Inhibition of mos-induced oocyte maturation by protein kinase A

The relationship between the mos protooncogene protein and cAMP- dependent protein kinase (PKA) during the maturation of Xenopus oocytes was investigated. Microinjection of the PKA catalytic subunit (PKAc) into Xenopus oocytes inhibited oocyte maturation induced by the mos product but did not markedly affect the autophosphorylation activity of injected mos protein. By contrast, PKAc did not inhibit maturation promoting factor (MPF) activation or germinal vesicle breakdown (GVBD) that was initiated by injecting crude MPF preparations. In addition, inhibiting endogenous PKA activity by microinjecting the PKA regulatory subunit (PKAr) induced oocyte maturation that was dependent upon the presence of the endogenous mos product. Moreover, PKAr potentiated mos protein-induced MPF activation in the absence of progesterone and protein synthesis. These data are consistent with the hypothesis that progesterone-induced release from G2/M is regulated via PKAc and that PKAc negatively regulates a downstream target that is positively regulated by mos.     

Small-molecule FRET probes for protein kinase activity monitoring in living cells

In this study, the applicability of fluorescently labeled adenosine analogue-oligoarginine conjugates (ARC-Photo probes) for monitoring of protein kinase A (PKA) activity in living cells was demonstrated. ARC-Photo probes possessing subnanomolar affinity towards the catalytic subunit of PKA (PKAc) and competitive with the regulatory subunit (PKAr), penetrate cell plasma membrane and associate with PKAc fused with yellow fluorescent protein (PKAc-YFP). Detection of inter-molecular Förster resonance energy transfer (FRET) efficiency between the fluorophores of the fusion protein and ARC-Photo probe can be used for both the evaluation of non-labeled inhibitors of PKAc and for monitoring of cAMP signaling via detection of changes in the activity of PKA as a cAMP downstream effector.     

Crosstalk between PKA and PKG controls pH‐dependent host cell egress of Toxoplasma gondii

Toxoplasma gondii encodes three protein kinase A catalytic (PKAc1‐3) and one regulatory (PKAr) subunits to integrate cAMP‐dependent signals. Here, we show that inactive PKAc1 is maintained at the parasite pellicle by interacting with acylated PKAr. Either a conditional knockdown of PKAr or the overexpression of PKAc1 blocks parasite division. Conversely, down‐regulation of PKAc1 or stabilisation of a dominant‐negative PKAr isoform that does not bind cAMP triggers premature parasite egress from infected cells followed by serial invasion attempts leading to host cell lysis. This untimely egress depends on host cell acidification. A phosphoproteome analysis suggested the interplay between cAMP and cGMP signalling as PKAc1 inactivation changes the phosphorylation profile of a putative cGMP‐phosphodiesterase. Concordantly, inhibition of the cGMP‐dependent protein kinase G (PKG) blocks egress induced by PKAc1 inactivation or environmental acidification, while a cGMP‐phosphodiesterase inhibitor circumvents egress repression by PKAc1 or pH neutralisation. This indicates that pH and PKAc1 act as balancing regulators of cGMP metabolism to control egress. These results reveal a crosstalk between PKA and PKG pathways to govern egress in T. gondii.     

Inhibitors and fluorescent probes for protein kinase PKAcβ and its S54L mutant,identified in a patient with cortisol producing adenoma

ABSTRACT

Recently, a mutation was discovered in the gene PRKACB encoding the catalytic subunit β of PKA (PKAcβ) from a patient with severe Cushing’s syndrome. This mutation, S54L, leads to a structural change in the glycine-rich loop of the protein. In the present study, an inhibitor with six-fold selectivity toward S54L-PKAcβ mutant over the wild-type enzyme was constructed. Moreover, we developed a fluorescent assay allowing to determine side by side the affinity of commercially available PKA inhibitors, newly synthesized compounds, and fluorescent probes toward PKAcβ and S54L-PKAcβ.     

Transmodulation of epidermal growth factor receptor function by cyclic AMP-dependent protein kinase.

Binding of epidermal growth factor (EGF) to its receptor (EGFR) augments the tyrosine kinase activity of the receptor and autophosphorylation. Exposure of some tissues and cells to EGF also stimulates adenylyl cyclase activity and results in an increase in cyclic AMP (cAMP) levels. Because cAMP activates the cAMP-dependent protein kinase A (PKA), we investigated the effect of PKA on the EGFR. The purified catalytic subunit of PKA (PKAc) stoichiometrically phosphorylated the purified full-length wild type (WT) and kinase negative (K721M) forms of the EGFR. PKAc phosphorylated both WT-EGFR as well as a mutant truncated form of EGFR (Delta1022-1186) exclusively on serine residues. Moreover, PKAc also phosphorylated the cytosolic domain of the EGFR (EGFRKD). Phosphorylation of the purified WT as well as EGFRDelta1022-1186 and EGFRKD was accompanied by decreased autophosphorylation and diminished tyrosine kinase activity. Pretreatment of REF-52 cells with the nonhydrolyzable cAMP analog, 8-(4-chlorophenylthio)-cAMP, decreased EGF-induced tyrosine phosphorylation of cellular proteins as well as activation of the WT-EGFR. Similar effects were also observed in B82L cells transfected to express the Delta1022-1186 form of EGFR. Furthermore, activation of PKAc in intact cells resulted in serine phosphorylation of the EGFR. The decreased phosphorylation of cellular proteins and diminished activation of the EGFR in cells treated with the cAMP analog was not the result of altered binding of EGF to its receptors or changes in receptor internalization. Therefore, we conclude that PKA phosphorylates the EGFR on Ser residues and decreases its tyrosine kinase activity and signal transduction both in vitro and in vivo.     

Subcellular localization and biological actions of activated RSK1 are determined by its interactions with subunits of cyclic AMP-dependent protein kinase

Cyclic AMP (cAMP)-dependent protein kinase (PKA) and ribosomal S6 kinase 1 (RSK1) share several cellular proteins as substrates. However, to date no other similarities between the two kinases or interactions between them have been reported. Here, we describe novel interactions between subunits of PKA and RSK1 that are dependent upon the activation state of RSK1 and determine its subcellular distribution and biological actions. Inactive RSK1 interacts with the type I regulatory subunit (RI) of PKA. Conversely, active RSK1 interacts with the catalytic subunit of PKA (PKAc). Binding of RSK1 to RI decreases the interactions between RI and PKAc, while the binding of active RSK1 to PKAc increases interactions between PKAc and RI and decreases the ability of cAMP to stimulate PKA. The RSK1/PKA subunit interactions ensure the colocalization of RSK1 with A-kinase PKA anchoring proteins (AKAPs). Disruption of the interactions between PKA and AKAPs decreases the nuclear accumulation of active RSK1 and, thus, increases its cytosolic content. This subcellular redistribution of active RSK1 is manifested by increased phosphorylation of its cytosolic substrates tuberous sclerosis complex 2 and BAD by epidermal growth factor along with decreased cellular apoptosis.     

Isolation and Characterization of the PKAr Gene From a Plant Pathogen,Curvularia lunata

By using EST database from a full-length cDNA library of Curvularia lunata, we have isolated a 2.9 kb cDNA, termed PKAr. An ORF of 1,383 bp encoding a polypeptide of 460 amino acids with molecular weight 50.1 kDa, (GeneBank Acc. No. KF675744) was cloned. The deduced amino acid sequence of the PKAr shows 90 and 88 % identity with cAMP-dependent protein kinase A regulatory subunit from Alternaria alternate and Pyrenophora tritici-repentis Pt-1C-BFP, respectively. Database analysis revealed that the deduced amino acid sequence of PKAr shares considerable similarity with that of PKA regulatory subunits in other organisms, particularly in the conserved regions. No introns were identified within the 1,383 bp of ORF compared with PKAr genomic DNA sequence. Southern blot indicated that PKAr existed as a single copy per genome. The mRNA expression level of PKAr in different development stages were demonstrated using real-time quantitative PCR. The results showed that the level of PKAr expression was highest in vegetative growth mycelium, which indicated it might play an important role in the vegetative growth of C. lunata. These results provided a fundamental supporting research on the function of PKAr in plant pathogen, C. lunata.     

Hedgehog-PKA Signaling and gnrh3 Regulate the Development of Zebrafish gnrh3 Neurons

GnRH neurons secrete GnRH that controls the development of the reproduction system. Despite many studies, the signals controlling the development of GnRH neurons from its progenitors have not been fully established. To understand the development of GnRH neurons, we examined the development of gnrh3-expressing cells using a transgenic zebrafish line that expresses green fluorescent protein (GFP) and LacZ driven by the gnrh3 promoter. GFP and LacZ expression recapitulated that of gnrh3 in the olfactory region, olfactory bulb and telencephalon. Depletion of gnrh3 by morpholinos led to a reduction of GFP- and gnrh3-expressing cells, while over-expression of gnrh3 mRNA increased the number of these cells. This result indicates a positive feed-forward regulation of gnrh3 cells by gnrh3. The gnrh3 cells were absent in embryos that lack Hedgehog signaling, but their numbers were increased in embryos overexpressing shhb. We manipulated the amounts of kinase that antagonizes the Hedgehog signaling pathway, protein kinase A (PKA), by treating embryos with PKA activator forskolin or by injecting mRNAs encoding its constitutively active catalytic subunit (PKA*) and dominant negative regulatory subunit (PKI) into zebrafish embryos. PKA* misexpression or forskolin treatment decreased GFP cell numbers, while PKI misexpression led to ectopic production of GFP cells. Our data indicate that the Hedgehog-PKA pathway participates in the development of gnrh3-expressing neurons during embryogenesis.     

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.     

Thrombin and Collagen Induce a Feedback Inhibitory Signaling Pathway in Platelets Involving Dissociation of the Catalytic Subunit of Protein Kinase A from an NFκB-IκB Complex

Protein kinase A (PKA) activation by cAMP phosphorylates multiple target proteins in numerous platelet inhibitory pathways that have a very important role in maintaining circulating platelets in a resting state. Here we show that in thrombin- and collagen-stimulated platelets, PKA is activated by cAMP-independent mechanisms involving dissociation of the catalytic subunit of PKA (PKAc) from an NFκB-IκBα-PKAc complex. We demonstrate mRNA and protein expression for most of the NFκB family members in platelets. From resting platelets, PKAc was co-immunoprecipitated with IκBα, and conversely, IκBα was also co-immunoprecipitated with PKAc. This interaction was significantly reduced in thrombin- and collagen-stimulated platelets. Stimulation of platelets with thrombin- or collagen-activated IKK, at least partly by PI3 kinase-dependent pathways, leading to phosphorylation of IκBα, disruption of an IκBα-PKAc complex, and release of free, active PKAc, which phosphorylated VASP and other PKA substrates. IKK inhibitor inhibited thrombin-stimulated IkBα phosphorylation, PKA-IkBα dissociation, and VASP phosphorylation, and potentiated integrin αIIbβ3 activation and the early phase of platelet aggregation. We conclude that thrombin and collagen not only cause platelet activation but also appear to fine-tune this response by initiating downstream NFκB-dependent PKAc activation, as a novel feedback inhibitory signaling mechanism for preventing undesired platelet activation.     

cAMP-dependent protein kinase and the disruption of learning in transgenic flies.

A molecular genetic approach was used to test for a role of cAMP-dependent protein kinase (PKA) in learning and memory in Drosophila. We used genes encoding a peptide inhibitor of PKA, an N-terminal regulatory subunit fragment containing a pseudosubstrate inhibitory domain, and a wild-type catalytic subunit. These dominantly acting genes were placed under control of the hsp70 promoter and transformed into wild-type flies. Induction of the transgenes by 1 hr heat shock results in overproduction of their RNA in adult flies. The same heat shock treatment disrupts the ability of the flies to learn in an odor discrimination task reinforced with electric shock. The results demonstrate the involvement of PKA in the associative learning of Drosophila.     

Genes encoding catalytic subunits of protein kinase A and risk of spina bifida

BACKGROUND: PRKACA and PRKACB are genes encoding the cAMP-dependent protein kinase A (PKA) catalytic subunits alpha and beta, respectively. PKA is known to be involved in embryonic development, as it down-regulates the Hedgehog (Hh) signaling pathway, which is critical to normal pattern formation and morphogenesis. The PKA-deficient mouse model, which has only a single catalytic subunit, provided intriguing evidence demonstrating a relationship between decreased PKA activity and risk for posterior neural tube defects (NTDs) in the thoracic to sacral regions of gene-knockout mice. Unlike most other mutant mouse models of NTDs, the PKA-deficient mice develop spina bifida with 100% penetrance. We hypothesized that sequence variations in human genes encoding the catalytic subunits may alter the PKA activity and similarly increase the risk of spina bifida. METHODS: We sequenced the coding regions and the exon/intron boundaries of PRKACA and PRKACB. We also examined 3 common single-nucleotide polymorphisms (SNPs) of these 2 genes by allele discrimination. RESULTS: Five sequence variants in coding region and 2 intronic sequence variants proximal to exons were detected. None of the 3 SNPs examined in the association study appeared to be associated with substantially increased risk for spina bifida. CONCLUSIONS: Our results did not reveal a strong association between these PKA SNPs and spina bifida risk. Nonetheless, it is important to examine the possible gene-gene interactions between PRKACA and PRKACB when evaluating the risk for NTDs, as well as genes encoding regulatory subunits of PKA. In addition, interactions with other genes such as Sonic Hedgehog (SHH) should also be considered for future investigations.     

Involvement of protein kinase A and A kinase anchoring protein in the progesterone-initiated human sperm acrosome reaction

The signal transduction pathways involved in the progesterone (P(4))-initiated mammalian sperm acrosome reaction (AR) are not fully understood. To investigate the role of the protein kinase A (PKA) pathway in the P(4)-initiated AR, we probed this pathway by pretreating capacitated human sperm with reagents designed to either inhibit PKA activation or disrupt PKA/A kinase anchoring protein (AKAP) interactions. Preincubation with the stearated (membrane permeable) PKA inhibitor, PKI alpha 5-24 (S-PKI alpha 5-24), significantly inhibited the P(4)-initiated AR at 10 microM as compared to stearated control peptide. In contrast, preincubation with 100 microM nonstearated PKI alpha 5-24 did not significantly inhibit versus solvent control. Preincubation with the PKA inhibitor Rp-8-Br-cAMP at 500 microM and 150 microM significantly inhibited the P(4)-initiated AR versus 8-Br-cAMP and versus solvent. Preincubation with the anchoring inhibitory peptide S-Ht-31 significantly stimulated the P(4)-initiated AR at 10, 3, and 1 microM versus inactive control peptide. The stimulation of the P(4)-initiated AR by 3 microM S-Ht31 was significantly inhibited by the addition of 30 microM S-PKI alpha 5-24 prior to the addition of S-Ht31. Preincubation with S-PKI alpha 5-24 (30 microM) partially inhibited the ionomycin (50 microM)-initiated AR. A role for PKA in the P(4)-initiated AR may exist both upstream and downstream of Ca(2+) entry. Our studies present the first evidence for the participation of PKA in the P(4)-initiated AR and also suggest that AKAPs are involved in the PKA-mediated events.