at least two mechanisms involved in regulating the timing of development in D. discoideum. Measurements of cell-associated and extracellular phosphodiesterase activities, and intracellular and total cAMP levels revealed that cAMP metabolism in both groups is significantly altered during development. Group A mutants showed precocious and excessive production of phosphodiesterase and cAMP during the entire course of development; intracellular cAMP levels in group C mutants were extremely low, and spore and stalk cell differentiation occurred without an apparent increase in these levels. Thus, while cAMP metabolism is abnormal in all the rde mutants studied, there exist several distinct types of derangement, not necessarily involving the overproduction of cAMP.     

Genetic interactions of the E3 ubiquitin ligase component FbxA with cyclic AMP metabolism and a histidine kinase signaling pathway during Dictyostelium discoideum development

Dictyostelium discoideum amoebae with an altered fbxA gene, which is thought to encode a component of an SCF E3 ubiquitin ligase, have defective regulation of cell type proportionality. In chimeras with wild-type cells, the mutant amoebae form mainly spores, leaving the construction of stalks to wild-type cells. To examine the role of fbxA and regulated proteolysis, we have recovered the promoter of fbxA and shown that it is expressed in a pattern resembling that of a prestalk-specific gene until late in development, when it is also expressed in developing spore cells. Because fbxA cells are developmentally deficient in pure culture, we were able to select suppressor mutations that promote sporulation of the original mutant. One suppressor mutation resides within the gene regA, which encodes a cyclic AMP (cAMP) phosphodiesterase linked to an activating response regulator domain. In another suppressor, there has been a disruption of dhkA, a gene encoding a two-component histidine kinase known to influence Dictyostelium development. RegA appears precociously and in greater amounts in the fbxA mutant than in the wild type, but in an fbxA/dhkA double mutant, RegA is restored to wild-type levels. Because the basis of regA suppression might involve alterations in cAMP levels during development, the concentrations of cAMP in all strains were determined. The levels of cAMP are relatively constant during multicellular development in all strains except the dhkA mutant, in which it is reduced at least sixfold. The level of cAMP in the double mutant dhkA/fbxA is relatively normal. The levels of cAMP in the various mutants do not correlate with spore formation, as would be expected on the basis of our present understanding of the signaling pathway leading to the induction of spores. Altered amounts of RegA and cAMP early in the development of the mutants suggest that both fbxA and dhkA genes act earlier than previously thought.