Requirements for the adenylyl cyclases in the development of Dictyostelium

It has been suggested that all intracellular signaling by cAMP during development of Dictyostelium is mediated by the cAMP-dependent protein kinase, PKA, since cells carrying null mutations in the acaA gene that encodes adenylyl cyclase can develop so as to form fruiting bodies under some conditions if PKA is made constitutive by overexpressing the catalytic subunit. However, a second adenylyl cyclase encoded by acrA has recently been found that functions in a cell autonomous fashion during late development. We have found that expression of a modified acaA gene rescues acrA- mutant cells indicating that the only role played by ACR is to produce cAMP. To determine whether cells lacking both adenylyl cyclase genes can develop when PKA is constitutive we disrupted acrA in a acaA- PKA-C(over) strain. When developed at high cell densities, acrA- acaA- PKA-C(over) cells form mounds, express cell type-specific genes at reduced levels and secrete cellulose coats but do not form fruiting bodies or significant numbers of viable spores. Thus, it appears that synthesis of cAMP is required for spore differentiation in Dictyostelium even if PKA activity is high.     

Role of cAMP-dependent protein kinase during growth and early development of Dictyostelium discoideum

cAMP-dependent protein kinase (PKA) is an essential regulator of gene expression and cell differentiation during multicellular development of Dictyostelium discoideum. Here we show that PKA activity also regulates gene expression during the growth phase and at the transition from growth to development. Overexpression of PKA leads to overexpression of the discoidinIgamma promoter, while expression of the discoidinIgamma promoter is reduced when PKA activity is reduced, either by expression of a dominant negative mutant of the regulatory subunit or by disruption of the gene for the catalytic subunit (PKA-C). The discoidin phenotype of PKA-C null cells is cell autonomous. In particular, normal secretion of discoidin-inducing factors was demonstrated. In addition, PKA-C null cells are able to respond to media conditioned by PSF and CMF. We conclude that PKA is a major activator of discoidin expression. However, it is not required for production or transduction of the inducing extracellular signals. Therefore, PKA-dependent and PKA-independent pathways regulate the expression of the discoidin genes.     

Multiple roles for cAMP-dependent protein kinase during Dictyostelium development.

The cAMP-dependent protein kinase (PKA) holoenzyme of Dictyostelium comprises a single regulatory (R) and catalytic (C) subunit, and both proteins increase in concentration during cellular aggregation. In order to determine the role of the kinase, we have constructed mutants of the R subunit that are defective in cAMP binding, in inhibition of the C subunit, or in both functions. Analysis of these mutants suggests that overexpression of the unmutated R subunit, which is known to block development, occurs by direct inactivation of the C subunit rather than by an effect on intracellular cAMP levels. Cells with an inactive C subunit (PKA- cells) are defective in cAMP relay, the production of cAMP in response to extracellular cAMP stimulation. This presumably accounts for their inability to undertake aggregation. When mixed with wild-type cells, PKA- cells migrate toward the signalling centre but remain confined to the periphery of the tight aggregate and are lost from the back of the migratory slug. This suggests that PKA may be required during the late, multicellular stages of development. Consistent with this, we find that a number of postaggregative genes are not expressed in PKA- cells, even when they are allowed to synergise with normal cells.     

Stimulus-induced translocation of the protein kinase A catalytic subunit to the apical membrane in blowfly salivary glands

Secretion in blowfly (Calliphora vicina) salivary glands is regulated by the neurohormone serotonin (5-HT), which activates the InsP₃/Ca²⁺ pathway and the cAMP/protein kinase A (PKA) pathway in the secretory cells. The latter signaling cascade induces the activation of a vacuolar H⁺-ATPase on the apical membrane. Here, we have determined the distribution of PKA by using antibodies against the PKA regulatory subunit-II (PKA-RII) and the PKA catalytic subunit (PKA-C) of Drosophila. PKA is present in high concentrations within the secretory cells. PKA-RII and PKA-C co-distribute in non-stimulated glands, being enriched in the basal portion of the secretory cells. Exposure to 8-CPT-cAMP or 5-HT induces the translocation of PKA-C to the apical membrane, whereas the PKA-RII distribution remains unchanged. The recruitment of PKA-C to the apical membrane corroborates our hypothesis that vacuolar H⁺-ATPase, which is enriched in this membrane domain, is a target protein for PKA. This work was supported by grants Wa463/9–5 and GRK837 from the Deutsche Forschungsgemeinschaft.     

Development of platelet inhibition by cAMP during megakaryocytopoiesis

Prostacyclin is a potent inhibitor of agonist-induced Ca2+ increases in platelets, but in the megakaryocytic cell line MEG-01 this inhibition is absent. Using human megakaryocytic cell lines representing different stages in megakaryocyte (Mk) maturation as well as stem cells and immature and mature megakaryocytes, we show that the inhibition by prostacyclin develops at a late maturation stage shortly before platelets are formed. This late appearance is not caused by insufficient cAMP formation or absent protein kinase A (PKA) activity in immature cells. Instead, the appearance of Ca2+ inhibition by prostacyclin is accompanied by a sharp increase in the expression of the catalytic subunit of PKA (PKA-C) but not by changes in the expression of the PKA-regulatory subunits Ialpha/beta, IIalpha, and IIbeta. Overexpression of PKA-C in the megakaryocytic cell line CHRF-288-11 potentiates the Ca2+ inhibition by prostacyclin. Thus, up-regulation of PKA-C appears to be a key step in the development of Ca2+ inhibition by prostacyclin in platelets.     

Overproduction of the regulatory subunit of the cAMP-dependent protein kinase blocks the differentiation of Dictyostelium discoideum.

During the aggregation of Dictyostelium discoideum extracellular cAMP is known to act as a chemotractant and as an inducer of cellular differentiation. However, its intracellular role as a second messenger remains obscure. We have constructed a fusion gene consisting of the cDNA encoding the regulatory subunit (R) of the cAMP-dependent protein kinase fused to the promoter and N-terminal-proximal sequences of a Dictyostelium actin gene. Stable transformants, containing multiple copies of this gene, overproduce the R subunit which accumulates prematurely relative to the endogenous protein. These transformants fail to aggregate. Detailed analysis has shown that they are blocked at interphase, the period prior to aggregation, and that they are severely defective in most responses to cAMP including the induction of gene expression. Our observations suggest that intracellular cAMP acts, presumably by activation of the catalytic subunit of the cAMP-dependent protein kinase, to facilitate early development.     

Interaction of gdt1 and protein kinase A (PKA) in the growth-differentiation-transition in Dictyostelium

The gdt1 gene is a negative regulator of the growth-differentiation-transition (GDT) in Dictyostelium. gdt1- cells express the GDT marker discoidin earlier and at higher levels and prematurely enter the differentiation pathway. Protein kinase A is a positive regulator of the GDT and is required for multicellular development. Disruption of the PKA catalytic subunit or overexpression of a constitutively active mutant of the regulatory subunit results in cells which do not form multicellular aggregates and which show strongly reduced levels of discoidin. We have created PKA-/gdt1- double mutants and show that these display high levels of discoidin expression but no aggregation, suggesting that gdt1 may be a downstream target of PKA in a branched signaling cascade initiating differentiation. Data obtained with the PKA inhibitor H89 support these result: in wild type cells H89 inhibits discoidin expression while in gdt1- mutants there is no obvious effect. However, since PKA-/gdt1- cells display less discoidin expression than the single gdt1 mutant, we propose that PKA and gdt1 are in two parallel interacting pathways. To get insight into the mechanism how PKA may block gdt1, we have tested two putative PKA phosphorylation sites in the protein and found that one of them is efficiently phosphorylated by PKA in vitro. A model for the interplay between PKA and gdt1 during the growth-differentiation-transition is discussed.     

Identification of a protein kinase A regulatory subunit from Leishmania having importance in metacyclogenesis through induction of autophagy

cAMP‐mediated responses act as modulators of environmental sensing and cellular differentiation of many kinetoplastidae parasites including Leishmania. Although cAMP synthesizing (adenylate cyclase) and degrading (phosphodiesterase) enzymes have been cloned and characterized from Leishmania, no cAMP‐binding effector molecule has yet been identified from this parasite. In this study, a regulatory subunit of cAMP‐dependent protein kinase (Ldpkar1), homologous to mammalian class I cAMP‐dependent protein kinase regulatory subunit, has been identified from L. donovani. Further characterization suggested possible interaction of LdPKAR1 with PKA catalytic subunits and inhibition of PKA activity. This PKA regulatory subunit is expressed in all life cycle stages and its expression attained maximum level in stationary phase promastigotes, which are biochemically similar to the infective metacyclic promastigotes. Starvation condition, the trigger for metacyclogenesis in the parasite, elevates LdPKAR1 expression and under starvation condition promastigotes overexpressing Ldpkar1 attained metacyclic features earlier than normal cells. Furthermore, Ldpkar1 overexpression accelerates autophagy, a starvation‐induced cytological event necessary for metacyclogenesis and amastigote formation. Conditional silencing of Ldpkar1 delays the induction of autophagy in the parasite. The study, for the first time, reports the identification of a functional cAMP‐binding effector molecule from Leishmania that may modulate important cytological events affecting metacyclogenesis.     

A cAMP-dependent protein kinase is present in differentiating Dictyostelium discoideum cells

We demonstrate the occurrence of a cAMP-dependent protein kinase in Dictyostelium discoideum cells at the terminal stage of differentiation. A cAMP-binding component was purified to homogeneity by affinity chromatography. This subunit inhibits the activity of purified catalytic subunit from beef heart protein kinase; the inhibition is reversed upon addition of cAMP. The protein is highly specific for cAMP and has a dissociation constant of 4 nM. The isolated regulatory subunit is a monomer of 39 K, with a sedimentation coefficient of 3.5S and a frictional coefficient of 1.24. The differences between this regulatory subunit and regulatory subunits of protein kinases from other sources are discussed.     

Cyclic nucleotides as affinity tools: phosphorothioate cAMP analogues address specific PKA subproteomes

cAMP (adenosine-3',5'-cyclic monophosphate) is a general second messenger controlling distinct targets in eukaryotic cells. In a (sub)proteomic approach, two classes of phosphorothioate cAMP affinity tools were used to isolate and to identify signalling complexes of the main cAMP target, cAMP dependent protein kinase (PKA). Agonist analogues (here: Sp-cAMPS) bind to the regulatory subunits of PKA (PKA-R), together with their interaction partners, and cause dissociation of a holoenzyme complex comprising PKA-R and catalytic subunits of PKA (PKA-C). Antagonist analogues (here: Rp-cAMPS) bind to the holoenzyme without dissociating the complex and were developed to identify interaction partners that bind to the entire complex or to PKA-C. More than 80 different proteins were isolated from tissue extracts including several PKA isoforms and known as well as potentially new interaction partners. Nevertheless, unspecific binding of general nucleotide binding proteins limited the outcome of this chemical proteomics approach. Surface plasmon resonance (SPR) was employed to optimise the entire workflow of pull down proteomics and to quantify the effects of different nucleotides (ATP, ADP, GTP and NADH) on PKA-R binding to affinity material. We could demonstrate that the addition of NADH to lysates improved specificity in pull down experiments. Using a combination of SPR studies and pull down experiments it was shown unambiguously that it is possible to specifically elute protein complexes with cAMP or cGMP from cAMPS analogue matrices. The side-by-side analysis of the PKA-R interactome and the holoenzyme complexed with interacting proteins will contribute to a further dissection of the multifaceted PKA signalling network.     

An adenylyl cyclase that functions during late development of Dictyostelium.

A variety of extracellular signals lead to the accumulation of cAMP which can act as a second message within cells by activating protein kinase A (PKA). Expression of many of the essential developmental genes in Dictyostelium discoideum are known to depend on PKA activity. Cells in which the receptor-coupled adenylyl cyclase gene, acaA, is genetically inactivated grow well but are unable to develop. Surprisingly, acaA(-) mutant cells can be rescued by developing them in mixtures with wild-type cells, suggesting that another adenylyl cyclase is present in developing cells that can provide the internal cAMP necessary to activate PKA. However, the only other known adenylyl cyclase gene in Dictyostelium, acgA, is only expressed during germination of spores and plays no role in the formation of fruiting bodies. By screening morphological mutants generated by Restriction Enzyme Mediated Integration (REMI) we discovered a novel adenylyl cyclase gene, acrA, that is expressed at low levels in growing cells and at more than 25-fold higher levels during development. Growth and development up to the slug stage are unaffected in acrA(-) mutant strains but the cells make almost no viable spores and produce unnaturally long stalks. Adenylyl cyclase activity increases during aggregation, plateaus during the slug stage and then increases considerably during terminal differentiation. The increase in activity following aggregation fails to occur in acrA(-) cells. As long as ACA is fully active, ACR is not required until culmination but then plays a critical role in sporulation and construction of the stalk.     

Interaction of gdt1 and protein kinase A (PKA) in the growth-differentiation-transition in Dictyostelium

Abstract The gdt1 gene is a negative regulator of the growth-differentiation-transition (GDT) in Dictyostelium . gdt1 ⁻ cells express the GDT marker discoidin earlier and at higher levels and prematurely enter the differentiation pathway. Protein kinase A is a positive regulator of the GDT and is required for multicellular development. Disruption of the PKA catalytic subunit or overexpression of a constitutively active mutant of the regulatory subunit results in cells which do not form multicellular aggregates and which show strongly reduced levels of discoidin. We have created PKA ⁻/gdt1⁻ double mutants and show that these display high levels of discoidin expression but no aggregation, suggesting that gdt1 may be a downstream target of PKA in a branched signalling cascade initiating differentiation. Data obtained with the PKA inhibitor H89 support these result: in wild type cells H89 inhibits discoidin expression while in gdt1 ⁻ mutants there is no obvious effect. However, since PKA⁻/gdt1⁻ cells display less discoidin expression than the single gdt1 mutant, we propose that PKA and gdt1 are in two parallel interacting pathways.
To get insight into the mechanism how PKA may block gdt1, we have tested two putative PKA phosphorylation sites in the protein and found that one of them is efficiently phosphorylated by PKA in vitro. A model for the interplay between PKA and gdt1 during the growth-differentiation-transition is discussed.     

An increase of cAMP-dependent protein kinase during development in Polysphondylium pallidum

Polysphondylium pallidum is a cellular slime mold in which, unlike in Dictyostelium discoideum, cAMP is not the chemotactic agent. The occurrence of a cAMP-dependent protein kinase in D. discoideum was demonstrated earlier and we suggested that it may mediate the intracellular effects of cAMP on the development of the organism, particularly since an increase in the amount of the enzyme during development was noted. In D. discoideum cAMP plays a dual role insofar as it serves both as chemotactic agent and as second messenger; it was of interest therefore, to determine whether a cAMP-dependent protein kinase occurred in P. pallidum. We found a cAMP-dependent protein kinase in P. pallidum using Kemptide as substrate. The regulatory subunit of the enzyme has an apparent molecular weight of 41,000 and seems to be similar in its properties with that isolated earlier from D. discoideum. The cAMP-dependent protein kinase catalytic subunits from the two species are also similar. Furthermore, there is a developmentally regulated, parallel, two- to threefold increase in the two subunits of the cAMP-dependent protein kinase in P. pallidum. The increase occurs before aggregates are formed. These findings are compatible with a role of the intracellular cAMP and of the cAMP-dependent protein kinase in the development of P. pallidum.     

The cdk5 homologue,crp, regulates endocytosis and secretion in dictyostelium and is necessary for optimum growth and differentiation

Dictyostelium Crp is a member of the cyclin-dependent kinase (Cdk) family of proteins. It is most related in sequence to mammalian Cdk5, which unlike other members of the family, has functions that are unrelated to the cell cycle. In order to better understand the function of Crp in Dictyostelium, we overexpressed a dominant negative form, Crp-D144N, under the control of the actin 15 promoter. Cells overexpressing Crp-D144N exhibit a reduced growth rate in suspension culture and reduced rates of fluid-phase endocytosis and phagocytosis. There is no reduction in Cdc2 kinase activity in extracts from cells overexpressing Crp-D144N, suggesting that the growth defect is not due to inhibition of Cdc2. In addition to the growth defect, the act15::crp-D144N transformants aggregate at a slower rate than wild-type cells and form large aggregation streams. These eventually break up to form small aggregates and most of these do not produce mature fruiting bodies. The aggregation defect is fully reversed in the presence of wild-type cells but terminal differentiation is only partially rescued. In act15::crp-D144N transformants, the countin component of the counting factor, a secreted protein complex that regulates the breakup of streams, mostly appears outside the cell as degradation products and the reduced level of the intact protein may at least partially account for the initial formation of the large aggregation streams. Our observations indicate that Crp is important for both endocytosis and efflux and that defects in these functions lead to reduced growth and aberrant development.     

Starvation promotes Dictyostelium development by relieving PufA inhibition of PKA translation through the YakA kinase pathway.

When nutrients are depleted, Dictyostelium cells undergo cell cycle arrest and initiate a developmental program that ensures survival. The YakA protein kinase governs this transition by regulating the cell cycle, repressing growth-phase genes and inducing developmental genes. YakA mutants have a shortened cell cycle and do not initiate development. A suppressor of yakA that reverses most of the developmental defects of yakA- cells, but none of their growth defects was identified. The inactivated gene, pufA, encodes a member of the Puf protein family of translational regulators. Upon starvation, pufA- cells develop precociously and overexpress developmentally important proteins, including the catalytic subunit of cAMP-dependent protein kinase, PKA-C. Gel mobility-shift assays using a 200-base segment of PKA-C's mRNA as a probe reveals a complex with wild-type cell extracts, but not with pufA- cell extracts, suggesting the presence of a potential PufA recognition element in the PKA-C mRNA. PKA-C protein levels are low at the times of development when this complex is detectable, whereas when the complex is undetectable PKA-C levels are high. There is also an inverse relationship between PufA and PKA-C protein levels at all times of development in every mutant tested. Furthermore, expression of the putative PufA recognition elements in wild-type cells causes precocious aggregation and PKA-C overexpression, phenocopying a pufA mutation. Finally, YakA function is required for the decline of PufA protein and mRNA levels in the first 4 hours of development. We propose that PufA is a translational regulator that directly controls PKA-C synthesis and that YakA regulates the initiation of development by inhibiting the expression of PufA. Our work also suggests that Puf protein translational regulation evolved prior to the radiation of metazoan species.     

Spatial expression patterns of genes involved in cyclic AMP responses in Dictyostelium discoideum development

The spatial expression patterns of genes involved in cyclic adenosine monophosphate (cAMP) responses during morphogenesis in Dictyostelium discoideum were analyzed by in situ hybridization. Genes encoding adenylyl cyclase A (ACA), cAMP receptor 1, G-protein alpha2 and beta subunits, cytosolic activator of ACA (CRAC and Aimless), catalytic subunit of protein kinase A (PKA-C) and cAMP phosphodiesterases (PDE and REG-A) were preferentially expressed in the anterior prestalk (tip) region of slugs, which acts as an organizing center. MAP kinase ERK2 (extracellular signal-regulated kinase-2) mRNA, however, was enriched in the posterior prespore region. At the culmination stage, the expression of ACA, CRAC and PKA-C mRNA increased in prespore cells in contrast with the previous stage. However, no alteration in the site of expression was observed for the other mRNA analyzed. Based on these findings, two and four classes of expression patterns were catalogued for these genes during the slug and culmination stages, respectively. Promoter analyses of genes in particular classes should enhance understanding of the regulation of dynamic and coordinated gene expression during morphogenesis.     

cAMP-dependent protein kinase from Dictyostelium discoideum

The cAMP-dependent protein kinase (cAK) from Dictyostelium discoideum is an enzyme composed of one catalytic and one regulatory subunit. Upon binding of cAMP, the holoenzyme dissociates to liberate free active catalytic subunits. The cAK is developmentally regulated, ranging from very little activity in vegetative cells to maximal expression in postaggregative cells. Although there is no immunological cross-reaction between the subunits of cAKs from Dictyostelium and from other organisms, they share several biochemical properties. A complete cDNA for the regulatory subunit has been cloned and sequenced. Only one copy of the gene for the regulatory subunit is present per haploid genome. On the basis of the comparison of the structure of the cAK from Dictyostelium with its counterparts in yeast and higher eukaryotes, we propose a model for the evolution of cyclic-nucleotide-binding proteins.