Cyclic AMP-dependent protein kinase controls virulence of the fungal pathogen Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic fungal pathogen that infects the human central nervous system. This pathogen elaborates two specialized virulence factors: the antioxidant melanin and an antiphagocytic immunosuppressive polysaccharide capsule. A signaling cascade controlling mating and virulence was identified. The PKA1 gene encoding the major cyclic AMP (cAMP)-dependent protein kinase catalytic subunit was identified and disrupted. pka1 mutant strains were sterile, failed to produce melanin or capsule, and were avirulent. The PKR1 gene encoding the protein kinase A (PKA) regulatory subunit was also identified and disrupted. pkr1 mutant strains overproduced capsule and were hypervirulent in animal models of cryptococcosis. pkr1 pka1 double mutant strains exhibited phenotypes similar to that of pka1 mutants, providing epistasis evidence that the Pka1 catalytic subunit functions downstream of the Pkr1 regulatory subunit. The PKA pathway was also shown to function downstream of the Galpha protein Gpa1 and to regulate cAMP production by feedback inhibition. These findings define a Galpha protein-cAMP-PKA signaling pathway regulating differentiation and virulence of a human fungal pathogen.     

Complex formation, phosphorylation, and localization of protein kinase A of Schizosaccharomyces pombe upon glucose starvation

Nine sam mutants that undergo sexual differentiation without requiring starvation in Schizosaccharomyces pombe were previously isolated. In this study, we identified a nonsense mutation on the pka1 locus in the sam6 mutant. pka1 encodes a catalytic subunit of protein kinase A (PKA). Replacement and overexpression of pka1 suppressed the KCl sensitivity and hyper-mating phenotype of sam6, confirming that sam6 is an allele of pka1. To characterize further the regulation of Pka1, we tested the physical interaction between Pka1 and Cgs1 (a regulatory subunit of PKA). Pka1 and Cgs1 physically interacted under glucose-limited conditions but not under glucose-rich conditions. In addition, the formation of a Pka1-Cgs1 complex was detected under glucose-limited conditions by Blue Native PAGE. Furthermore, the Pka1 protein was found to be phosphorylated under glucose-starved conditions, and at the same time its localization shifted from the nucleus towards the cytoplasm (mainly the vacuoles), suggesting a strong relationship among phosphorylation, complex formation, and the cytoplasmic distribution of Pka1.     

Cyclic AMP-dependent protein kinase catalytic subunits have divergent roles in virulence factor production in two varieties of the fungal pathogen Cryptococcus neoformans

Our earlier findings established that cyclic AMP-dependent protein kinase functions in a signaling cascade that regulates mating and virulence of Cryptococcus neoformans var. grubii (serotype A). Mutants lacking the serotype A protein kinase A (PKA) catalytic subunit Pka1 are unable to mate, fail to produce melanin or capsule, and are avirulent in animal models, whereas mutants lacking the PKA regulatory subunit Pkr1 overproduce capsule and are hypervirulent. Because other mutations have been observed to confer different phenotypes in two diverged varieties of C. neoformans (grubii variety [serotype A] and neoformans variety [serotype D]), we analyzed the functions of the PKA genes in the serotype D neoformans variety. Surprisingly, the Pka1 catalytic subunit was not required for mating, haploid fruiting, or melanin or capsule production of serotype D strains. Here we identify a second PKA catalytic subunit gene, PKA2, that is present in both serotype A and D strains of C. neoformans. The divergent Pka2 catalytic subunit was found to regulate mating, haploid fruiting, and virulence factor production in serotype D strains. In contrast, Pka2 has no role in mating, melanin production, or capsule formation in serotype A strains. Our studies illustrate how different components of signaling pathways can be co-opted and functionally specialized during the evolution of related but distinct varieties or subspecies of a human fungal pathogen.     

Complex Formation,Phosphorylation, and Localization of Protein Kinase A of Schizosaccharomyces pombe upon Glucose Starvation

Nine sam mutants that undergo sexual differentiation without requiring starvation in Schizosaccharomyces pombe were previously isolated. In this study, we identified a nonsense mutation on the pka1 locus in the sam6 mutant. pka1 encodes a catalytic subunit of protein kinase A (PKA). Replacement and overexpression of pka1 suppressed the KCl sensitivity and hyper-mating phenotype of sam6, confirming that sam6 is an allele of pka1. To characterize further the regulation of Pka1, we tested the physical interaction between Pka1 and Cgs1 (a regulatory subunit of PKA). Pka1 and Cgs1 physically interacted under glucose-limited conditions but not under glucose-rich conditions. In addition, the formation of a Pka1-Cgs1 complex was detected under glucose-limited conditions by Blue Native PAGE. Furthermore, the Pka1 protein was found to be phosphorylated under glucose-starved conditions, and at the same time its localization shifted from the nucleus towards the cytoplasm (mainly the vacuoles), suggesting a strong relationship among phosphorylation, complex formation, and the cytoplasmic distribution of Pka1.     

Divergence of protein kinase A catalytic subunits in Cryptococcus neoformans and Cryptococcus gattii illustrates evolutionary reconfiguration of a signaling cascade

Gene duplication and divergence via both the loss and gain of gene activities are powerful evolutionary forces underlying the origin of new biological functions. Here a comparative genetics approach was applied to examine the roles of protein kinase A (PKA) catalytic subunits in three closely related varieties or sibling species of the pathogenic fungus genus Cryptococcus. Previous studies revealed that two PKA catalytic subunits, Pka1 and Pka2, control virulence factor production and mating. However, only one of the two plays the predominant physiological role, and this function has been exchanged between Pka1 and Pka2 in strains of the Cryptococcus neoformans var. grubii serotype A lineage compared to divergent C. neoformans var. neoformans serotype D isolates. To understand the basis for this functional plasticity, here the activities of Pka1 and Pka2 were defined in the two varieties and the related sibling species Cryptococcus gattii by gene disruption and characterization, heterologous complementation, and analysis of serotype AD hybrid mutant strains. The findings provide evidence for a shared ancestral role of PKA in governing mating and virulence factor production and indicate that the exchange of catalytic subunit roles is attributable to loss of function. Our studies illustrate the plasticity of signaling networks enabling rapid rewiring during speciation of a clade of common human fungal pathogens.     

mAKAP assembles a protein kinase A/PDE4 phosphodiesterase cAMP signaling module

Spatiotemporal regulation of protein kinase A (PKA) activity involves the manipulation of compartmentalized cAMP pools. Now we demonstrate that the muscle-selective A-kinase anchoring protein, mAKAP, maintains a cAMP signaling module, including PKA and the rolipram-inhibited cAMP-specific phosphodiesterase (PDE4D3) in heart tissues. Functional analyses indicate that tonic PDE4D3 activity reduces the activity of the anchored PKA holoenzyme, whereas kinase activation stimulates mAKAP-associated phosphodiesterase activity. Disruption of PKA- mAKAP interaction prevents this enhancement of PDE4D3 activity, suggesting that the proximity of both enzymes in the mAKAP signaling complex forms a negative feedback loop to restore basal cAMP levels.     

Two phosphodiesterase genes, PDEL and PDEH, regulate development and pathogenicity by modulating intracellular cyclic AMP levels in Magnaporthe oryzae

Cyclic AMP (cAMP) signaling plays an important role in regulating multiple cellular responses, such as growth, morphogenesis, and/or pathogenicity of eukaryotic organisms such as fungi. As a second messenger, cAMP is important in the activation of downstream effector molecules. The balance of intracellular cAMP levels depends on biosynthesis by adenylyl cyclases (ACs) and hydrolysis by cAMP phosphodiesterases (PDEases). The rice blast fungus Magnaporthe oryzae contains a high-affinity (PdeH/Pde2) and a low-affinity (PdeL/Pde1) PDEases, and a previous study showed that PdeH has a major role in asexual differentiation and pathogenicity. Here, we show that PdeL is required for asexual development and conidial morphology, and it also plays a minor role in regulating cAMP signaling. This is in contrast to PdeH whose mutation resulted in major defects in conidial morphology, cell wall integrity, and surface hydrophobicity, as well as a significant reduction in pathogenicity. Consistent with both PdeH and PdeL functioning in cAMP signaling, disruption of PDEH only partially rescued the mutant phenotype of ΔmagB and Δpka1. Further studies suggest that PdeH might function through a feedback mechanism to regulate the expression of pathogenicity factor Mpg1 during surface hydrophobicity and pathogenic development. Moreover, microarray data revealed new insights into the underlying cAMP regulatory mechanisms that may help to identify potential pathogenicity factors for the development of new disease management strategies.     

Phosphorylation of the protein kinase A catalytic subunit is induced by cyclic AMP deficiency and physiological stresses in the fission yeast, Schizosaccharomyces pombe

In the fission yeast, Schizosaccharomyces pombe, cyclic AMP (cAMP)-dependent protein kinase (PKA) is not essential for viability under normal culturing conditions, making this organism attractive for investigating mechanisms of PKA regulation. Here we show that S. pombe cells carrying a deletion in the adenylate cyclase gene, cyr1, express markedly higher levels of the PKA catalytic subunit, Pka1, than wild type cells. Significantly, in cyr1Δ cells, but not wild type cells, a substantial proportion of Pka1 protein is hyperphosphorylated. Pka1 hyperphosphorylation is strongly induced in cyr1Δ cells, and to varying degrees in wild type cells, by both glucose starvation and stationary phase stresses, which are associated with reduced cAMP-dependent PKA activity, and by KCl stress, the cellular adaptation to which is dependent on PKA activity. Interestingly, hyperphosphorylation of Pka1 was not detected in either cyr1⁺ or cyr1Δ S. pombe strains carrying a deletion in the PKA regulatory subunit gene, cgs1, under any of the tested conditions. Our results demonstrate the existence of a cAMP-independent mechanism of PKA catalytic subunit phosphorylation, which we propose could serve as a mechanism for inducing or maintaining specific PKA functions under conditions in which its cAMP-dependent activity is downregulated.     

Requirement of a Tsp2-type tetraspanin for laccase repression and stress resistance in the basidiomycete Cryptococcus neoformans

Fungal laccases have been widely used in industry. The expression of laccase often is repressible by the primary carbon source glucose in many fungi. The underlying basis is largely unclear. We demonstrate here that a gene, TSP2-1, was required for laccase repression by glucose in the basidiomycete Cryptococcus neoformans. TSP2-1 encodes a Tsp2-type tetraspanin. The disruption of TSP2-1 resulted in constant melanin formation and the expression of the laccase gene LAC1. This derepression phenotype was restorable by 10 mM exogenous cyclic AMP (cAMP). A capsule defect in the mutant tsp2-1Δ also was restored by cAMP. The results indicate an interaction of Tsp2-1 with the cAMP-dependent protein kinase A (PKA) pathway that has been shown to modulate laccase repression and capsule biosynthesis in this fungus. Other roles of TSP2-1, e.g., in maintaining cell membrane integrity and stress resistance, also were defined. This work reveals a Tsp2-1-dependent glucose repression in C. neoformans. The function of Tsp2-type tetraspanin Tsp2-1 is described for the first time.     

Regulated expression of cyclic AMP-dependent protein kinase A reveals an influence on cell size and the secretion of virulence factors in Cryptococcus neoformans

Cyclic AMP-dependent protein kinase A (PKA) regulates elaboration of the virulence factors melanin and polysaccharide capsule in Cryptococcus neoformans. A mutation in PKA1 encoding the catalytic subunit is known to reduce virulence in mice while a defect in PKR1 encoding the regulatory subunit enhances disease. Here, we constructed strains with galactose-inducible and glucose-repressible versions of PKA1 and PKR1 by inserting the GAL7 promoter upstream of the genes. As expected, no capsule was found in dextrose-containing media for the P(GAL7) :PKA1 strain, whereas a large capsule was formed on cells grown in galactose. Along with capsule thickness, high PKA activity also influenced cell size, ploidy and vacuole enlargement, as observed in previous reports of giant/titan cell formation. We employed the regulated strains to test the hypothesis that PKA influences secretion and found that elevated PKA expression positively regulates extracellular protease activity and negatively regulates urease secretion. Furthermore, proper PKA regulation and activity were required for wild-type levels of melanization and laccase activity, as well as correct localization of the enzyme. The latter phenotype is consistent with the discovery that PKA regulates the organization of intracellular membrane compartments. Overall, these results indicate that PKA influences secretion pathways directly related to virulence factor elaboration.     

Glucose limitation and pka1 deletion rescue aberrant mitotic spindle formation induced by Mal3 overexpression in Schizosaccharomyces pombe

ABSTRACT

The cAMP-dependent protein kinase Pka1 is known as a regulator of glycogenesis, transition into meiosis, proper chromosome segregation, and stress responses in Schizosaccharomyces pombe. We demonstrated that both the cAMP/PKA pathway and glucose limitation play roles in appropriate spindle formation. Overexpression of Mal3 (1–308), an EB1 family protein, caused growth defects, increased 4C DNA content, and induced monopolar spindle formation. Overproduction of a high-affinity microtubule binding mutant (Q89R) and a recombinant protein possessing the CH and EB1 domains (1–241) both resulted in more severe phenotypes than Mal3 (1–308). Loss of functional Pka1 and glucose limitation rescued the phenotypes of Mal3-overexpressing cells, whereas deletion of Tor1 or Ssp2 did not. Growth defects and monopolar spindle formation in a kinesin-5 mutant, cut7-446, was partially rescued by pka1 deletion or glucose limitation. These findings suggest that Pka1 and glucose limitation regulate proper spindle formation in Mal3-overexpressing cells and the cut7-446 mutant.     

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.     

An anchored PKA and PDE4 complex regulates subplasmalemmal cAMP dynamics

The spatiotemporal regulation of cAMP can generate microdomains just beneath the plasma membrane where cAMP increases are larger and more dynamic than those seen globally. Real-time measurements of cAMP using mutant cyclic nucleotide-gated ion channel biosensors, pharmacological tools and RNA interference (RNAi) were employed to demonstrate a subplasmalemmal cAMP signaling module in living cells. Transient cAMP increases were observed upon stimulation of HEK293 cells with prostaglandin E1. However, pretreatment with selective inhibitors of type 4 phosphodiesterases (PDE4), protein kinase A (PKA) or PKA/A-kinase anchoring protein (AKAP) interaction blocked an immediate return of subplasmalemmal cAMP to basal levels. Knockdown of specific membrane-associated AKAPs using RNAi identified gravin (AKAP250) as the central organizer of the PDE4 complex. Co-immunoprecipitation confirmed that gravin maintains a signaling complex that includes PKA and PDE4D. We propose that gravin-associated PDE4D isoforms provide a means to rapidly terminate subplasmalemmal cAMP signals with concomitant effects on localized ion channels or enzyme activities.     

Expression of phosphodiesterase 4D (PDE4D) is regulated by both the cyclic AMP-dependent protein kinase and mitogen-activated protein kinase signaling pathways. A potential mechanism allowing for the coordinated regulation of PDE4D activity and expression in cells

Multiple families of cyclic nucleotide phosphodiesterases (PDE) have been described, and the regulated expression of these genes in cells is complex. Although cAMP is known to control the expression of certain PDE in cells, presumably reflecting a system of feedback on cAMP signaling, relatively little is known about the influence of non-cAMP signaling systems on PDE expression. In this study, we describe a novel mechanism by which activators of the protein kinase C (PKC)-Raf-MEK-ERK cascade regulate phosphodiesterase 4D (PDE4D) expression in vascular smooth muscle cells (VSMC) and assess the functional consequences of this effect. Whereas a prolonged elevation of cAMP in VSMC resulted in a protein kinase A (PKA)-dependent induction of expression of two PDE4D variants (PDE4D1 and PDE4D2), simultaneous activation of both the cAMP-PKA and PKC-Raf-MEK-ERK signaling cascades blunted this cAMP-mediated increase in PDE4D expression. By using biochemical, molecular biological, and pharmacological approaches, we demonstrate that this PDE4D-selective effect of activators of the PKC-Raf-MEK-ERK cascade was mediated through a mechanism involving altered PDE4D mRNA stability and markedly attenuated the cAMP-mediated desensitization that results from prolonged activation of the cAMP signaling system in cells. The data are presented in the context of activators of the PKC-Raf-MEK-ERK cascade having both short and long term effects on PDE4D activity and expression in cells that may influence cAMP signaling.     

Regulation of the subcellular localization of cyclic AMP-dependent protein kinase in response to physiological stresses and sexual differentiation in the fission yeast Schizosaccharomyces pombe

We describe regulation of the subcellular localization of cyclic AMP (cAMP)-dependent protein kinase (PKA) regulatory (Cgs1p) and catalytic (Pka1p) subunits in the fission yeast Schizosaccharomyces pombe in response to physiological stresses and during sexual differentiation as determined by fluorescence microscopy of the Cgs1-green fluorescent protein (GFP) and Pka1-GFP fusion proteins, respectively. In wild-type S. pombe cells cultured to log phase under normal growth conditions, Cgs1p and Pka1p are concentrated in the nucleus and more diffusely present in the cytoplasm. Nuclear localization of both proteins is dependent on cAMP, since in cells lacking adenylate cyclase they are detectable only in the cytoplasm. In cells lacking Cgs1p or both Cgs1p and adenylate cyclase, Pka1p is concentrated in the nucleus, demonstrating a role for Cgs1p in the nuclear exclusion of Pka1p. Nuclear-cytoplasmic redistribution of Cgs1p and Pka1p is triggered by growth in glucose-limited or hyperosmotic media and in response to stationary-phase growth. In addition, both proteins are excluded from the nucleus in mating cells undergoing karyogamy and subsequently concentrated in postmeiotic spores. Cgs1p is required for subcellular redistribution of Pka1p induced by growth in glucose-limited and hyperosmotic media and during karyogamy but is not required for Pka1p redistribution triggered by stationary-phase growth or for the enrichment of Pka1p in spores. Our results demonstrate that PKA localization is regulated by cAMP and regulatory subunit-dependent and -independent mechanisms in S. pombe.