Subunit Of cAMP-dependent Protein Kinase Research Articles

Insights into the phosphoryl transfer catalyzed by cAMP-dependent protein kinase

Protein kinases are involved in a number of cell signaling pathways. They catalyze phosphorylation of proteins and regulate the majority of cellular processes (such as growth, differentiation, lipid metabolism, regulation of sugar, nucleic acid synthesis, etc.). Chemically, protein kinases covalently transfer the gamma-phosphate group of a nucleoside triphosphate (e.g. ATP) to a hydroxyl group of a Ser, Thr or Tyr residue of substrate protein or peptide. The reaction involves moving hydrogen atoms between the enzyme, substrate and nucleoside. The unanswered question is whether the proton transfer from the Ser residue happens before the phosphoryl transfer using the general acid-base catalyst, Asp166, or after the reaction went through the transition state by directly protonating the phosphate group. To address this key question about the phosphoryl transfer, we determined a number of X-ray structures of ternary complexes of catalytic subunit of cAMP-dependent protein kinase (PKAc) with various substrates, nucleotides and cofactors. Importantly, we were able to trap and mimic the initial (Michaelis complex) and final (product complex) stages of the reaction. The results demonstrate that Mg2+, Ca2+, Sr2+, and Ba2+ metal ions bind to the active site and facilitate the reaction to produce ADP and a phosphorylated peptide. The study also revealed that metal-free PKAc can facilitate the phosphoryl transfer reaction; a result that was confirmed with single turnover enzyme kinetics measurements. Comparison of the product and the pseudo-Michaelis complex structures, in conjunction with molecular dynamics simulations, reveals conformational, coordination, and hydrogen bonding changes that help further our understanding of the mechanism, roles of metals, and active site residues involved in PKAc activity.

Estradiol determines the effects of PTH on ERα-dependent transcription in MC3T3-E1 cells

Bone remodeling is a continuous process regulated by several hormones such as estrogens and parathyroid hormone (PTH). Here we investigated the influence of PTH on estrogen receptor alpha (ERα)-dependent transcriptional activity in MC3T3-E1 osteoblasts. Cells that were transfected with an ER-responsive reporter plasmid and treated with PTH showed increased luciferase activity. However, in the presence of 17β-estradiol, we observed that PTH inhibited ERα-mediated transcription. cAMP mimicked the effects by PTH, and the findings were confirmed in COS-1 cells transfected with expression vector encoding the catalytic subunit of cAMP-dependent protein kinase (PKA). Furthermore, PTH exhibited specific effects on the mRNA expression of the decoy receptor osteoprotegerin (OPG) and the receptor activator of NF kappa-B ligand (RANKL) in MC3T3-E1 osteoblasts. In the absence of 17β-estradiol, PTH and cAMP enhanced the OPG/RANKL ratio, whereas, OPG/RANKL was suppressed when estradiol was present. In conclusion, our results indicate that the presence of estradiol determines whether PTH and cAMP stimulates or inhibits ERα-dependent activity and the OPG/RANKL mRNA expression in an osteoblastic cell line.

Metal-free cAMP-dependent protein kinase can catalyze phosphoryl transfer.

X-ray structures of several ternary product complexes of the catalytic subunit of cAMP-dependent protein kinase (PKAc) have been determined with no bound metal ions and with Na+ or K+ coordinated at two metal-binding sites. The metal-free PKAc and the enzyme with alkali metals were able to facilitate the phosphoryl transfer reaction. In all studied complexes, the ATP and the substrate peptide (SP20) were modified into the products ADP and the phosphorylated peptide. The products of the phosphotransfer reaction were also found when ATP-γS, a nonhydrolyzable ATP analogue, reacted with SP20 in the PKAc active site containing no metals. Single turnover enzyme kinetics measurements utilizing 32P-labeled ATP confirmed the phosphotransferase activity of the enzyme in the absence of metal ions and in the presence of alkali metals. In addition, the structure of the apo-PKAc binary complex with SP20 suggests that the sequence of binding events may become ordered in a metal-free environment, with SP20 binding first to prime the enzyme for subsequent ATP binding. Comparison of these structures reveals conformational and hydrogen bonding changes that might be important for the mechanism of catalysis.

Research in brief

Survivors of childhood cancer are at increased risk of developing endocrine disorders, according to data from a recent study. In the population-based study, data were analysed for 31 723 survivors of childhood cancer from Denmark, Finland, Iceland, Norway, and Sweden. Using registry data, follow-up begain 1 year after a childhood cancer diagnosis (median follow-up of 10 years, range 0–42). Survivors of childhood cancer had a 4·8-times increased risk for having at least one hospital contact for an endocrine disorder. Pituitary hypofunction, hypothyroidism, and testicular and ovarian dysfunction comprised 61% (655 of 1078) of all excess disease-induced and treatment-induced endocrine disorders. Survivors of childhood leukaemia and CNS cancer were at especially high risk of developing an endocrine disorder. Gastritis can lead to malabsorption of levothyroxine as a result of altering gastric juice pH. Whether taking levothyroxine with ascorbic acid (vitamin C) could improve thyroid hormone profile was tested in a small study. 31 patients with poorly controlled hypothyroidism and gastritis entered a 2 month control period, followed by a 2 month intervention phase, and a final 2 month control period. In the intervention phase, patients ingested their levothyroxine dose in 120ml of water containing vitamin C. Serum thyroid-stimulating hormone decreased in all patients during the intervention period, with an average decrease of 69·2%. Serum T4 was higher with the intervention in 30 of 31 patients. With malabsorption of levothyroxine posing a clinical barrier to adequate management of hypothyroidism, methods to overcome this are needed. Around 35% of adults in the USA are obese, according to results from the National Health and Nutrition Examination Survey. The authors used time-trend analysis to compare rates of obesity between 2003 and 2012. In the 2011–12 study period, almost 17% of 2–19-year-olds were obese, and the prevalence of high weight for recumbent length in infants and toddlers was 8%. Although no significant changes occurred between 2003–04 and 2011–12 in high weight for recumbent length among infants and toddlers or obesity in young people and adults, overall rates of obesity were high and surveillance is a key priority. Results of a proof-of-concept study suggest that SGLT2 inhibition holds some promise for management of type 1 diabetes as an adjunct to insulin. In a single-group, open-label trial, 40 adults with type 1 diabetes were given empagliflozin 25 mg daily for 8 weeks. Mean HbA1c decreased from 8·0% to 7·6% (p<0·0001), and was associated with a reduction in daily insulin dose (p<0·0001) and weight loss (p<0·0001). Two patients were withdrawn from the study because of diabetic ketoacidosis (one had severe gastroenteritis and the other insulin pump failure). This small study lends support to the design of large-scale studies investigating the effect of SGLT2 inhibition in type 1 diabetes. A new study has shed some light on the molecular basis of corticotropin-independent Cushing's syndrome. The investigators analysed samples from patients with adrenal cortex hyperplasia and a range of adrenal tumours. Exome sequencing showed that eight of 10 cortisol-producing adenomas had somatic mutations in PRKACA—a gene encoding the catalytic subunit of cAMP-dependent protein kinase. Comparative genomic hybridisation showed germline duplications of the PRKACA gene in five of 35 samples from patients with bilateral adrenal hyperplasias. All patients with PRKACA defects had overt Cushing's syndrome; patients with subclinical disease or other adrenal tumours showed no such defects. This study draws attention to the role of constitutive PKA activation in corticotropin-independent Cushing's syndrome. The opening or expansion of Native American-owned casinos can decrease the risk of overweight/obesity in childhood, note the authors of an ecological study. Anthropometric data were obtained from repeated cross-sectional fitness testing of Native American children between 2001 and 2012, from 117 Californian school districts. Of the 117 districts encompassing tribal lands, 57 gained or expanded a casino. For every slot machine gained per person, there was an associated increase of US$541 (95%CI $245–836) in annual income per person, and a decrease in BMI z-score (β=–0·003; 95% CI, −0·005 to −0·0002). In view of inherent limitations of this study, additional research is necessary to better appreciate the interplay between casino opening, economic resources, and childhood overweight/obesity.

NMR mapping of protein conformational landscapes using coordinated behavior of chemical shifts upon ligand binding.

Proteins exist as an ensemble of conformers that are distributed on free energy landscapes resembling folding funnels. While the most stable conformers populate low energy basins, protein function is often carried out through low-populated conformational states that occupy high energy basins. Ligand binding shifts the populations of these states, changing the distribution of these conformers. Understanding how the equilibrium among the states is altered upon ligand binding, interaction with other binding partners, and/or mutations and post-translational modifications is of critical importance for explaining allosteric signaling in proteins. Here, we propose a statistical analysis of the linear trajectories of NMR chemical shifts (CONCISE, COordiNated ChemIcal Shifts bEhavior) for the interpretation of protein conformational equilibria. CONCISE enables one to quantitatively measure the population shifts associated with ligand titrations and estimate the degree of collectiveness of the protein residues' response to ligand binding, giving a concise view of the structural transitions. The combination of CONCISE with thermocalorimetric and kinetic data allows one to depict a protein's approximate conformational energy landscape. We tested this method with the catalytic subunit of cAMP-dependent protein kinase A, a ubiquitous enzyme that undergoes conformational transitions upon both nucleotide and pseudo-substrate binding. When complemented with chemical shift covariance analysis (CHESCA), this new method offers both collective response and residue-specific correlations for ligand binding to proteins.

HIV-1-associated PKA acts as a cofactor for genome reverse transcription

BackgroundHost cell proteins, including cellular kinases, are embarked into intact HIV-1 particles. We have previously shown that the Cα catalytic subunit of cAMP-dependent protein kinase is packaged within HIV-1 virions as an enzymatically active form able to phosphorylate a synthetic substrate in vitro (Cartier et al. J. Biol. Chem. 278:35211 (2003)). The present study was conceived to investigate the contribution of HIV-1-associated PKA to the retroviral life cycle.ResultsNL4.3 viruses were produced from cells cultured in the presence of PKA inhibitors H89 (H89-NL4.3) or Myr-PKI (PKI-NL4.3) and analyzed for viral replication. Despite being mature and normally assembled, and containing expected levels of genomic RNA and RT enzymatic activity, such viruses showed poor infectivity. Indeed, infection generated reduced amounts of strong-strop minus strand DNA, while incoming RNA levels in target cells were unaffected. Decreased cDNA synthesis was also evidenced in intact H89-NL4.3 and PKI-NL4.3 cell free particles using endogenous reverse transcription (ERT) experiments. Moreover, similar defects were reproduced when wild type NL4.3 particles preincubated with PKA inhibitors were subjected to ERT reactions.ConclusionsAltogether, our results indicate that HIV-1-associated PKA is required for early reverse transcription of the retroviral genome both in cell free intact viruses and in target cells. Accordingly, virus-associated PKA behaves as a cofactor of an intraviral process required for optimal reverse transcription and for early post-entry events.

Crystal Structure of a Human IκB Kinase β Asymmetric Dimer

Phosphorylation of inhibitor of nuclear transcription factor κB (IκB) by IκB kinase (IKK) triggers the degradation of IκB and migration of cytoplasmic κB to the nucleus where it promotes the transcription of its target genes. Activation of IKK is achieved by phosphorylation of its main subunit, IKKβ, at the activation loop sites. Here, we report the 2.8 Å resolution crystal structure of human IKKβ (hIKKβ), which is partially phosphorylated and bound to the staurosporine analog K252a. The hIKKβ protomer adopts a trimodular structure that closely resembles that from Xenopus laevis (xIKKβ): an N-terminal kinase domain (KD), a central ubiquitin-like domain (ULD), and a C-terminal scaffold/dimerization domain (SDD). Although hIKKβ and xIKKβ utilize a similar dimerization mode, their overall geometries are distinct. In contrast to the structure resembling closed shears reported previously for xIKKβ, hIKKβ exists as an open asymmetric dimer in which the two KDs are further apart, with one in an active and the other in an inactive conformation. Dimer interactions are limited to the C-terminal six-helix bundle that acts as a hinge between the two subunits. The observed domain movements in the structures of IKKβ may represent trans-phosphorylation steps that accompany IKKβ activation.

Insights into the Phosphoryl Transfer Catalyzed by cAMP-Dependent Protein Kinase: An X-ray Crystallographic Study of Complexes with Various Metals and Peptide Substrate SP20

X-ray structures of several ternary substrate and product complexes of the catalytic subunit of cAMP-dependent protein kinase (PKAc) have been determined with different bound metal ions. In the PKAc complexes, Mg2+, Ca2+, Sr2+, and Ba2+ metal ions could bind to the active site and facilitate the phosphoryl transfer reaction. ATP and a substrate peptide (SP20) were modified, and the reaction products ADP and the phosphorylated peptide were found trapped in the enzyme active site. Finally, we determined the structure of a pseudo-Michaelis complex containing Mg2+, nonhydrolyzable AMP-PCP (β,γ-methyleneadenosine 5′-triphosphate) and SP20. The product structures together with the pseudo-Michaelis complex provide snapshots of different stages of the phosphorylation reaction. Comparison of these structures reveals conformational, coordination, and hydrogen bonding changes that might occur during the reaction and shed new light on its mechanism, roles of metals, and active site residues.

The catalytic subunit of cAMP-dependent protein kinase AStPKA-ccontributes to conidiation and early invasion in the phytopathogenic fungusSetosphaeria turcica

Cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) is an important mediator of signal transduction in eukaryotic cells. Thus, identifying its function is necessary to understand the cAMP signaling network. StPKA-c, the PKA catalytic subunit gene in Setosphaeria turcica, was investigated by RNA interference technology. Transformant strains M3, M5, and M9 with diverse StPKA-c silencing efficiency were confirmed by reverse transcription polymerase chain reaction and Northern blot. Compared with the wild-type strain 01-23, the transformant strains exhibited increased growth rate and significantly decreased conidium production. In addition, the ratios of spore germination and appressorium formation and penetration were slightly reduced. Relative to the wild-type strain, the transformants demonstrated different colony color, greatly reduced pathogenicity, and similar HT-toxin activity. Further studies showed that the content of intracellular melanin in the transformants significantly decreased, and the transcription of transcriptional factor StMR was down-regulated correspondingly. The transcription and enzyme activity of xylanase was also impaired. Thus, we proposed that StPKA-c was mainly involved in the mycelium growth, conidiation, and pathogenesis of S.turcica. Furthermore, it was positively correlated with the biosyntheses of melanin and xylanase but dispensable for the activity of HT-toxin.

Phosphoryl transfer by protein kinase A is captured in a crystal lattice.

The catalytic (C) subunit of cAMP-dependent protein kinase (PKA) is a serine/threonine kinase responsible for most of the effects of cAMP signaling, and PKA serves as a prototype for the entire kinase family. Despite multiple studies of PKA, the steps involved in phosphoryl transfer, the roles of the catalytically essential magnesium ions, and the processes that govern the rate-limiting step of ADP release are unresolved. Here we identified conditions that yielded slow phosphoryl transfer of the γ-phosphate from the generally nonhydrolyzable analog of ATP, adenosine-5'-(β,γ-imido)triphosphate (AMP-PNP), onto a substrate peptide within protein crystals. By trapping both products in the crystal lattice, we now have a complete resolution profile of all the catalytic steps. One crystal structure refined to 1.55 Å resolution shows two states of the protein with 55% displaying intact AMP-PNP and an unphosphorylated substrate and 45% displaying transfer of the γ-phosphate of AMP-PNP onto the substrate peptide yielding AMP-PN and a phosphorylated substrate. Another structure refined to 2.15 Å resolution displays complete phosphoryl transfer to the substrate. These structures, in addition to trapping both products in the crystal lattice, implicate one magnesium ion, previously termed Mg2, as the more stably bound ion. Following phosphoryl transfer, Mg2 recruits a water molecule to retain an octahedral coordination geometry suggesting the strong binding character of this magnesium ion, and Mg2 remains in the active site following complete phosphoryl transfer while Mg1 is expelled. Loss of Mg1 may thus be an important part of the rate-limiting step of ADP release.

Biological roles of cAMP: variations on a theme in the different kingdoms of life

Cyclic AMP (cAMP) plays a key regulatory role in most types of cells; however, the pathways controlled by cAMP may present important differences between organisms and between tissues within a specific organism. Changes in cAMP levels are caused by multiple triggers, most affecting adenylyl cyclases, the enzymes that synthesize cAMP. Adenylyl cyclases form a large and diverse family including soluble forms and others with one or more transmembrane domains. Regulatory mechanisms for the soluble adenylyl cyclases involve either interaction with diverse proteins, as happens in Escherichia coli or yeasts, or with calcium or bicarbonate ions, as occurs in mammalian cells. The transmembrane cyclases can be regulated by a variety of proteins, among which the α subunit and the βγ complex from G proteins coupled to membrane receptors are prominent. cAMP levels also are controlled by the activity of phosphodiesterases, enzymes that hydrolyze cAMP. Phosphodiesterases can be regulated by cAMP, cGMP or calcium-calmodulin or by phosphorylation by different protein kinases. Regulation through cAMP depends on its binding to diverse proteins, its proximal targets, this in turn causing changes in a variety of distal targets. Specifically, binding of cAMP to regulatory subunits of cAMP-dependent protein kinases (PKAs) affects the activity of substrates of PKA, binding to exchange proteins directly activated by cAMP (Epac) regulates small GTPases, binding to transcription factors such as the cAMP receptor protein (CRP) or the virulence factor regulator (Vfr) modifies the rate of transcription of certain genes, while cAMP binding to ion channels modulates their activity directly. Further studies on cAMP signalling will have important implications, not only for advancing fundamental knowledge but also for identifying targets for the development of new therapeutic agents.

Analyses of the Involvement of PKA Regulation Mechanism in Meiotic Incompetence of Porcine Growing Oocytes1

Mammalian growing oocytes (GOs) lack the ability to resume meiosis, although the molecular mechanism of this limitation is not fully understood. In the present study, we cloned cDNAs of cAMP-dependent protein-kinase (PKA) subunits from porcine oocytes and analyzed the involvement of the PKA regulation mechanism in the meiotic incompetence of GOs at the molecular level. We found a cAMP-independent high PKA activity in GOs throughout the in vitro culture using a porcine PKA assay system we established, and inhibition of the activity by injection of the antisense RNA of the PKA catalytic subunit (PKA-C) induced meiotic resumption in GOs. Then we examined the possibility that the amount of the PKA regulatory subunit (PKA-R), which can bind and inhibit PKA-C, was insufficient to suppress PKA activity in GOs because of the overexpression of two PKA-Rs, PRKAR1A and PRKAR2A. We found that neither of them affected PKA activity and induced meiotic resumption in GO although PRKAR2A could inhibit PKA activity and induce meiosis in cAMP-treated full-grown oocytes (FGOs). Finally, we analyzed the subcellular localization of PKA subunits and found that all the subunits were localized in the cytoplasm during meiotic arrest and that PKA-C and PRKAR2A, but not PRKAR1A, entered into the nucleus just before meiotic resumption in FGOs, whereas all of them remained in the cytoplasm in GOs throughout the culture period. Our findings suggest that the continuous high PKA activity is a primary cause of the meiotic incompetence of porcine GOs and that this PKA activity is not simply caused by an insufficient expression level of PKA-R, but can be attributed to more complex spatial-temporal regulation mechanisms.

Characterization and Solution Structure of Mouse Myristoylated Methionine Sulfoxide Reductase A

Methionine sulfoxide reductase A is an essential enzyme in the antioxidant system which scavenges reactive oxygen species through cyclic oxidation and reduction of methionine and methionine sulfoxide. The cytosolic form of the enzyme is myristoylated, but it is not known to translocate to membranes, and the function of myristoylation is not established. We compared the biochemical and biophysical properties of myristoylated and nonmyristoylated mouse methionine sulfoxide reductase A. These were almost identical for both forms of the enzyme, except that the myristoylated form reduced methionine sulfoxide in protein much faster than the nonmyristoylated form. We determined the solution structure of the myristoylated protein and found that the myristoyl group lies in a relatively surface exposed "myristoyl nest." We propose that this structure functions to enhance protein-protein interaction.

Engineering the central pathways in Lactococcus lactis: Functional expression of the phosphofructokinase (pfk) and alternative oxidase (aox1) genes from Aspergillus niger in Lactococcus lactis facilitates improved carbon conversion rates under oxidizing conditions

The present work describes a novel central pathway engineering method that has been designed with the aim to increase the carbon conversion rates under oxidizing conditions in L. lactis fermentations. The nisin producer L. lactis ATCC11454 strain has been genetically engineered by cloning a truncated version of the phosphofructokinase gene (pfk13), along with the pkaC, encoding for the catalytic subunit of cAMP-dependent protein kinase, and the alternative oxidase (aox1) genes of A. niger. Functional expression of the above genes resulted in enhanced PFK activity and the introduction of AOX activity and alternative respiration in the presence of a source of heme in the substrate, under fully aerobic growth conditions. The constructed strain is capable of fermenting high concentrations of glucose as was demonstrated in a series of glucostat fed-batch fermentations with glucose levels maintained at 55, 138 and 277mM. The high maximum specific uptake rate of glucose of 1.8mMs−1gCDW−1 at 277mM glucose is characteristic of the improved ability of the microorganism to handle elevated glucose concentrations under conditions otherwise causing severe reduction of PFK activity. The increased carbon flow through glycolysis led to increased protein synthesis that was reflected in increased biomass and nisin levels. The pfk13–pkaC–aox1-transformant strain's fermentation at 277mM glucose gave a final biomass concentration of 7.5g/l and nisin activity of 14,000IU/ml which is, compared to the parental strain's production levels at its optimal 55mM glucose, increased by a factor of 2.34 for biomass and 4.37 for nisin.

Role of N-Terminal Myristylation in the Structure and Regulation of cAMP-Dependent Protein Kinase

The catalytic (C) subunit of cAMP-dependent protein kinase [protein kinase A (PKA)] is a major target of cAMP signaling, and its regulation is of fundamental importance to biological processes. One mode of regulation is N-myristylation, which has eluded structural and functional characterization so far because most crystal structures are of the non-myristylated enzyme, are phosphorylated on Ser10, and generally lack electron density for the first 13 residues. We crystallized myristylated wild-type (WT) PKA and a K7C mutant as binary (bound to a substrate peptide) and ternary [bound to a substrate peptide and adenosine-5'-(β,γ-imido)triphosphate] complexes. There was clear electron density for the entire N-terminus in the binary complexes, both refined to 2.0 Å, and K7C ternary complex, refined to 1.35 Å. The N-termini in these three structures display a novel conformation with a previously unseen helix from residues 1 to 7. The K7C mutant appears to have a more stable N-terminus, and this correlated with a significant decrease in the B-factors for the N-terminus in the myr-K7C complexes compared to the WT binary complex. The N-terminus of the myristylated WT ternary complex, refined to 2.0 Å, was disordered as in previous structures. In addition to a more ordered N-terminus, the myristylated K7C mutant exhibited a 53% increase in k(cat). The effect of nucleotide binding on the structure of the N-terminus in the WT protein and the kinetic changes in the K7C protein suggest that myristylation or occupancy of the myristyl binding pocket may serve as a site for allosteric regulation in the C-subunit.

Abstract 954: COX-2 inhibition reduces bone tumor growth in animal models:A role for celecoxib treatment in cAMP/protein kinase A-induced tumors

Abstract Background: Carney Complex (CNC) is a multiple neoplasia syndrome characterized by skin lesions, endocrine and other tumors, including osteochondromyxomas. It is caused by inactivating mutations in the PRKAR1A gene encoding the regulatory type 1A subunit of cAMP-dependent protein kinase A (PKA). This subunit is the main suppressor of activity of catalytic subunit (PRCKACA) of PKA which regulates cell proliferation and differentiation. Loss of PRKAR1A increases PKA activity and causes tumors in CNC-affected tissues. Mice with deletion of one prkar1a allele (prkar1a+/−) develop various CNC-like tumors and multiple tail osteotic lesions. Further deletion of a prkaca allele on the prkar1a+/− background unexpectedly increases the number and severity of bone lesions and reduces their age of onset. Recent data indicate that increased PKA activity stimulates the inflammasome without inflammation in mouse and human cells. The released prostaglandin E2 (PGE2) further increases cAMP, which in turn activates PKA and Wnt signaling and promotes the growth of these bony tumors. These data led us to investigate celecoxib, which is a selective inhibitor of cyclooxygenase-2 (COX-2) and therefore of PGE2's release, in prkar1α+/−/prkacα+/− mice. Methods: Eleven out of twenty-two mice 6-12 months old were exposed to celecoxib-containing diet (1,500 mg/kg of NIH-31 diet). The remaining mice were fed the standard NIH-31 diet. After a six-week treatment, we looked at the number, the PKA and phosphodiesterase (PDE) activities, c AMP levels and histology of bony lesions. Sixteen and seven days before sacrifice, mice were injected with calcein which labels bone forming surfaces. From calcein fluorescence observed under microscope, the rate of apposition of cortical bone at periosteal side was measured. This rate reflects tumor growth rate because growing tumors remain encapsulated by cortical bone forming at periosteal side and resorbing at endosteal side. Results: Celecoxib reduced the rate of tumor growth by two-fold as measured by calcein labeling (p=0.016). Histological examination revealed decreased cellularity in the lesions of treated animals due to treatment effect on the tumor cells. Treatment reduced PKA enzymatic activity in the tail tumors as measured by high-performance liquid chromatography (4.0 vs 3.0 CMP/ug protein, p=0.07) whereas cAMP levels and total PDE activity did not change. The expression of all inflammasome-related genes (ets-1, caspase-1, il1b and cox2) was down-regulated by at least 70% in the bone tumors from treated animals compared to controls. Conclusion: COX-2 inhibition reduces PKA activity and bone tumor growth in prkar1α+/−/prkacα+/− mice. Since local tumor control is clinically meaningful for patients with cAMP-induced tumors, celecoxib may merit consideration as the first molecular targeted therapy in this setting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 954. doi:1538-7445.AM2012-954

Structural Basis for the Regulation of Protein Kinase A by Activation Loop Phosphorylation

The catalytic subunit of cAMP-dependent protein kinase (PKA) is a member of the AGC group of protein kinases. Whereas PKA has served as a structural model for the protein kinase superfamily, all previous structures of the catalytic subunit contain a phosphorylated activation loop. To understand the structural effects of activation loop phosphorylation at Thr-197 we used a PKA mutant that does not autophosphorylate at Thr-197. The enzyme crystallized in the apo-state, and the structure was solved to 3.0 Å. The N-lobe is rotated by 18° relative to the wild-type apoenzyme, which illustrates that the enzyme likely exists in a wide range of conformations in solution due to the uncoupling of the N- and C-lobes. Several regions of the protein including the activation loop are disordered in the structure, and there are alternate main chain conformations for the magnesium positioning loop and catalytic loop causing a complete loss of hydrogen bonding between these two active site structural elements. These alterations are reflected in a 20-fold decrease in the apparent phosphoryl transfer rate as measured by pre-steady-state kinetic methods.

Identification of a protein kinase A regulatory subunit from Leishmania having importance in metacyclogenesis through induction of autophagy

cAMP-mediated responses act as modulators of environmental sensing and cellular differentiation of many kinetoplastidae parasites including Leishmania. Although cAMP synthesizing (adenylate cyclase) and degrading (phosphodiesterase) enzymes have been cloned and characterized from Leishmania, no cAMP-binding effector molecule has yet been identified from this parasite. In this study, a regulatory subunit of cAMP-dependent protein kinase (Ldpkar1), homologous to mammalian class I cAMP-dependent protein kinase regulatory subunit, has been identified from L. donovani. Further characterization suggested possible interaction of LdPKAR1 with PKA catalytic subunits and inhibition of PKA activity. This PKA regulatory subunit is expressed in all life cycle stages and its expression attained maximum level in stationary phase promastigotes, which are biochemically similar to the infective metacyclic promastigotes. Starvation condition, the trigger for metacyclogenesis in the parasite, elevates LdPKAR1 expression and under starvation condition promastigotes overexpressing Ldpkar1 attained metacyclic features earlier than normal cells. Furthermore, Ldpkar1 overexpression accelerates autophagy, a starvation-induced cytological event necessary for metacyclogenesis and amastigote formation. Conditional silencing of Ldpkar1 delays the induction of autophagy in the parasite. The study, for the first time, reports the identification of a functional cAMP-binding effector molecule from Leishmania that may modulate important cytological events affecting metacyclogenesis.

Inactivation of the Catalytic Subunit of cAMP-Dependent Protein Kinase A Causes Delayed Appressorium Formation and Reduced Pathogenicity ofColletotrichum gloeosporioides

The cyclic AMP- (cAMP-) dependent protein kinase A signaling pathway is one of the major signaling pathways responsible for regulation of the morphogenesis and pathogenesis of several pathogenic fungi. To evaluate the role of this pathway in the plant pathogenic fungus, Colletotrichum gloeosporioides, the gene encoding the catalytic subunit of cAMP-dependent protein kinase A, CgPKAC, was cloned, inactivated, and the mutant was analyzed. Analysis of the Cgpkac mutant generated via gene replacement showed that the mutants were able to form appressoria; however, their formation was delayed compared to the wild type. In addition, the mutant conidia underwent bipolar germination after appressoria formation, but no appressoria were generated from the second germ tube. The mutants also showed reduced ability to adhere to a hydrophobic surface and to degrade lipids localized in the appressoria. Based on the number of lesions produced during a pathogenicity test, the mutant's ability to cause disease in healthy mango fruits was reduced, which may be due to failure to penetrate into the fruit. These findings indicate that cAMP-dependent protein kinase A has an important role in regulating morphogenesis and is required for pathogenicity of C. gloeosporioides.

Candidate genes in ocular dominance plasticity

Many studies have been devoted to the identification of genes involved in experience-dependent plasticity in the visual cortex. To discover new candidate genes, we have reexamined data from one such study on ocular dominance (OD) plasticity in recombinant inbred BXD mouse strains. We have correlated the level of plasticity with the gene expression data in the neocortex that have become available for these same strains. We propose that genes with a high correlation are likely to play a role in OD plasticity. We have tested this hypothesis for genes whose inactivation is known to affect OD plasticity. The expression levels of these genes indeed correlated with OD plasticity if their levels showed strong differences between the BXD strains. To narrow down our candidate list of correlated genes, we have selected only those genes that were previously found to be regulated by visual experience and associated with pathways implicated in OD plasticity. This resulted in a list of 32 candidate genes. The list contained unproven, but not unexpected candidates such as the genes for IGF-1, NCAM1, NOGO-A, the gamma2 subunit of the GABA(A) receptor, acetylcholine esterase, and the catalytic subunit of cAMP-dependent protein kinase A. This demonstrates the viability of our approach. More interestingly, the following novel candidate genes were identified: Akap7, Akt1, Camk2d, Cckbr, Cd44, Crim1, Ctdsp2, Dnajc5, Gnai1, Itpka, Mapk8, Nbea, Nfatc3, Nlk, Npy5r, Phf21a, Phip, Ppm1l, Ppp1r1b, Rbbp4, Slc1a3, Slit2, Socs2, Spock3, St8sia1, Zfp207. Whether all these novel candidates indeed function in OD plasticity remains to be established, but possible roles of some of them are discussed in the article.