scholarly journals Cyclic AMP-Dependent Protein Kinase Controls Virulence of the Fungal Pathogen Cryptococcus neoformans

2001 ◽  
Vol 21 (9) ◽  
pp. 3179-3191 ◽  
Author(s):  
Cletus A. D'Souza ◽  
J. Andrew Alspaugh ◽  
Changli Yue ◽  
Toshiaki Harashima ◽  
Gary M. Cox ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Cryptococcus Neoformans ◽  
Fungal Pathogen ◽  
Catalytic Subunit ◽  
Regulatory Subunit ◽  
Dependent Protein Kinase ◽  
Gene Encoding ◽  
Mutant Strains ◽  
Dependent Protein

ABSTRACT 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. ThePKA1 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 Gα protein Gpa1 and to regulate cAMP production by feedback inhibition. These findings define a Gα protein-cAMP-PKA signaling pathway regulating differentiation and virulence of a human fungal pathogen.

scholarly journals Adenosine 3':5'-cyclic monophosphate-binding proteins in bovine and rat tissues

1976 ◽  
Vol 159 (2) ◽  
pp. 423-427 ◽  
Author(s):  
P H Sugden ◽  
J D Corbin
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Binding Proteins ◽  
Binding Protein ◽  
Bovine Liver ◽  
Catalytic Subunit ◽  
Regulatory Subunit ◽  
Trypsin Treatment ◽  
Dependent Protein Kinase ◽  
Dependent Protein

1. At least two classes of high-affinity cyclic AMP-binding proteins have been identified: those derived from cyclic AMP-dependent protein kinases (regulatory subunits) and those that bind a wide range of adenine analogues (adenine analogue-binding proteins). 2. In fresh-tissue extracts, regulatory subunits could be further subdivided into ‘type I’ or ‘type II’ depending on whether they were derived from ‘type I’ or ‘type II’ protein kinase [see Corbin et al. (1975) J. Biol. Chem. 250, 218-225]. 3. The adenine analogue-binding protein was detected in crude tissue supernatant fractions of bovine and rat liver. It differed from the regulatory subunit of cyclic AMP-dependent protein kinase in many of its properties. Under the conditions of assay used, the protein accounted for about 45% of the binding of cyclic AMP to bovine liver supernatants. 4. The adenine analogue-binding protein from bovine liver was partially purified by DEAE-cellulose and Sepharose 6B chromatography. It had mol.wt. 185000 and was trypsin-sensitive. As shown by competition and direct binding experiments, it bound adenosine and AMP in addition to cyclic AMP. At intracellular concentrations of adenine nucleotides, binding of cyclic AMP was essentially completely inhibited in vitro. Adenosine binding was inhibited by only 30% under similar conditions. 5. Rat tissues were examined for the presence of the adenine analogue-binding protein, and, of those examined (adipose tissue, heart, brain, testis, kidney and liver), significant amounts were only found in the liver. The possible physiological role of the adenine analogue-binding protein is discussed. 6. Because the adenine analogue-binding protein or other cyclic AMP-binding proteins in tissues may be products of partial proteolysis of the regulatory subunit of cyclic AMP-dependent protein kinase, the effects of trypsin and aging on partially purified protein kinase and its regulatory subunit from bovine liver were investigated. In all studies, the effects of trypsin and aging were similar. 7. In fresh preparations, the cyclic AMP-dependent protein kinase had mol.wt. 150000. Trypsin treatment converted it into a form of mol.wt 79500. 8. The regulatory subunit of the protein kinase had mol.wt. 87000. It would reassociate with and inhibit the catalytic subunit of the enzyme. Trypsin treatment of the regulatory subunit produced a species of mol.wt. 35500 which bound cyclic AMP but did not reassociate with the catalytic subunit. Trypsin treatment of the protein kinase and dissociation of the product by cyclic AMP produced a regulatory subunit of mol.wt. 46500 which reassociated with the catalytic subunit. 9. These results may be explained by at least two trypsin-sensitive sites on the regulatory subunit. A model for the effects of trypsin is described.

scholarly journals Chemical cross-linking of cyclic AMP-dependent protein kinase and its dissimilar subunits

1983 ◽  
Vol 209 (3) ◽  
pp. 581-586 ◽  
Author(s):  
J P Charlton ◽  
C H Huang ◽  
L C Huang
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Ternary Complex ◽  
Kinetic Studies ◽  
Catalytic Subunit ◽  
Regulatory Subunit ◽  
Cross Linking ◽  
Dependent Protein Kinase ◽  
Native Form ◽  
Dependent Protein

Previous kinetic studies have demonstrated that the activation of cyclic AMP-dependent protein kinase by cyclic AMP involves the formation of a ternary complex of cyclic AMP, the regulatory subunit (R) and the catalytic subunit (C). It is suggested that only this ternary complex breaks down to liberate the enzymically active catalytic subunit. We have performed cross-linking experiments with the holoenzyme and its dissimilar subunits in the presence of MgATP and various concentrations of cyclic AMP. Results from these cross-linking studies indicate that regulatory subunits exist as dimers in the native form. Moreover, dissociation of the holoenzyme or the reconstituted enzyme is promoted by cyclic AMP, and the effect of MgATP is to stabilize the enzyme in the tetrameric form. The success in cross-linking the regulatory and the catalytic subunits of protein kinase with the lysine-specific bifunctional cross-linking reagent dimethyl suberimidate may be attributed to the presence of a large number of lysine residues in the enzyme.

scholarly journals Association of cyclic-AMP-dependent protein kinase with neurofilaments

1992 ◽  
Vol 282 (2) ◽  
pp. 477-481 ◽  
Author(s):  
A Dosemeci ◽  
H C Pant
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Kinase Activity ◽  
Inhibitory Effect ◽  
Catalytic Subunit ◽  
Regulatory Subunit ◽  
Dependent Manner ◽  
Dependent Protein Kinase ◽  
Bovine Heart ◽  
Dependent Protein

Neurofilament preparations isolated from bovine spinal cord contain cyclic-AMP-dependent protein kinase (PKA) activity. Treatment of this preparation with cyclic AMP, to dissociate the regulatory subunit of the kinase from the catalytic subunit, resulted in retention of the kinase activity but loss of cyclic AMP regulation. This suggests that PKA is associated via its catalytic subunit with the neurofilament preparation. The association of exogenous PKA from bovine heart with the neurofilament preparation and with neurofilaments reconstituted from purified neurofilament proteins was also investigated. Either the free catalytic subunit or combinations of the catalytic and regulatory subunits of PKA were incubated with the preparations, and the degree of association was determined as the level of kinase activity that co-sediments with neurofilaments. The results indicate that the free catalytic subunit of PKA co-sediments with neurofilaments reconstituted from purified proteins. The regulatory subunit of PKA from bovine heart, when pre-mixed with the catalytic subunit, decreased the level of kinase that co-sediments with the neurofilament fraction in a dose-dependent manner. This effect of the regulatory subunit was reversed by inclusion of cyclic AMP in the incubation medium before centrifugation. The above findings suggest that the regulatory subunit, when attached to the catalytic subunit, has an inhibitory effect on its association with neurofilaments, with the implication that the association may be a cyclic-AMP-regulated event.

scholarly journals Cyclic AMP-Dependent Protein Kinase Catalytic Subunits Have Divergent Roles in Virulence Factor Production in Two Varieties of the Fungal Pathogen Cryptococcus neoformans

2004 ◽  
Vol 3 (1) ◽  
pp. 14-26 ◽  
Author(s):  
Julie K. Hicks ◽  
Cletus A. D'Souza ◽  
Gary M. Cox ◽  
Joseph Heitman
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Virulence Factor ◽  
Fungal Pathogen ◽  
Catalytic Subunit ◽  
Dependent Protein Kinase ◽  
Serotype A ◽  
Dependent Protein ◽  
Virulence Factor Production ◽  
Pka Catalytic Subunit

ABSTRACT 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.

scholarly journals Regulation of cyclic AMP-dependent protein kinase levels during skeletal myogenesis

1989 ◽  
Vol 264 (1) ◽  
pp. 305-308 ◽  
Author(s):  
I A J Lorimer ◽  
B D Sanwal
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Catalytic Subunit ◽  
Regulatory Subunit ◽  
Type I ◽  
Skeletal Myogenesis ◽  
Dependent Protein Kinase ◽  
Skeletal Myoblasts ◽  
Dependent Protein ◽  
Degradation Studies

We showed previously that the levels of type I regulatory subunit of cyclic AMP-dependent protein kinase increase during differentiation of L6 skeletal myoblasts as a result of a specific decrease in its rate of degradation. Studies on the rates of degradation of the catalytic subunit show that unlike the type I regulatory subunit, catalytic subunit is degraded very slowly in myoblasts (t1/2 = 29 h) and more rapidly in myotubes (t1/2 = 14 h). As with the regulatory subunit, the degradation of catalytic subunit is increased by treatment of myoblasts with cyclic AMP analogues. These results suggest that the overall increase in the amount of type I cyclic AMP-dependent protein kinase holoenzyme during myogenesis is due to the increase in levels of mRNA for the catalytic subunit. This probably leads to an increase in the amount of catalytic subunit, which then stabilizes the regulatory subunit, thereby causing an increase in the levels of this protein also.

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