14–3-3 Inhibits the Dictyostelium Myosin II Heavy-Chain-specific Protein Kinase C Activity by a Direct Interaction: Identification of the 14–3-3 Binding Domain

Myosin II heavy chain (MHC) specific protein kinase C (MHC-PKC), isolated from Dictyostelium discoideum, regulates myosin II assembly and localization in response to the chemoattractant cyclic AMP. Immunoprecipitation of MHC-PKC revealed that it resides as a complex with several proteins. We show herein that one of these proteins is a homologue of the 14–3-3 protein (Dd14–3-3). This protein has recently been implicated in the regulation of intracellular signaling pathways via its interaction with several signaling proteins, such as PKC and Raf-1 kinase. We demonstrate that the mammalian 14–3-3 ζ isoform inhibits the MHC-PKC activity in vitro and that this inhibition is carried out by a direct interaction between the two proteins. Furthermore, we found that the cytosolic MHC-PKC, which is inactive, formed a complex with Dd14–3-3 in the cytosol in a cyclic AMP-dependent manner, whereas the membrane-bound active MHC-PKC was not found in a complex with Dd14–3-3. This suggests that Dd14–3-3 inhibits the MHC-PKC in vivo. We further show that MHC-PKC binds Dd14–3-3 as well as 14–3-3ζ through its C1 domain, and the interaction between these two proteins does not involve a peptide containing phosphoserine as was found for Raf-1 kinase. Our experiments thus show an in vivo function for a member of the 14–3-3 family and demonstrate that MHC-PKC interacts directly with Dd14–3-3 and 14–3-3ζ through its C1 domain both in vitro and in vivo, resulting in the inhibition of the kinase.     

Myosin heavy chain kinase inactivated by Ca2+/calmodulin from aggregating cells of Dictyostelium discoideum.

Soluble myosin heavy chain kinases (MHC kinases) were partially purified from growth phase and aggregation-competent cells of Dictyostelium discoideum. In the aggregation-competent cells, two MHC kinases were distinguishable. One of these enzymes, called MHC kinase II, was inactivated by Ca2+ and calmodulin in a highly temperature-dependent reaction. A MHC kinase found in growth phase cells did not have these regulatory properties. Substrate specificities were analysed for MHC kinase II and for the MHC kinase from growth phase cells. Both enzymes phosphorylated threonine residues of the myosin heavy chains of D. discoideum and Physarum polycephalum. Phosphopeptide mapping of D. discoideum myosin and determination of the stoichiometry of its phosphorylation suggested the presence of two phosphorylation sites per heavy chain. Both sites were contained within a 38-kd chymotryptic fragment. The inactivation of MHC kinase II by Ca2+ plus calmodulin suggests this enzyme has a role in the regulation of myosin functions during the chemotactic response of a cell. The phosphorylated myosin had about one third the actin-activated Mg2+-ATPase activity of the non-phosphorylated myosin. Previous findings indicated that stimulation of D. discoideum cells with the chemo-attractant cAMP increases the cytoplasmic Ca2+ concentration. Under these conditions MHC kinase II might be inhibited and the dephosphorylated, more active form of myosin would accumulate.     

Recruitment of a myosin heavy chain kinase to actin-rich protrusions in Dictyostelium

Nonmuscle myosin II plays fundamental roles in cell body translocation during migration and is typically depleted or absent from actin-based cell protrusions such as lamellipodia, but the mechanisms preventing myosin II assembly in such structures have not been identified [1-3]. In Dictyostelium discoideum, myosin II filament assembly is controlled primarily through myosin heavy chain (MHC) phosphorylation. The phosphorylation of sites in the myosin tail domain by myosin heavy chain kinase A (MHCK A) drives the disassembly of myosin II filaments in vitro and in vivo [4]. To better understand the cellular regulation of MHCK A activity, and thus the regulation of myosin II filament assembly, we studied the in vivo localization of native and green fluorescent protein (GFP)-tagged MHCK A. MHCK A redistributes from the cytosol to the cell cortex in response to stimulation of Dictyostelium cells with chemoattractant in an F-actin-dependent manner. During chemotaxis, random migration, and phagocytic/endocytic events, MHCK A is recruited preferentially to actin-rich leading-edge extensions. Given the ability of MHCK A to disassemble myosin II filaments, this localization may represent a fundamental mechanism for disassembling myosin II filaments and preventing localized filament assembly at sites of actin-based protrusion.     

Polarization of myosin II heavy chain-protein kinase C in chemotaxing dictyostelium cells

Eukaryotic cells need morphological polarity to carry out chemotaxis (Parent, C. A., Blacklock, B. J., Froehlich, W. M., Murphy, D. B., and Devreotes, P. N. (1998) Cell 95, 81-91; Jin, T., Zhang, N., Long, Y., Parent, C., and Devreotes, P. N. (2000) Science 287, 1034-1036; Servant, G., Weiner, O. D., Herzmark, P., Balla, T., Sedat, J. W., and Bourne, H. R. (2000) Science 287, 1037-1040), but sensing direction does not require polarization of chemoattractant receptors. When cells are exposed to a gradient of chemoattractant, activation occurs selectively at the stimulated edge. Such localized activation, transmitted by the recruitment of cytosolic proteins, may be a general mechanism for gradient sensing by G protein-linked chemotactic systems. Here we show that in Dictyostelium discoideum cells exposed to a cAMP gradient the myosin II heavy chain kinase (MHC-PKC) and myosin II translocate to opposite ends of the cell. We further show that MHC-PKC C1 domain is responsible for the localization of MHC-PKC to the cell leading edge, but it is not sufficient to promote cell polarization. Our findings suggest a mechanism by which MHC-PKC regulates myosin II, allowing cell polarization and movement in the direction of the cAMP source.     

Non-chemotactic Dictyostelium discoideum mutants with altered cGMP signal transduction

Folic acid and cAMP are chemoattractants in Dictyostelium discoideum, which bind to different surface receptors. The signal is transduced from the receptors via different G proteins into a common pathway which includes guanylyl cyclase and acto-myosin. To investigate this common pathway, ten mutants which do not react chemotactically to both cAMP and folic acid were isolated with a simple new chemotactic assay. Genetic analysis shows that one of these mutants (KI-10) was dominant; the other nine mutants were recessive, and comprise nine complementation groups. In wild-type cells, the chemoattractants activate adenylyl cyclase, phospholipase C, and guanylyl cyclase in a transient manner. In mutant cells the formation of cAMP and IP3 were generally normal, whereas the cGMP response was altered in most of the ten mutants. Particularly, mutant KI-8 has strongly reduced basal guanylyl cyclase activity; the enzyme is present in mutant KI-10, but can not be activated by cAMP or folic acid. The cGMP response of five other mutants is altered in either magnitude, dose dependency, or kinetics. These observations suggest that the second messenger cGMP plays a key role in chemotaxis in Dictyostelium.     

Biochemical characterization of a Dictyostelium myosin II heavy-chain phosphatase that promotes filament assembly.

In Dictyostelium cells, myosin II is found as cytosolic nonassembled monomers and cytoskeletal bipolar filaments. It is thought that the phosphorylation state of three threonine residues in the tail of myosin II heavy chain regulates the molecular motor's assembly state and localization. Phosphorylation of the myosin heavy chain at threonine residues 1823, 1833 and 2029 is responsible for maintaining myosin in the nonassembled state, and subsequent dephosphorylation of these residues is a prerequisite for assembly into the cytoskeleton. We report here the characterization of myosin heavy-chain phosphatase activities in Dictyostelium utilizing myosin II phosphorylated by myosin heavy-chain kinase A as a substrate. One of the myosin heavy-chain phosphatase activities was identified as protein phosphatase 2A and the purified holoenzyme was composed of a 37-kDa catalytic subunit, a 65-kDa A subunit and a 55-kDa B subunit. The protein phosphatase 2A holoenzyme displays two orders of magnitude higher activity towards myosin phosphorylated on the heavy chains than it does towards myosin phosphorylated on the regulatory light chains, consistent with a role in the control of filament assembly. The purified myosin heavy-chain phosphatase activity promotes bipolar filament assembly in vitro via dephosphorylation of the myosin heavy chain. This system should provide a valuable model for studying the regulation and localization of protein phosphatase 2A in the context of cytoskeletal reorganization.     

EppA, a putative substrate of DdERK2, regulates cyclic AMP relay and chemotaxis in Dictyostelium discoideum

The mitogen-activated protein kinase DdERK2 is critical for cyclic AMP (cAMP) relay and chemotaxis to cAMP and folate, but the details downstream of DdERK2 are unclear. To search for targets of DdERK2 in Dictyostelium discoideum, 32PO4(3-)-labeled protein samples from wild-type and Dderk2- cells were resolved by 2-dimensional electrophoresis. Mass spectrometry was used to identify a novel 45-kDa protein, named EppA (ERK2-dependent phosphoprotein A), as a substrate of DdERK2 in Dictyostelium. Mutation of potential DdERK2 phosphorylation sites demonstrated that phosphorylation on serine 250 of EppA is DdERK2 dependent. Changing serine 250 to alanine delayed development of Dictyostelium and reduced Dictyostelium chemotaxis to cAMP. Although overexpression of EppA had no significant effect on the development or chemotaxis of Dictyostelium, disruption of the eppA gene led to delayed development and reduced chemotactic responses to both cAMP and folate. Both eppA gene disruption and overexpression of EppA carrying the serine 250-to-alanine mutation led to inhibition of intracellular cAMP accumulation in response to chemoattractant cAMP, a pivotal process in Dictyostelium chemotaxis and development. Our studies indicate that EppA regulates extracellular cAMP-induced signal relay and chemotaxis of Dictyostelium.     

A cell number-counting factor regulates group size in Dictyostelium by differentially modulating cAMP-induced cAMP and cGMP pulse sizes

A secreted counting factor (CF), regulates the size of Dictyostelium discoideum fruiting bodies in part by regulating cell-cell adhesion. Aggregation and the expression of adhesion molecules are mediated by relayed pulses of cAMP. Cells also respond to cAMP with a short cGMP pulse. We find that CF slowly down-regulates the cAMP-induced cGMP pulse by inhibiting guanylyl cyclase activity. A 1-min exposure of cells to purified CF increases the cAMP-induced cAMP pulse. CF does not affect the cAMP receptor or its interaction with its associated G proteins or the translocation of the cytosolic regulator of adenylyl cyclase to the membrane in response to cAMP. Pulsing streaming wild-type cells with a high concentration of cAMP results in the formation of small groups, whereas reducing cAMP pulse size with exogenous cAMP phosphodiesterase during stream formation causes cells to form large groups. Altering the extracellular cAMP pulse size does not phenocopy the effects of CF on the cAMP-induced cGMP pulse size or cell-cell adhesion, indicating that CF does not regulate cGMP pulses and adhesion via CF's effects on cAMP pulses. The results suggest that regulating cell-cell adhesion, the cGMP pulse size, or the cAMP pulse size can control group size and that CF regulates all three of these independently.     

The regulation of myosin II in Dictyostelium

Dictyostelium conventional myosin (myosin II) is an abundant protein that plays a role in various cellular processes such as cytokinesis, cell protrusion and development. This review will focus on the signal transduction pathways that regulate myosin II during cell movement. Myosin II appears to have two modes of action in Dictyostelium: local stabilization of the cytoskeleton by myosin filament association to the actin meshwork (structural mode) and force generation by contraction of actin filaments (motor mode). Some processes, such as cell movement under restrictive environment, require only the structural mode of myosin. However, cytokinesis in suspension and uropod retraction depend on motor activity as well. Myosin II can self-assemble into bipolar filaments. The formation of these filaments is negatively regulated by heavy chain phosphorylation through the action of a set of novel alpha kinases and is relatively well understood. However, only recently it has become clear that the formation of bipolar filaments and their translocation to the cortex are separate events. Translocation depends on filamentous actin, and is regulated by a cGMP pathway and possibly also by the cAMP phosphodiesterase RegA and the p21-activated kinase PAKa. Myosin motor activity is regulated by phosphorylation of the regulatory light chain through myosin light chain kinase A. Unlike conventional light chain kinases, this enzyme is not regulated by calcium but is activated by cGMP-induced phosphorylation via an upstream kinase and subsequent autophosphorylation.     

Expression of a myosin regulatory light chain phosphorylation site mutant complements the cytokinesis and developmental defects of Dictyostelium RMLC null cells

In a number of systems phosphorylation of the regulatory light chain (RMLC) of myosin regulates the activity of myosin. In smooth muscle and vertebrate nonmuscle systems RMLC phosphorylation is required for contractile activity. In Dictyostelium discoideum phosphorylation of the RMLC regulates both ATPase activity and motor function. We have determined the site of phosphorylation on the Dictyostelium RMLC and used site-directed mutagenesis to replace the phosphorylated serine with an alanine. The mutant light chain was then expressed in RMLC null Dictyostelium cells (mLCR-) from an actin promoter on an integrating vector. The mutant RMLC was expressed at high levels and associated with the myosin heavy chain. RMLC bearing a ser13ala substitution was not phosphorylated in vitro by purified myosin light chain kinase, nor could phosphate be detected on the mutant RMLC in vivo. The mutant myosin had reduced actin-activated ATPase activity, comparable to fully dephosphorylated myosin. Unexpectedly, expression of the mutant RMLC rescued the primary phenotypic defects of the mlcR- cells to the same extent as did expression of wild-type RMLC. These results suggest that while phosphorylation of the Dictyostelium RMLC appears to be tightly regulated in vivo, it is not essential for myosin-dependent cellular functions.     

A novel cGMP signalling pathway mediating myosin phosphorylation and chemotaxis in Dictyostelium

Chemotactic stimulation of Dictyostelium cells results in a transient increase in cGMP levels, and transient phosphorylation of myosin II heavy and regulatory light chains. In Dictyostelium, two guanylyl cyclases and four candidate cGMP-binding proteins (GbpA- GbpD) are implicated in cGMP signalling. GbpA and GbpB are homologous proteins with a Zn2+-hydrolase domain. A double gbpA/gbpB gene disruption leads to a reduction of cGMP-phosphodiesterase activity and a 10-fold increase of basal and stimulated cGMP levels. Chemotaxis in gbpA(-)B(-) cells is associated with increased myosin II phosphorylation compared with wild-type cells; formation of lateral pseudopodia is suppressed resulting in enhanced chemotaxis. GbpC is homologous to GbpD, and contains Ras, MAPKKK and Ras-GEF domains. Inactivation of the gbp genes indicates that only GbpC harbours high affinity cGMP-binding activity. Myosin phosphorylation, assembly of myosin in the cytoskeleton as well as chemotaxis are severely impaired in mutants lacking GbpC and GbpD, or mutants lacking both guanylyl cyclases. Thus, a novel cGMP signalling cascade is critical for chemotaxis in Dictyostelium, and plays a major role in myosin II regulation during this process.     

Actin activation of myosin heavy chain kinase A in Dictyostelium: a biochemical mechanism for the spatial regulation of myosin II filament disassembly

Studies in Dictyostelium discoideum have established that the cycle of myosin II bipolar filament assembly and disassembly controls the temporal and spatial localization of myosin II during critical cellular processes, such as cytokinesis and cell locomotion. Myosin heavy chain kinase A (MHCK A) is a key enzyme regulating myosin II filament disassembly through myosin heavy chain phosphorylation in Dictyostelium. Under various cellular conditions, MHCK A is recruited to actin-rich cortical sites and is preferentially enriched at sites of pseudopod formation, and thus MHCK A is proposed to play a role in regulating localized disassembly of myosin II filaments in the cell. MHCK A possesses an aminoterminal coiled-coil domain that participates in the oligomerization, cellular localization, and actin binding activities of the kinase. In the current study, we show that the interaction between the coiled-coil domain of MHCK A and filamentous actin leads to an approximately 40-fold increase in the initial rate of kinase catalytic activity. Actin-mediated activation of MHCK A involves increased rates of kinase autophosphorylation and requires the presence of the coiled-coil domain. Structure-function analyses revealed that the coiled-coil domain alone binds to actin filaments (apparent K(D) = 0.9 microm) and thus mediates the direct interaction with F-actin required for MHCK A activation. Collectively, these results indicate that MHCK A recruitment to actin-rich sites could lead to localized activation of the kinase via direct interaction with actin filaments, and thus this mode of kinase regulation may represent an important mechanism by which the cell achieves localized disassembly of myosin II filaments required for specific changes in cell shape.     

Lithium, an inhibitor of cAMP-induced inositol 1,4,5-trisphosphate accumulation in Dictyostelium discoideum, inhibits activation of guanine-nucleotide-binding regulatory proteins, reduces activation of adenylylcyclase, but potentiates activation of guanylyl cyclase by cAMP.

Li+ drastically alters pattern formation in Dictyostelium by inhibiting cAMP-induced prespore-gene expression and promoting cAMP-induced prestalk-gene expression. We reported previously that Li+ inhibits inositol monophosphatases in this organism and strongly reduces basal and cAMP-stimulated inositol 1,4,5-trisphosphate levels. We show here that Li+ also reduces cAMP-induced accumulation of cAMP, but promotes cAMP-induced accumulation of cGMP. This effect is not due to inhibition of cGMP hydrolysis or inhibition of adaptation and may therefore reflect stimulation of guanylyl-cyclase activation. Li+ does not affect the binding of cAMP to surface receptors but interferes with the interaction between receptors and guanine-nucleotide-binding regulatory (G) proteins. These effects are complex; in the absence of Mg2+, Li+ increases guanosine 5'-[gamma-thio]triphosphate(GTP[S])-binding activity to similar levels as 1 mM Mg2+. However, while Mg2+ potentiates cAMP-induced stimulation of GTP[S]-binding activity, Li+ effectively inhibits stimulation. Li+ also inhibits cAMP-stimulated, but not basal high-affinity GTP-ase activity, indicating an inhibitory effect on cAMP-induced activation of G-proteins. Our data suggest that in addition to inositolphosphate metabolism, the activation of G-proteins may be a second biochemical target for Li+ effects on pattern formation and signal transduction in Dictyostelium.     

Multiple effects of differentiation-inducing factor on prespore differentiation and cyclic-AMP signal transduction in Dictyostelium

Abstract. The effects of the differentiation inducing factor (DIF) on several cAMP-induced responses in Dictyostelium were investigated. It was found that DIF reduces the apparent affinity of cell-surface cAMP receptors. DIF does not affect the cAMP-induced cGMP response, but it is a potent inhibitor of the cAMP-relay response. DIF also inhibits the induction of prespore differentiation by cAMP in aggregation-competent cells. We also compared the effects of DIF on cAMP-induced responses with those of the relay inhibitor, caffeine, and the morphogen, adenosine.     

Dictyostelium myosin heavy chain kinase A regulates myosin localization during growth and development

Phosphorylation of the Dictyostelium myosin II heavy chain (MHC) has a key role in regulating myosin localization in vivo and drives filament disassembly in vitro. Previous molecular analysis of the Dictyostelium myosin II heavy chain kinase (MHCK A) gene has demonstrated that the catalytic domain of this enzyme is extremely novel, showing no significant similarity to the known classes of protein kinases (Futey, L. M., Q. G. Medley, G. P. Cote, and T. T. Egelhoff. 1995. J. Biol. Chem. 270:523-529). To address the physiological roles of this enzyme, we have analyzed the cellular consequences of MHCK A gene disruption (mhck A- cells) and MHCK A overexpression (MHCK A++ cells). The mhck A- cells are viable and competent for tested myosin-based contractile events, but display partial defects in myosin localization. Both growth phase and developed mhck A- cells show substantially reduced MHC kinase activity in crude lysates, as well as significant overassembly of myosin into the Triton-resistant cytoskeletal fractions. MHCK A++ cells display elevated levels of MHC kinase activity in crude extracts, and show reduced assembly of myosin into Triton-resistant cytoskeletal fractions. MHCK A++ cells show reduced growth rates in suspension, becoming large and multinucleated, and arrest at the mound stage during development. These results demonstrate that MHCK A functions in vivo as a protein kinase with physiological roles in regulating myosin II localization and assembly in Dictyostelium cells during both growth and developmental stages.     

Myosin heavy chain kinase B participates in the regulation of myosin assembly into the cytoskeleton

Myosin II plays critical roles in events such as cytokinesis, chemotactic migration, and morphological changes during multicellular development. The amoeba Dictyostelium discoideum provides a simple system for the study of this contractile protein. In this system, myosin II filament assembly is regulated by myosin heavy chain (MHC) phosphorylation in the tail region of the molecule. Earlier studies identified an alpha-kinase, MHC kinase A (MHCK A), which phosphorylates three mapped threonine residues in the myosin tail, driving myosin disassembly. Using molecular and genomic approaches, we have identified a series of related kinases in Dictyostelium. The enzyme MHCK B shares with MHCK A a domain organization that includes a highly novel catalytic domain coupled to a carboxyl-terminal WD repeat domain. We have engineered, expressed, and purified a FLAG-tagged version of the novel kinase. In the present study, we report detailed biochemical and cellular studies documenting that MHCK B plays a physiological role in the control of Dictyostelium myosin II assembly and disassembly during the vegetative life of Dictyostelium amoebae. The presented data supports a model of multiple related MHCKs in this system, with different regulatory mechanisms and pathways controlling each enzyme.     

Myosin light chain kinase A is activated by cGMP-dependent and cGMP-independent pathways

Stimulation of Dictyostelium cells with the chemoattractant cAMP results in transient phosphorylation of the myosin regulatory light chain (RLC). We show that myosin light chain kinase A (MLCK-A) is responsible for RLC phosphorylation during chemotaxis, and that MLCK-A itself is transiently phosphorylated on threonine-166, dramatically increasing its catalytic activity. MLCK-A activation during chemotaxis is highly responsive to cellular cGMP levels and the cGMP-binding protein GbpC. MLCK-A- cells have a partial cytokinesis defect, and do not phosphorylate RLC in response to concanavalin A (conA), but cells lacking cGMP or GbpC divide normally and phosphorylate in response to conA. Thus MLCK-A is activated by a cGMP/GbpC-independent mechanism activated during cytokinesis or by conA, and a cGMP/GbpC-dependent pathway during chemotaxis.     

Multiple myosin II heavy chain kinases: roles in filament assembly control and proper cytokinesis in Dictyostelium

Myosin II filament assembly in Dictyostelium discoideum is regulated via phosphorylation of residues located in the carboxyl-terminal portion of the myosin II heavy chain (MHC) tail. A series of novel protein kinases in this system are capable of phosphorylating these residues in vitro, driving filament disassembly. Previous studies have demonstrated that at least three of these kinases (MHCK A, MHCK B, and MHCK C) display differential localization patterns in living cells. We have created a collection of single, double, and triple gene knockout cell lines for this family of kinases. Analysis of these lines reveals that three MHC kinases appear to represent the majority of cellular activity capable of driving myosin II filament disassembly, and reveals that cytokinesis defects increase with the number of kinases disrupted. Using biochemical fractionation of cytoskeletons and in vivo measurements via fluorescence recovery after photobleaching (FRAP), we find that myosin II overassembly increases incrementally in the mutants, with the MHCK A(-)/B(-)/C(-) triple mutant showing severe myosin II overassembly. These studies suggest that the full complement of MHC kinases that significantly contribute to growth phase and cytokinesis myosin II disassembly in this organism has now been identified.     

Cdc42 antagonizes inductive action of cAMP on cell shape,via effects of the myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) on myosin light chain phosphorylation

Rho GTPases play pivotal roles in regulating cell morphology. We previously showed that RhoA acts via ROKalpha to counteract the effects of the classical second messenger cyclic AMP on cell shape changes. Here we show that active Cdc42V12 also competes against the cAMP-induced stellate morphology in SH-EP cells. This Cdc42 effect is not mediated by the RhoA/ ROK pathway but rather the related MRCKalpha, a myotonic dystrophy kinase-related Cdc42-binding kinase. Co-expression of a dominant inhibitory MRCKalpha mutant with Cdc42V12 blocks the ability of the GTPase to counteract cAMP, suggesting that MRCK acts downstream of Cdc42 in this process. Cdc42V12 enhances the phosphorylation of myosin light chain (MLC) at the cell periphery and sustains focal adhesion complexes, while MLC kinase inhibitors destroy focal adhesion complexes and impair the Cdc42V12 protective effect. The data suggest that the maintenance of focal adhesion complexes via the regulation of myosin II activity underlies the ability of Cdc42 to protect against the effect of elevated cAMP.     

The cyclase-associated protein CAP as regulator of cell polarity and cAMP signaling in Dictyostelium

Cyclase-associated protein (CAP) is an evolutionarily conserved regulator of the G-actin/F-actin ratio and, in yeast, is involved in regulating the adenylyl cyclase activity. We show that cell polarization, F-actin organization, and phototaxis are altered in a Dictyostelium CAP knockout mutant. Furthermore, in complementation assays we determined the roles of the individual domains in signaling and regulation of the actin cytoskeleton. We studied in detail the adenylyl cyclase activity and found that the mutant cells have normal levels of the aggregation phase-specific adenylyl cyclase and that receptor-mediated activation is intact. However, cAMP relay that is responsible for the generation of propagating cAMP waves that control the chemotactic aggregation of starving Dictyostelium cells was altered, and the cAMP-induced cGMP production was significantly reduced. The data suggest an interaction of CAP with adenylyl cyclase in Dictyostelium and an influence on signaling pathways directly as well as through its function as a regulatory component of the cytoskeleton.