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.     

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.     

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.     

Pde1 phosphodiesterase modulates cyclic AMP levels through a protein kinase A-mediated negative feedback loop in Cryptococcus neoformans

The virulence of the human pathogenic fungus Cryptococcus neoformans is regulated by a cyclic AMP (cAMP)-dependent protein kinase A (PKA) signaling cascade that promotes mating and the production of melanin and capsule. In this study, genes encoding homologs of the Saccharomyces cerevisiae low- and high-affinity phosphodiesterases, PDE1 and PDE2, respectively, were deleted in serotype A strains of C. neoformans. The resulting mutants exhibited moderately elevated levels of melanin and capsule production relative to the wild type. Epistasis experiments indicate that Pde1 functions downstream of the Galpha subunit Gpa1, which initiates cAMP-dependent signaling in response to an extracellular signal. Previous work has shown that the PKA catalytic subunit Pka1 governs cAMP levels via a negative feedback loop. Here we show that a pde1Delta pka1Delta mutant strain exhibits cAMP levels that are dramatically increased ( approximately 15-fold) relative to those in a pka1Delta single mutant strain and that a site-directed mutation in a consensus PKA phosphorylation site reduces Pde1 function. These data provide evidence that fluctuations in cAMP levels are modulated by both Pka1-dependent regulation of Pde1 and another target that comprise a robust negative feedback loop to tightly constrain intracellular cAMP levels.     

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.     

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.     

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.     

In vivo activation of protein kinase A in Schizosaccharomyces pombe requires threonine phosphorylation at its activation loop and is dependent on PDK1

Phosphoinositide-dependent protein kinase 1 (PDK1) plays a central role in cellular signaling by phosphorylating members of the AGC family of kinases. This family includes protein kinase C (PKC), protein kinase B (PKB), p70/p90 ribosomal S6 kinases (RSK and S6K), and the catalytic subunit of cAMP-dependent protein kinase (PKA). Although PDK1 phosphorylates and activates PKC, PKB, and RSK in vivo, PDK1 regulation of PKA remains controversial. We isolated ksg1, the fission yeast ortholog of mammalian PDK1, as a suppressor of growth defects caused by loss of the stress-activated MAP kinase, Spc1. Here, we demonstrate that Ksg1 is required for activation of PKA. Cells containing the ksg1.12 thermolabile allele exhibit pleiotropic phenotypes, including the failure to arrest in G(1) and an inability to conjugate. The ksg1.12 allele strongly suppresses defects associated with unregulated PKA. Pka1, the catalytic subunit of cAMP-dependent protein kinase, is phosphorylated in vivo at Thr-356, which is located in the activation loop of the kinase and corresponds to Thr-197 in mammalian PKA. Phosphorylation of Thr-356 is required for in vivo activation of Pka1 and is dependent upon Ksg1. These data provide experimental evidence that PKA is a physiological substrate for PDK1.     

Cellular stress induces cytoplasmic RNA granules in fission yeast

Severe stress causes plant and animal cells to form large cytoplasmic granules containing RNA and proteins. Here, we demonstrate the existence of stress-induced cytoplasmic RNA granules in Schizosaccharomyces pombe. Homologs to several known protein components of mammalian processing bodies and stress granules are found in fission yeast RNA granules. In contrast to mammalian cells, poly(A)-binding protein (Pabp) colocalizes in stress-induced granules with decapping protein. After glucose deprivation, protein kinase A (PKA) is required for accumulation of Pabp-positive granules and translational down-regulation. This is the first demonstration of a role for PKA in RNA granule formation. In mammals, the translation initiation protein eIF2α is a key regulator of formation of granules containing poly(A)-binding protein. In S. pombe, nonphosphorylatable eIF2α does not block but delays granule formation and subsequent clearance after exposure to hyperosmosis. At least two separate pathways in S. pombe appear to regulate stress-induced granules: pka1 mutants are fully proficient to form granules after hyperosmotic shock; conversely, eIF2α does not affect granule formation in glucose starvation. Further, we demonstrate a Pka1-dependent link between calcium perturbation and RNA granules, which has not been described earlier in any organism.     

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.     

AMY-1 interacts with S-AKAP84 and AKAP95 in the cytoplasm and the nucleus,respectively, and inhibits cAMP-dependent protein kinase activity by preventing binding of its catalytic subunit to A-kinase-anchoring protein (AKAP) complex

We have reported that a novel c-Myc-binding protein, AMY-1, binds to cAMP-dependent protein kinase-anchoring protein 149 (AKAP149) and its splicing variant, AKAP84 and is localized in the mitochondria in a complex with RII, a regulatory subunit of cAMP-dependent protein kinase (PKA) (Furusawa, M., Ohnishi, T., Taira, T., Iguchi-Ariga, S. M. M., and Ariga, H. (2001) J. Biol. Chem. 276, 36647-36651). In this study, we further found that AMY-1 competitively bound to either AKAP95 or AKAP84 in the nucleus and the cytoplasm, respectively, in a concentration-dependent manner of either AKAP. Like AKAP84, AMY-1 was found to bind to the RII-binding region of AKAP95 in vivo and in vitro and to make a ternary complex with RII. It was also found that the formation of the complex of AMY-1 with AKAP84/95 and RII prevented a catalytic subunit from binding to this AKAP complex, leading to suppression of PKA activity. These findings suggest that AMY-1 is an important modulator of PKA.     

Cyclic AMP-Dependent Protein Kinase Decreases γ-Aminobutyric AcidA Receptor-Mediated 36Q1− Uptake by Brain Microsacs

The effect of cyclic AMP (cAMP)-dependent protein phosphorylation on gamma-aminobutyric acidA (GABAA) receptor function was examined using isolated brain membrane vesicles (microsacs). Muscimol-stimulated 36Cl- uptake was studied in mouse brain microsacs permeabilized to introduce the catalytic subunit of cAMP-dependent protein kinase (PKA). At both submaximal and maximally effective concentrations of muscimol, PKA inhibited muscimol-stimulated 36Cl- uptake by approximately 25%. In parallel experiments, PKA and [gamma-32P]ATP were introduced into the microsacs, and we attempted to immunoprecipitate the entire GABAA receptor complex, under nondenaturing conditions, using an anti-alpha 1-subunit antibody. Data from such experiments show that PKA increases the phosphorylation of several microsac proteins, including a 66-kDa polypeptide specifically immunoprecipitated with the GABAA receptor anti-alpha 1 subunit antibody. Phosphopeptide mapping of the 66-kDa polypeptide demonstrated a 14-kDa fragment similar to that obtained with the purified, PKA-phosphorylated GABAA receptor. These results provide evidence that the catalytic subunit of PKA inhibits the function of brain GABAA receptors and demonstrate that this functional change is concomitant with an increase in protein phosphorylation.     

Catalytic subunit of cAMP-dependent protein kinase from a catch muscle of the bivalve mollusk Mytilus galloprovincialis: purification, characterization, and phosphorylation of muscle proteins

cAMP-dependent protein kinase (PKA) plays a crucial role in the release of the catch state of molluskan muscles, but the nature of the enzyme in such tissues is unknown. In this paper, we report the purification of the catalytic (C) subunit of PKA from the posterior adductor muscle (PAM) of the sea mussel Mytilus galloprovincialis. It is a monomeric protein with an apparent molecular mass of 40.0+/-2.0kDa and Stoke's radius 25.1+/-0.3A. The protein kinase activity of the purified enzyme was inhibited by both isoforms of the PKA regulatory (R) subunit that we had previously characterized in the mollusk, and also by the inhibitor peptide PKI(5-24). On the other hand, the main proteins of the contractile apparatus of PAM were partially purified and their ability to be phosphorylated in vitro by purified PKA C subunit was analyzed. The results showed that twitchin, a high molecular mass protein associated with thick filaments, was the better substrate for endogenous PKA. It was rapidly phosphorylated with a stoichiometry of 3.47+/-0.24mol Pmol(-1) protein. Also, catchin, paramyosin, and actin were phosphorylated, although more slowly and to a lesser extent. On the contrary, myosin heavy chain (MHC) and tropomyosin were not phosphorylated under the conditions used.     

Presence of a complex containing vesicle-associated membrane protein 2 in rat parotid acinar cells and its disassembly upon activation of cAMP-dependent protein kinase.

Amylase release from parotid acinar cells is mainly induced by the accumulation of intracellular cAMP, presumably through the phosphorylation of substrates by cAMP-dependent protein kinase (PKA). However, the molecular mechanisms of this process are not clear. In a previous study (Fujita-Yoshigaki, J., Dohke, Y., Hara-Yokoyama, M., Kamata, Y., Kozaki, S., Furuyama, S., and Sugiya, H. (1996) J. Biol. Chem. 271, 13130-13134), we reported that vesicle-associated membrane protein 2 (VAMP2) is localized at the secretory granule membrane and is involved in cAMP-induced amylase secretion. To study the formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex containing VAMP2 in parotid acinar cells, we prepared rabbit polyclonal antibody against the peptide corresponding to Arg(47)-Asp(64) of VAMP2 (anti-SER4256). The recognition site of anti-SER4256 overlaps the domain involved in binding target membrane SNAREs (t-SNARES). Then we examined the condition of VAMP2 by immunoprecipitation with anti-SER4256. VAMP2 was not included in the immunoprecipitate from solubilized granule membrane fraction under the control conditions, but incubation with cytosolic fraction and cAMP caused immunoprecipitation of VAMP2. The effect of cytosolic fraction and cAMP was reduced by addition of PKA inhibitor H89. Addition of both the catalytic subunit of PKA and the cytosolic fraction allowed immunoprecipitation of VAMP2, whereas the PKA catalytic subunit alone did not. These results suggest that () the t-SNARE binding region of VAMP2 is masked by some protein X and activation of PKA caused the dissociation of X from VAMP2; and () the effect of PKA is not direct phosphorylation of X, but works through phosphorylation of some other cytosolic protein.     

A transforming growth factor beta-induced Smad3/Smad4 complex directly activates protein kinase A

Transforming growth factor beta (TGFbeta) interacts with cell surface receptors to initiate a signaling cascade critical in regulating growth, differentiation, and development of many cell types. TGFbeta signaling involves activation of Smad proteins which directly regulate target gene expression. Here we show that Smad proteins also regulate gene expression by using a previously unrecognized pathway involving direct interaction with protein kinase A (PKA). PKA has numerous effects on growth, differentiation, and apoptosis, and activation of PKA is generally initiated by increased cellular cyclic AMP (cAMP). However, we found that TGFbeta activates PKA independent of increased cAMP, and our observations support the conclusion that there is formation of a complex between Smad proteins and the regulatory subunit of PKA, with release of the catalytic subunit from the PKA holoenzyme. We also found that the activation of PKA was required for TGFbeta activation of CREB, induction of p21(Cip1), and inhibition of cell growth. Taken together, these data indicate an important and previously unrecognized interaction between the TGFbeta and PKA signaling pathways.