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3',5'-cyclic adenosine monophosphate (cAMP) dependent protein kinase or protein kinase A (PKA) has served as a prototype for the large family of protein kinases that are crucially important for signal transduction in eukaryotic cells. The PKA catalytic subunits Ca and Cß, encoded by the two genes PRKACA and PRKACB, respectively, are among the best understood and characterized human kinases. Here we have studied the evolution of this gene family in chordates, arthropods, mollusks and other animals employing probabilistic methods and show that Ca and Cß arose by duplication of an ancestral PKA catalytic subunit in a common ancestor of vertebrates. The two genes have subsequently been duplicated in teleost fishes. The evolution of the PRKACG retroposon in simians was also investigated. Although the degree of sequence conservation in the PKA Ca/Cß kinase family is exceptionally high, a small set of signature residues defining Ca and Cß subfamilies were identified. These conserved residues might be important for functions that are unique to the Ca or Cß clades. This study also provides a good example of a seemingly simple phylogenetic problem which, due to a very high degree of sequence conservation and corresponding weak phylogenetic signals, combined with problematic nonphylogenetic signals, is nontrivial for state-of-the-art probabilistic phylogenetic methods. Copyright: 2013 Søberg et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Although the modulation of Ca(2+) channel activity by extremely low-frequency electromagnetic fields (ELF-EMF) has been studied previously, few reports have addressed the effects of such fields on the activity of voltage-activated Na(+) channels (Na(v)). Here, we investigated the effects of ELF-EMF on Na(v) activity in rat cerebellar granule cells (GCs). Our results reveal that exposing cerebellar GCs to ELF-EMF for 10-60 min significantly increased Na(v) currents (I(Na)) by 30-125% in a time- and intensity-dependent manner. The Na(v) channel steady-state activation curve, but not the steady-state inactivation curve, was significantly shifted (by 5.2 mV) towards hyperpolarization by ELF-EMF stimulation. This phenomenon is similar to the effect of intracellular application of arachidonic acid (AA) and prostaglandin E(2) (PGE(2)) on I(Na) in cerebellar GCs. Increases in intracellular AA, PGE(2) and phosphorylated PKA levels in cerebellar GCs were observed following ELF-EMF exposure. Western blottings indicated that the Na(V) 1.2 protein on the cerebellar GCs membrane was increased, the total expression levels of Na(V) 1.2 protein were not affected after exposure to ELF-EMF. Cyclooxygenase inhibitors and PGE(2) receptor (EP) antagonists were able to eliminate this ELF-EMF-induced increase in phosphorylated PKA and I(Na). In addition, ELF-EMF exposure significantly enhanced the activity of PLA(2) in cerebellar GCs but did not affect COX-1 or COX-2 activity. Together, these data demonstrate for the first time that neuronal I(Na) is significantly increased by ELF-EMF exposure via a cPLA2 AA PGE(2) EP receptors PKA signaling pathway.

Background Long-term and unresolved airway inflammation and airway remodeling, characteristic features of chronic asthma, if not treated could lead to permanent structural changes in the airways. Aldose reductase (AR), an aldo-sugar and lipid aldehyde metabolizing enzyme, mediates allergen-induced airway inflammation in mice, but its role in the airway remodeling is not known. In the present study, we have examined the role of AR on airway remodeling using ovalbumin (OVA)-induced chronic asthma mouse model and cultured human primary airway epithelial cells (SAECs) and mouse lung fibroblasts (mLFs). Methods Airway remodeling in chronic asthma model was established in mice sensitized and challenged twice a week with OVA for 6 weeks. AR inhibitor, fidarestat, was administered orally in drinking water after first challenge. Inflammatory cells infiltration in the lungs and goblet cell metaplasia, airway thickening, collagen deposition and airway hyper-responsiveness (AHR) in response to increasing doses of methacholine were assessed. The TGFβ1-induced epithelial-mesenchymal transition (EMT) in SAECs and changes in mLFs were examined to investigate AR-mediated molecular mechanism(s) of airway remodeling. Results In the OVA-exposed mice for 6 wks inflammatory cells infiltration, levels of inflammatory cytokines and chemokines, goblet cell metaplasia, collagen deposition and AHR were significantly decreased by treatment with AR inhibitor, fidarestat. Further, inhibition of AR prevented TGFβ1-induced altered expression of E-cadherin, Vimentin, Occludin, and MMP-2 in SAECs, and alpha-smooth muscle actin and fibronectin in mLFs. Further, in SAECs, AR inhibition prevented TGFβ1- induced activation of PI3K/AKT/GSK3β pathway but not the phosphorylation of Smad2/3. Conclusion Our results demonstrate that allergen-induced airway remodeling is mediated by AR and its inhibition blocks the progression of remodeling via inhibiting TGFβ1-induced Smad-independent and PI3K/AKT/GSK3β-dependent pathway.

Author(s): Spindler, Matthew J; Burmeister, Brian T; Huang, Yu; Hsiao, Edward C; Salomonis, Nathan; Scott, Mark J; Srivastava, Deepak; Carnegie, Graeme K; Conklin, Bruce R | Abstract: BackgroundA-kinase anchoring proteins (AKAPs) are scaffolding molecules that coordinate and integrate G-protein signaling events to regulate development, physiology, and disease. One family member, AKAP13, encodes for multiple protein isoforms that contain binding sites for protein kinase A (PKA) and D (PKD) and an active Rho-guanine nucleotide exchange factor (Rho-GEF) domain. In mice, AKAP13 is required for development as null embryos die by embryonic day 10.5 with cardiovascular phenotypes. Additionally, the AKAP13 Rho-GEF and PKD-binding domains mediate cardiomyocyte hypertrophy in cell culture. However, the requirements for the Rho-GEF and PKD-binding domains during development and cardiac hypertrophy are unknown.Methodology/principal findingsTo determine if these AKAP13 protein domains are required for development, we used gene-trap events to create mutant mice that lacked the Rho-GEF and/or the protein kinase D-binding domains. Surprisingly, heterozygous matings produced mutant mice at Mendelian ratios that had normal viability and fertility. The adult mutant mice also had normal cardiac structure and electrocardiograms. To determine the role of these domains during β-adrenergic-induced cardiac hypertrophy, we stressed the mice with isoproterenol. We found that heart size was increased similarly in mice lacking the Rho-GEF and PKD-binding domains and wild-type controls. However, the mutant hearts had abnormal cardiac contractility as measured by fractional shortening and ejection fraction.ConclusionsThese results indicate that the Rho-GEF and PKD-binding domains of AKAP13 are not required for mouse development, normal cardiac architecture, or β-adrenergic-induced cardiac hypertrophic remodeling. However, these domains regulate aspects of β-adrenergic-induced cardiac hypertrophy.

The protein kinase A (PKA)/Cdc25B pathway plays a critical role in maintaining meiotic arrest in mouse oocytes. However, the molecular mechanism underlying this interchange is not known. In this study, we assessed the role of 14-3-3e interaction with phosphorylated Cdc25B at its Ser321 as the mouse oocyte is released from prophase I arrest. The 14-3-3e isoform is a highly conserved protein with various regulatory roles, including maintenance of meiotic arrest. Cdc25B phosphatase is also a key cell cycle regulator. 14-3-3e binds to Cdc25B-WT, which was abrogated when Ser321 of Cdc25B was mutated to Ala. In addition, we found that 14-3-3e and Cdc25B were co-localized. Cdc25B was translocated from the cytoplasm to the nucleus shortly before germinal vesicle breakdown (GVBD) during the primary oocyte stage of oogenesis. However, mutation of Ser321 to Ala completely abolished the cytoplasmic localization of Cdc25B. Furthermore, oocytes co-expressing of Cdc25B-WT or Cdc25B-Ser321D and 14-3-3e were unable to undergo GVBD. In contrast, co-expression of 14-3-3e and Cdc25B-Ser321A induced GVBD and allowed the process to continue. Down-regulation of 14-3-3e caused partial meiotic resumption. Taken together, these data indicate that Ser321 of Cdc25B is the specific binding site for 14-3-3e binding, and that 14-3-3e is the significant factor in Cdc25B regulation during meiotic resumption of GV stage.

Light in the visible range can be stressful to non-photosynthetic organisms. The yeast Saccharomyces cerevisiae has earlier been reported to respond to blue light via activation of the stress-regulated transcription factor Msn2p. Environmental changes also induce activation of calcineurin, a Ca(2+)/calmodulin dependent phosphatase, which in turn controls gene transcription by dephosphorylating the transcription factor Crz1p. We investigated the connection between cellular stress caused by blue light and Ca(2+) signalling in yeast by monitoring the nuclear localization dynamics of Crz1p, Msn2p and Msn4p. The three proteins exhibit distinctly different stress responses in relation to light exposure. Msn2p, and to a lesser degree Msn4p, oscillate rapidly between the nucleus and the cytoplasm in an apparently stochastic fashion. Crz1p, in contrast, displays a rapid and permanent nuclear localization induced by illumination, which triggers Crz1p-dependent transcription of its target gene CMK2. Moreover, increased extracellular Ca(2+) levels stimulates the light-induced responses of all three transcription factors, e.g. Crz1p localizes much quicker to the nucleus and a larger fraction of cells exhibits permanent Msn2p nuclear localization at higher Ca(2+) concentration. Studies in mutants lacking Ca(2+) transporters indicate that influx of extracellular Ca(2+) is crucial for the initial stages of light-induced Crz1p nuclear localization, while mobilization of intracellular Ca(2+) stores appears necessary for a sustained response. Importantly, we found that Crz1p nuclear localization is dependent on calcineurin and the carrier protein Nmd5p, while not being affected by increased protein kinase A activity (PKA), which strongly inhibits light-induced nuclear localization of Msn2/4p. We conclude that the two central signalling pathways, cAMP-PKA-Msn2/4 and Ca(2+)-calcineurin-Crz1, are both activated by blue light illumination.

A striking and clinically relevant virulence trait of the human fungal pathogen Candida albicans is its ability to grow and switch reversibly among different morphological forms. Inhibition of yeast-to-hypha transition in C. albicans represents a new paradigm for antifungal intervention. We have previously demonstrated the novel antifungal activity of purpurin against Candida fungi. In this study, we extended our investigation by examining the in vitro effect of purpurin on C. albicans morphogenesis and biofilms. The susceptibility of C. albicans biofilms to purpurin was examined quantitatively by 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide reduction assay. Hyphal formation and biofilm ultrastructure were examined qualitatively by scanning electron microscopy (SEM). Quantitative reverse transcription-PCR (qRT-PCR) was used to evaluate the expression of hypha-specific genes and hyphal regulator in purpurin-treated fungal cells. The results showed that, at sub-lethal concentration (3 µg/ml), purpurin blocked the yeast-to-hypha transition under hypha-inducing conditions. Purpurin also inhibited C. albicans biofilm formation and reduced the metabolic activity of mature biofilms in a concentration-dependent manner. SEM images showed that purpurin-treated C. albicans biofilms were scanty and exclusively consisted of aggregates of blastospores. qRT-PCR analyses indicated that purpurin downregulated the expression of hypha-specific genes (ALS3, ECE1, HWP1, HYR1) and the hyphal regulator RAS1. The data strongly suggested that purpurin suppressed C. albicans morphogenesis and caused distorted biofilm formation. By virtue of the ability to block these two virulence traits in C. albicans, purpurin may represent a potential candidate that deserves further investigations in the development of antifungal strategies against this notorious human fungal pathogen in vivo.

International audience; Astrocytes synthesize and release endozepines, a family of regulatory peptides, including the octadecaneuropeptide (ODN) an endogenous ligand of both central-type benzodiazepine (CBR) and metabotropic receptors. We have recently shown that ODN exerts a protective effect against hydrogen peroxide (H(2)O(2))-induced oxidative stress in astrocytes. The purpose of the present study was to determine the type of receptor and the transduction pathways involved in the protective effect of ODN in cultured rat astrocytes. We have first observed a protective activity of ODN at very low concentrations that was abrogated by the metabotropic ODN receptor antagonist cyclo(1-8)[DLeu(5)]OP, but not by the CBR antagonist flumazenil. We have also found that the metabotropic ODN receptor is positively coupled to adenylyl cyclase in astrocytes and that the glioprotective action of ODN upon H(2)O(2)-induced astrocyte death is PKA- and MEK-dependent, but PLC/PKC-independent. Downstream of PKA, ODN induced ERK phosphorylation, which in turn activated the expression of the anti-apoptotic gene Bcl-2 and blocked the stimulation by H(2)O(2) of the pro-apoptotic gene Bax. The effect of ODN on the Bax/Bcl-2 balance contributed to abolish the deleterious action of H(2)O(2) on mitochondrial membrane integrity and caspase-3 activation. Finally, the inhibitory effect of ODN on caspase-3 activity was shown to be PKA and MEK-dependent. In conclusion, the present results demonstrate that the potent glioprotective action of ODN against oxidative stress involves the metabotropic ODN receptor coupled to the PKA/ERK-kinase pathway to inhibit caspase-3 activation.

Administration of glucocorticoids and caffeine is a common therapeutic intervention in the neonatal period, but possible interactions between these substances are still unclear. The present study investigated the effect of caffeine and different glucocorticoids on expression of surfactant protein (SP)-B, crucial for the physiological function of pulmonary surfactant. We measured expression levels of SP-B, various SP-B transcription factors including erythroblastic leukemia viral oncogene homolog 4 (ErbB4) and thyroid transcription factor-1 (TTF-1), as well as the glucocorticoid receptor (GR) after administering different doses of glucocorticoids, caffeine, cAMP, or the phosphodiesterase-4 inhibitor rolipram in the human airway epithelial cell line NCI-H441. Administration of dexamethasone (1 µM) or caffeine (5 mM) stimulated SP-B mRNA expression with a maximal of 38.8±11.1-fold and 5.2±1.4-fold increase, respectively. Synergistic induction was achieved after co-administration of dexamethasone (1 mM) in combination with caffeine (10 mM) (206±59.7-fold increase, p

Amino acids can induce yeast cell adhesion but how amino acids are sensed and signal the modulation of the FLO adhesion genes is not clear. We discovered that the budding yeast Saccharomyces cerevisiae CEN.PK evolved invasive growth ability under prolonged nitrogen limitation. Such invasive mutants were used to identify amino acid transporters as regulators of FLO11 and invasive growth. One invasive mutant had elevated levels of FLO11 mRNA and a Q320STOP mutation in the SFL1 gene that encodes a protein kinase A pathway regulated repressor of FLO11. Glutamine-transporter genes DIP5 and GNP1 were essential for FLO11 expression, invasive growth and biofilm formation in this mutant. Invasive growth relied on known regulators of FLO11 and the Ssy1-Ptr3-Ssy5 complex that controls DIP5 and GNP1, suggesting that Dip5 and Gnp1 operates downstream of the Ssy1-Ptr3-Ssy5 complex for regulation of FLO11 expression in a protein kinase A dependent manner. The role of Dip5 and Gnp1 appears to be conserved in the S. cerevisiae strain ∑1278b since the dip5 gnp1 ∑1278b mutant showed no invasive phenotype. Secondly, the amino acid transporter gene GAP1 was found to influence invasive growth through FLO11 as well as other FLO genes. Cells carrying a dominant loss-of-function PTR3(647::CWNKNPLSSIN) allele had increased transcription of the adhesion genes FLO1, 5, 9, 10, 11 and the amino acid transporter gene GAP1. Deletion of GAP1 caused loss of FLO11 expression and invasive growth. However, deletions of FLO11 and genes encoding components of the mitogen-activated protein kinase pathway or the protein kinase A pathway were not sufficient to abolish invasive growth, suggesting involvement of other FLO genes and alternative pathways. Increased intracellular amino acid pools in the PTR3(647::CWNKNPLSSIN)-containing strain opens the possibility that Gap1 regulates the FLO genes through alteration of the amino acid pool sizes.

Nuclear factor erythroid-derived 2 (NF-E2), a heterodimer composed of p45 and p18, is a transcriptional activator in hematopoietic progenitors. The transcriptional activity of NF-E2 is not only upregulated by SUMOylation but also stimulated by the cAMP-dependent protein kinase A (PKA). However, the relationship between SUMOylation and phosphorylation in the activation of NF-E2 is unclear. In the present studies, we have demonstrated that PKA enhances NF-E2 SUMOylation in an in vitro system using purified proteins, suggesting a possible mechanism for PKA-dependent activation of the NF-E2 transcription factor through SUMOylation.

The extent of damage following spinal cord injury (SCI) can be reduced by various neuroprotective regimens that include maintaining levels of cyclic adenosine monophosphate (cyclic AMP), via administration of the phosphodiesterase 4 (PDE4) inhibitor Rolipram. The current study sought to determine the optimal neuroprotective dose, route and therapeutic window for Rolipram following contusive SCI in rat as well as its prominent PDE target and putative mechanism of protection. Rolipram or vehicle control (10% ethanol) was given subcutaneously (s.c.) daily for 2 wk post-injury (PI) after which the preservation of oligodendrocytes, neurons and central myelinated axons was stereologically assessed. Doses of 0.1 mg/kg to 1.0 mg/kg (given at 1 h PI) increased neuronal survival; 0.5 mg to 1.0 mg/kg protected oligodendrocytes and 1.0 mg/kg produced optimal preservation of central myelinated axons. Ethanol also demonstrated significant neuronal and oligo-protection; though the preservation provided was significantly less than Rolipram. Subsequent use of this optimal Rolipram dose, 1.0 mg/kg, via different routes (i.v., s.c. or oral, 1 h PI), demonstrated that i.v. administration produced the most significant and consistent cyto- and axo- protection, although all routes were effective. Examination of the therapeutic window for i.v. Rolipram (1.0 mg/kg), when initiated between 1 and 48 h after SCI, revealed maximal neuroprotection at 2 h post-SCI, although the protective efficacy of Rolipram could still be observed when administration was delayed for up to 48 h PI. Importantly, use of the optimal Rolipram regimen significantly improved locomotor function after SCI as measured by the BBB score. Lastly we show SCI-induced changes in PDE4A, B and D expression and phosphorylation as well as cytokine expression and immune cell infiltration. We demonstrate that Rolipram abrogates SCI-induced PDE4B1 and PDE4A5 production, PDE4A5 phosphorylation, MCP-1 expression and immune cell infiltration, while preventing post-injury reductions in IL-10. This work supports the use of Rolipram as an acute neuroprotectant following SCI and defines an optimal administration protocol and target for its therapeutic application.

The genes PRKACA and PRKACB encode the principal catalytic (C) subunits of protein kinase A (PKA) Cα and Cβ, respectively. Cα is expressed in all eukaryotic tissues examined and studies of Cα knockout mice demonstrate a crucial role for Cα in normal physiology. We have sequenced exon 2 through 10 of PRKACA from the genome of 498 Norwegian donors and extracted information about PRKACA mutations from public databases. We identified four interesting nonsynonymous point mutations, Arg45Gln, Ser109Pro, Gly186Val, and Ser263Cys, in the Cα1 splice variant of the kinase. Cα variants harboring the different amino acid mutations were analyzed for kinase activity and regulatory (R) subunit binding. Whereas mutation of residues 45 and 263 did not alter catalytic activity or R subunit binding, mutation of Ser109 significantly reduced kinase activity while R subunit binding was unaltered. Mutation of Cα Gly186 completely abrogated kinase activity and PKA type I but not type II holoenzyme formation. Gly186 is located in the highly conserved DFG motif of Cα and mutation of this residue to Val was predicted to result in loss of binding of ATP and Mg2+, which may explain the kinetic inactivity. We hypothesize that individuals born with mutations of Ser109 or Gly186 may be faced with abnormal development and possibly severe disease. © 2012 Søberg et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

A-kinase anchoring proteins (AKAPs) bind the regulatory subunits of protein kinase A (PKA) and localize the holoenzyme to discrete signaling microdomains in multiple subcellular compartments. Despite emerging evidence for a nuclear pool of PKA that rapidly responds to activation of the PKA signaling cascade, only a few AKAPs have been identified that localize to the nucleus. Here we show a PKA-binding domain in the amino terminus of Chd8, and demonstrate subcellular colocalization of Chd8 with RII. RII overlay and immunoprecipitation assays demonstrate binding between Chd8-S and RIIα. Binding is abrogated upon dephosphorylation of RIIα. By immunofluorescence, we identified nuclear and perinuclear pools of Chd8 in HeLa cells and rat neonatal cardiomyocytes. We also show high levels of Chd8 mRNA in RNA extracted from post-natal rat hearts. These data add Chd8 to the short list of known nuclear AKAPs, and implicate a function for Chd8 in post-natal rat cardiac development.

The molecular pathways by which long chain polyunsaturated fatty acids (LCPUFA) influence skeletal health remain elusive. Both LCPUFA and parathyroid hormone type 1 receptor (PTH1R) are known to be involved in bone metabolism while any direct link between the two is yet to be established. Here we report that LCPUFA are capable of direct, PTH1R dependent activation of extracellular ligand-regulated kinases (ERK). From a wide range of fatty acids studied, varying in chain length, saturation, and position of double bonds, eicosapentaenoic (EPA) and docosahexaenoic fatty acids (DHA) caused the highest ERK phosphorylation. Moreover, EPA potentiated the effect of parathyroid hormone (PTH(1-34)) in a superagonistic manner. EPA or DHA dependent ERK phosphorylation was inhibited by the PTH1R antagonist and by knockdown of PTH1R. Inhibition of PTH1R downstream signaling molecules, protein kinases A (PKA) and C (PKC), reduced EPA and DHA dependent ERK phosphorylation indicating that fatty acids predominantly activate G-protein pathway and not the β-arrestin pathway. Using picosecond time-resolved fluorescence microscopy and a genetically engineered PTH1R sensor (PTH-CC), we detected conformational responses to EPA similar to those caused by PTH(1-34). PTH1R antagonist blocked the EPA induced conformational response of the PTH-CC. Competitive binding studies using fluorescence anisotropy technique showed that EPA and DHA competitively bind to and alter the affinity of PTH1 receptor to PTH(1-34) leading to a superagonistic response. Finally, we showed that EPA stimulates protein kinase B (Akt) phosphorylation in a PTH1R-dependent manner and affects the osteoblast survival pathway, by inhibiting glucocorticoid-induced cell death. Our findings demonstrate for the first time that LCPUFAs, EPA and DHA, can activate PTH1R receptor at nanomolar concentrations and consequently provide a putative molecular mechanism for the action of fatty acids in bone.

Yin Yang 1 (YY1) is a ubiquitously expressed and highly conserved multifunctional transcription factor that is involved in a variety of cellular processes. Many YY1-regulated genes have crucial roles in cell proliferation, differentiation, apoptosis, and cell cycle regulation. Numerous mechanisms have been shown to regulate the function of YY1, such as DNA binding affinity, subcellular localization, and posttranslational modification including phosphorylation. Polo-like kinase 1(Plk1) and Casein kinase 2α (CK2 α) were the first two kinases identified to phosphorylate YY1. In this study, we identify a third kinase. We report that YY1 is a novel substrate of the Aurora B kinase both in vitro and in vivo. Serine 184 phosphorylation of YY1 by Aurora B is cell cycle regulated and peaks at G2/M and is rapidly dephosphorylated, likely by protein phosphatase 1 (PP1) as the cells enter G1. Aurora A and Aurora C can also phosphorylate YY1 in vitro, but at serine/threonine residues other than serine 184. We present evidence that phosphorylation of YY1 in the central glycine/alanine (G/A)-rich region is important for DNA binding activity, with a potential phosphorylation/acetylation interplay regulating YY1 function. Given their importance in mitosis and overexpression in human cancers, Aurora kinases have been identified as promising therapeutic targets. Increasing our understanding of Aurora substrates will add to the understanding of their signaling pathways.

Background Elevated levels of interleukin-6 (IL-6), prostaglandin (PG)E(2), PGD(2) and its dehydration end product 15-deoxy-Δ(12,14)-PGJ(2) (15d-PGJ(2)) have been detected in joint synovial fluids from patients with rheumatoid arthritis (RA). PGE(2) directly stimulates IL-6 production in human articular chondrocytes. However, the effects of PGD(2) and 15d-PGJ(2) in the absence or presence of PGE(2) on IL-6 synthesis in human chondrocytes have yet to be determined. It is believed that dysregulated overproduction of IL-6 is responsible for the systemic inflammatory manifestations and abnormal laboratory findings in RA patients. Methodology/principal findings Using the T/C-28a2 chondrocyte cell line as a model system, we report that exogenous PGE(2) and PGD(2)/15d-PGJ(2) exert antagonistic effects on IL-6 synthesis in human T/C-28a2 chondrocytes. Using a synthesis of sophisticated molecular biology techniques, we determined that PGE(2) stimulates Toll-like receptor 4 (TLR4) synthesis, which is in turn responsible for the activation of the ERK1/2, PI3K/Akt and PKA/CREB pathways that phosphorylate the NF-κB p65 subunit leading to NF-κB activation. Binding of the activated NF-κB p65 subunit to IL-6 promoter induces IL-6 synthesis in human T/C28a2 chondrocytes. PGD(2) or 15d-PGJ(2) concurrently downregulates TLR4 and upregulates caveolin-1, which in turn inhibit the PGE(2)-dependent ERK1/2, PI3-K and PKA activation, and ultimately with NF-κB-dependent IL-6 synthesis in chondrocytes. Conclusions/significance We have delineated the signaling cascade by which PGE(2) and PGD(2)/15d-PGJ(2) exert opposing effects on IL-6 synthesis in human chondrocytes. Elucidation of the molecular pathway of IL-6 synthesis and secretion by chondrocytes will provide insights for developing strategies to reduce inflammation and pain in RA patients.

Checkpoint pathways regulate genomic integrity in part by blocking anaphase until all chromosomes have been completely replicated, repaired, and correctly aligned on the spindle. In Saccharomyces cerevisiae, DNA damage and mono-oriented or unattached kinetochores trigger checkpoint pathways that bifurcate to regulate both the metaphase to anaphase transition and mitotic exit. The sensor-associated kinase, Mec1, phosphorylates two downstream kinases, Chk1 and Rad53. Activation of Chk1 and Rad53 prevents anaphase and causes inhibition of the mitotic exit network. We have previously shown that the PKA pathway plays a role in blocking securin and Clb2 destruction following DNA damage. Here we show that the Mec1 DNA damage checkpoint regulates phosphorylation of the regulatory (R) subunit of PKA following DNA damage and that the phosphorylated R subunit has a role in restraining mitosis following DNA damage. In addition we found that proteins known to regulate PKA in response to nutrients and stress either by phosphorylation of the R subunit or regulating levels of cAMP are required for the role of PKA in the DNA damage checkpoint. Our data indicate that there is cross-talk between the DNA damage checkpoint and the proteins that integrate nutrient and stress signals to regulate PKA., Author Summary Previous studies showed that phosphorylation of a subset of regulatory (R) subunits of the cAMP-dependent protein kinase (PKA) occurred under conditions that down-regulate global PKA activity, including growth on non-fermentable carbon sources. However, the role of the phosphorylation sites has not been elucidated. Addition of glucose to cells growing on a non-fermentable carbon source causes a transient increase of cAMP and PKA activity, which drives cells into S phase. A second peak in cAMP was proposed to restrain mitosis if the daughter cell had not reached an appropriate size. We identified a role for PKA in restraining mitosis following DNA damage. Here we provide evidence of cross-talk between the DNA damage checkpoint and PKA by phosphorylation of the R subunit. The R subunit phosphorylation sites and cAMP are necessary for the role of PKA following DNA damage. We propose that activation of PKA in response to DNA damage occurs in two steps: the phosphorylation of a subset of R subunits, probably to allow localized activation of these complexes, and cAMP to activate PKA. Our work suggests that the checkpoint and nutrient-sensing pathways share a signaling node to restrain mitosis following nutrient-induced rapid transition through the cell cycle and DNA damage.

The mechanism of PKAc-dependent NF-κB activation and subsequent translocation into the nucleus is not well defined. Previously, we showed that A kinase interacting protein 1 (AKIP1) was important for binding and retaining PKAc in the nucleus. Since then, other groups have demonstrated that AKIP1 binds the p65 subunit of NF-κB and regulates its transcriptional activity through the phosphorylation at Ser 276 by PKAc. However, little is known about the formation and activation of the PKAc/AKIP1/p65 complex and the rate at which it enters the nucleus. Initially, we found that the AKIP1 isoform (AKIP 1A) simultaneously binds PKAc and p65 in resting and serum starved cells. Using peptide arrays, we refined the region of AKIP 1A binding on PKAc and mapped the non-overlapping regions on AKIP 1A where PKAc and p65 bind. A peptide to the amino-terminus of PKAc (CAT 1-29) was generated to specifically disrupt the interaction between AKIP 1A and PKAc to study nuclear import of the complex. The rate of p65 nuclear translocation was monitored in the presence or absence of overexpressed AKIP 1A and/or (CAT 1-29). Enhanced nuclear translocation of p65 was observed in the presence of overexpressed AKIP1 and/or CAT 1-29 in cells stimulated with TNFα, and this correlated with decreased phosphorylation of serine 276. To determine whether PKAc phosphorylation of p65 in the cytosol regulated nuclear translocation, serine 276 was mutated to alanine or aspartic acid. Accelerated nuclear accumulation of p65 was observed in the alanine mutant, while the aspartic acid mutation displayed slowed nuclear translocation kinetics. In addition, enhanced nuclear translocation of p65 was observed when PKAc was knocked-down by siRNA. Taken together, these results suggest that AKIP 1A acts to scaffold PKAc to NF-κB in the cytosol by protecting the phosphorylation site and thereby regulating the rate of nuclear translocation of p65.

Background Understanding drivers for metastasis in human cancer is important for potential development of therapies to treat metastases. The role of loss of TGFβ tumor suppressor activities in the metastatic process is essentially unknown. Methodology/Principal Findings Utilizing in vitro and in vivo techniques, we have shown that loss of TGFβ tumor suppressor signaling is necessary to allow the last step of the metastatic process - colonization of the metastatic site. This work demonstrates for the first time that TGFβ receptor reconstitution leads to decreased metastatic colonization. Moreover, we have identified a novel TGFβ/PKA tumor suppressor pathway that acts directly on a known cell survival mechanism that responds to stress with the survivin/XIAP dependent inhibition of caspases that effect apoptosis. The linkage between the TGFβ/PKA transduceome signaling and control of metastasis through induction of cell death was shown by TGFβ receptor restoration with reactivation of the TGFβ/PKA pathway in receptor deficient metastatic colon cancer cells leading to control of aberrant cell survival. Conclusion/Significance This work impacts our understanding of the possible mechanisms that are critical to the growth and maintenance of metastases as well as understanding of a novel TGFβ function as a metastatic suppressor. These results raise the possibility that regeneration of attenuated TGFβ signaling would be an effective target in the treatment of metastasis. Our work indicates the clinical potential for developing anti-metastasis therapy based on inhibition of this very important aberrant cell survival mechanism by the multifaceted TGFβ/PKA transduceome induced pathway. Development of effective treatments for metastatic disease is a pressing need since metastases are the major cause of death in solid tumors.

Cystic fibrosis (CF) is the most common life-limiting recessive genetic disease among Caucasians caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) with over 95% male patients infertile. However, whether CFTR mutations could affect spermatogenesis and result in azoospermia remains an open question. Here we report compromised spermatogenesis, with significantly reduced testicular weight and sperm count, and decreased cAMP-responsive element binding protein (CREB) expression in the testes of CFTR knockout mice. The involvement of CFTR in HCO(3) (-) transport and the expression of the HCO(3) (-) sensor, soluble adenylyl cyclase (sAC), are demonstrated for the first time in the primary culture of rat Sertoli cells. Inhibition of CFTR or depletion of HCO(3) (-) could reduce FSH-stimulated, sAC-dependent cAMP production and phosphorylation of CREB, the key transcription factor in spermatogenesis. Decreased CFTR and CREB expression are also observed in human testes with azoospermia. The present study reveals a previously undefined role of CFTR and sAC in regulating the cAMP-CREB signaling pathway in Sertoli cells, defect of which may result in impaired spermatogenesis and azoospermia. Altered CFTR-sAC-cAMP-CREB functional loop may also underline the pathogenesis of various CF-related diseases.

Cyclic AMP (cAMP) inhibits the proliferation of several tumor cells. We previously reported an antiproliferative effect of PKA I-selective cAMP analogs (8-PIP-cAMP and 8-HA-cAMP) on two human cancer cell lines of different origin. 8-Cl-cAMP, another cAMP analog with known antiproliferative properties, has been investigated as a potential anticancer drug. Here, we compared the antiproliferative effect of 8-Cl-cAMP and the PKA I-selective cAMP analogs in three human cancer cell lines (ARO, NPA and WRO). 8-Cl-cAMP and the PKA I-selective cAMP analogs had similarly potent antiproliferative effects on the BRAF-positive ARO and NPA cells, but not on the BRAF-negative WRO cells, in which only 8-Cl-cAMP consistently inhibited cell growth. While treatment with the PKA I-selective cAMP analogs was associated with growth arrest, 8-Cl-cAMP induced apoptosis. To further investigate the actions of 8-Cl-cAMP and the PKA I-selective cAMP analogs, we analyzed their effects on signaling pathways involved in cell proliferation and apoptosis. Interestingly, the PKA I-selective cAMP analogs, but not 8-Cl-cAMP, inhibited ERK phosphorylation, whereas 8-Cl-cAMP alone induced a progressive phosphorylation of the p38 mitogen-activated protein kinase (MAPK), via activation of AMPK by its metabolite 8-Cl-adenosine. Importantly, the pro-apoptotic effect of 8-Cl-cAMP could be largely prevented by pharmacological inhibition of the p38 MAPK. Altogether, these data suggest that 8-Cl-cAMP and the PKA I-selective cAMP analogs, though of comparable antiproliferative potency, act through different mechanisms. PKA I-selective cAMP analogs induce growth arrest in cells carrying the BRAF oncogene, whereas 8-Cl-cAMP induce apoptosis, apparently through activation of the p38 MAPK pathway.

o,p'-DDT is an infamous xenoestrogen as well as a ubiquitous and persistent pollutant. Biomonitoring studies show that women have been internally exposed to o,p'-DDT at range of 0.3-500 ng/g (8.46×10(-10) M-1.41×10(-6) M) in blood and other tissues. However, very limited studies have investigated the biological effects and mechanism(s) of o,p'-DDT at levels equal to or lower than current exposure levels in human. In this study, using primary cultures of rat ovarian granulosa cells, we determined that very low doses of o,p'-DDT (10(-12)-10(-8) M) suppressed the expression of ovarian genes and production of prostaglandin E2 (PGE2). In vivo experiments consistently demonstrated that o,p'-DDT at 0.5-1 mg/kg inhibited the gene expression and PGE2 levels in rat ovary. The surprising results from the receptor inhibitors studies showed that these inhibitory effects were exerted independently of either classical estrogen receptors (ERs) or G protein-coupled receptor 30 (GPR30). Instead, o,p'-DDT altered gene expression or hormone action via inhibiting the activation of protein kinase A (PKA), rather than protein kinase C (PKC). We further revealed that o,p'-DDT directly interfered with the PKA catalytic subunit. Our novel findings support the hypothesis that exposure to low concentrations of o,p'-DDT alters gene expression and hormone synthesis through signaling mediators beyond receptor binding, and imply that the current exposure levels of o,p'-DDT observed in the population likely poses a health risk to female reproduction.

Angiogenesis inhibition is an important therapeutic strategy for advanced stage prostate cancer. Previous work from our laboratory showed that sustained stimulation of Rap1 by 8-pCPT-2'-O-Me-cAMP (8CPT) via activation of Epac, a Rap1 GEF, or by expression of a constitutively active Rap1 mutant (cRap1) suppresses endothelial cell chemotaxis and subsequent angiogenesis. When we tested this model in the context of a prostate tumor xenograft, we found that 8CPT had no significant effect on prostate tumor growth alone. However, in cells harboring cRap1, 8CPT dramatically inhibited not only prostate tumor growth but also VEGF expression and angiogenesis within the tumor microenvironment. Subsequent analysis of the mechanism revealed that, in prostate tumor epithelial cells, 8CPT acted via stimulation of PKA rather than Epac/Rap1. PKA antagonizes Rap1 and hypoxic induction of 1α protein expression, VEGF production and, ultimately, angiogenesis. Together these findings provide evidence for a novel interplay between Rap1, Epac, and PKA that regulates tumor-stromal induction of angiogenesis.

β(2) adrenergic receptor (β(2)AR) is a prototypical G-protein coupled receptor that stimulates the classic cAMP-protein kinase A (PKA) signaling pathway. Recent studies indicate that the cAMP-PKA activities are spatiotemporally regulated in part due to dynamic association of β(2)AR with phosphodiesterase 4D (PDE4D), a group of cAMP degradation enzymes. Here, we demonstrate that in cardiomyocytes, palmitoylation of β(2)AR, the covalent acylation of cysteine residue 341, plays a critical role in shaping subcellular cAMP-PKA activities in cardiomyocytes via regulating β(2)AR association with arrestin/PDE4D. Replacing cysteine 341 on β(2)AR with alanine (C341A) leads to an impaired binding to β arrestin 2. Surprisingly, the C341A mutant is able to internalize via an arrestin-independent pathway at saturated concentration of agonist stimulation; the internalization becomes caveolae-dependent and requires dynamin GTPase. However, the impaired binding to β arrestin 2 also leads to an impaired recruitment of PDE4D to the C341A mutant. Thus, the mutant C341A β(2)AR is transported alone from the plasma membrane to the endosome without recruiting PDE4D. This alteration leads to an enhanced cytoplasmic cAMP signal for PKA activation under β(2)AR stimulation. Functionally, Mutation of the C341 residue or inhibition of palmitoylation modification of β(2)AR enhances the receptor-induced PKA activities in the cytoplasm and increases in myocyte contraction rate. Our data reveal a novel function of palmitoylation in shaping subcellular cAMP-PKA signaling in cardiomyocytes via modulating the recruitment of β arrestin 2-PDE4D complexes to the agonist-stimulated β(2)AR.

In obesity, there is an increase in reactive oxygen species (ROS) within adipose tissue caused by increases in inflammation and overnutrition. Hormone sensitive lipase (HSL) is part of the canonical lipolytic pathway and critical for complete lipolysis. This study hypothesizes that ROS is a signal that integrates regulation of lipolysis by targeting HSL. Experiments were performed with human differentiated adipocytes from the subcutaneous depot. Antioxidants were employed as a tool to decrease ROS, and it was found that scavenging ROS with diphenyliodonium, N-acetyl cysteine, or resveratrol decreased lipolysis in adipocytes. HSL phosphorylation of a key serine residue, Ser552, as well as translocation of this enzyme from the cytosol to the lipid droplet upon lipolytic stimulation were both abrogated by scavenging ROS. The phosphorylation status of other serine residues on HSL were not affected. These findings are significant because they document that ROS contributes to the physiological regulation of lipolysis via an effect on translocation. Such regulation could be useful in developing new obesity therapies.

Lysophosphatidic acid (LPA), a lipid mediator enriched in serum, stimulates cell migration, proliferation and other functions in many cell types. LPA acts on six known G protein-coupled receptors, termed LPA(1-6), showing both overlapping and distinct signaling properties. Here we show that, unexpectedly, LPA and serum almost completely inhibit the transwell migration of B16 melanoma cells, with alkyl-LPA(18:1) being 10-fold more potent than acyl-LPA(18:1). The anti-migratory response to LPA is highly polarized and dependent on protein kinase A (PKA) but not Rho kinase activity; it is associated with a rapid increase in intracellular cAMP levels and PIP3 depletion from the plasma membrane. B16 cells express LPA(2), LPA(5) and LPA(6) receptors. We show that LPA-induced chemorepulsion is mediated specifically by the alkyl-LPA-preferring LPA(5) receptor (GPR92), which raises intracellular cAMP via a noncanonical pathway. Our results define LPA(5) as an anti-migratory receptor and they implicate the cAMP-PKA pathway, along with reduced PIP3 signaling, as an effector of chemorepulsion in B16 melanoma cells.

Cell division is controlled in part by the timely activation of the CDK, Cdc28, through its association with G1 and G2 cyclins. Cdc28 complexes are regulated in turn by the ubiquitin conjugating enzyme Cdc34 and SCF ubiquitin ligase complexes of the ubiquitin-proteasome system (UPS) to control the initiation of DNA replication. Here we demonstrate that the nutrient sensing kinases PKA and Sch9 phosphorylate S97 of Cdc34. S97 is conserved across species and restricted to the catalytic domain of Cdc34/Ubc7-like E2s. Cdc34-S97 phosphorylation is cell cycle regulated, elevated during active cell growth and division and decreased during cell cycle arrest. Cell growth and cell division are orchestrated to maintain cell size homeostasis over a wide range of nutrient conditions. Cells monitor changes in their environment through nutrient sensing protein kinases. Thus Cdc34 phosphorylation by PKA and Sch9 provides a direct tether between G1 cell division events and cell growth.

Huntingtin is a large HEAT repeat protein first identified in humans, where a polyglutamine tract expansion near the amino terminus causes a gain-of-function mechanism that leads to selective neuronal loss in Huntington's disease (HD). Genetic evidence in humans and knock-in mouse models suggests that this gain-of-function involves an increase or deregulation of some aspect of huntingtin's normal function(s), which remains poorly understood. As huntingtin shows evolutionary conservation, a powerful approach to discovering its normal biochemical role(s) is to study the effects caused by its deficiency in a model organism with a short life-cycle that comprises both cellular and multicellular developmental stages. To facilitate studies aimed at detailed knowledge of huntingtin's normal function(s), we generated a null mutant of hd, the HD ortholog in Dictyostelium discoideum. Dictyostelium cells lacking endogenous huntingtin were viable but during development did not exhibit the typical polarized morphology of Dictyostelium cells, streamed poorly to form aggregates by accretion rather than chemotaxis, showed disorganized F-actin staining, exhibited extreme sensitivity to hypoosmotic stress, and failed to form EDTA-resistant cell–cell contacts. Surprisingly, chemotactic streaming could be rescued in the presence of the bivalent cations Ca2+ or Mg2+ but not pulses of cAMP. Although hd − cells completed development, it was delayed and proceeded asynchronously, producing small fruiting bodies with round, defective spores that germinated spontaneously within a glassy sorus. When developed as chimeras with wild-type cells, hd − cells failed to populate the pre-spore region of the slug. In Dictyostelium, huntingtin deficiency is compatible with survival of the organism but renders cells sensitive to low osmolarity, which produces pleiotropic cell autonomous defects that affect cAMP signaling and as a consequence development. Thus, Dictyostelium provides a novel haploid organism model for genetic, cell biological, and biochemical studies to delineate the functions of the HD protein., Author Summary Genetic evidence in humans and mouse models of Huntington's disease suggests that the disease mutation confers a deleterious gain-of-function on huntingtin that acts through the deregulation of some aspect of the protein's normal function(s). While huntingtin's function is poorly understood, its evolutionary conservation makes investigation of its physiological role in lower organisms an attractive route that has yet to be fully exploited. Therefore, we have used Dictyostelium discoideum to study the consequences of huntingtin (hd) deficiency. Developing Dictyostelium cells chemotax to form a multicellular slug that forms a fruiting body, comprising dormant spores encased above dead stalk cells. We found that hd − cells were hypersensitive to hypoosmotic stress. When starved, hd − cells aggregate by accretion, showed disorganized F-actin, and failed to form EDTA-resistant cell–cell contacts. Surprisingly, chemotactic signaling was rescued with Ca2+ or Mg2+ but not pulses of cAMP. Development of hd − mutants produced small fruiting bodies with round, defective spores, and when mixed with wild-type cells they didn't differentiate into spores. Our results are consistent with mammalian studies that show huntingtin is a multifunctional protein involved in many biochemical processes; and, importantly, they establish Dictyostelium as a valuable experimental organism for exploring in biochemical detail huntingtin's normal function(s).

Background Recently, a variant of ER-α, ER-α36 was identified and cloned. ER-α36 lacks intrinsic transcription activity and mainly mediates non-genomic estrogen signaling. The purpose of this study was to investigate the function and the underlying mechanisms of ER-α36 in growth regulation of endometrial Ishikawa cancer cells. Methods The cellular localization of ER-α36 and ER-α66 were determined by immunofluorescence in the Ishikawa cells. Ishikawa endometrial cancer control cells transfected with an empty expression vector, Ishikawa cells with shRNA knockdown of ER-α36 (Ishikawa/RNAiER36) and Ishikawa cells with shRNA knockdown of ER-α66 (Ishikawa/RNAiER66) were treated with E2 and E2-conjugated to bovine serum albumin (E2-BSA, membrane impermeable) in the absence and presence of different kinase inhibitors HBDDE, bisindolylmaleimide, rottlerin, H89 and U0126. The phosphorylation levels of signaling molecules and cyclin D1/cdk4 expression were examined with Western blot analysis and cell growth was monitored with the MTT assay. Results Immunofluorescence staining of Ishikawa cells demonstrated that ER-α36 was expressed mainly on the plasma membrane and in the cytoplasm, while ER-α66 was predominantly localized in the cell nucleus. Both E2 and E2-BSA rapidly activated PKCδ not PKCα in Ishikawa cells, which could be abrogated by ER-α36 shRNA expression. E2-and E2-BSA-induced ERK phosphorylation required ER-α36 and PKCδ. However, only E2 was able to induce Camp-dependent protein kinase A (PKA) phosphorylation. Furthermore, E2 enhances cyclin D1/cdk4 expression via ER-α36. Conclusion E2 activates the PKCδ/ERK pathway and enhances cyclin D1/cdk4 expression via the membrane-initiated signaling pathways mediated by ER-α36, suggesting a possible involvement of ER-α36 in E2-dependent growth-promoting effects in endometrial cancer cells.