Regulatory Subunit Of Protein Kinase A Research Articles

(007) TESTOSTERONE POSITIVELY REGULATES ADENOSINE-INDUCED RELAXATION IN THE VAGINA: AN EXPERIMENTAL STUDY IN RATS

Abstract Introduction It has been previously demonstrated that testosterone (T) has a pivotal role in controlling vaginal relaxation through the related molecular machinery, i.e., improving the nitric oxide (NO)/protein kinase G (PKG)/cyclic guanosine monophosphate (cGMP) vaginal signaling and maintaining the integrity of the muscular wall. Adenosine (ADO) is a purinergic neuromodulator that, in the male, shows a positive effect on penile erection by controlling smooth muscle relaxation and local blood flow increase. However, the role, if any, of T on the ADO-induced relaxation signaling is yet to be investigated. Objective The aim of this study was to evaluate the effect of sex steroids on ADO-mediated vaginal relaxation using a validated animal model of ovariectomized (OVX) and sex steroid supplemented Sprague-Dawley rats. Methods Subgroups of OVX rats were treated with 17β-estradiol (E2; 10 mg/kg/die), testosterone (T; 30 mg/kg/week), or testosterone and letrozole (T+L, 2.5 mg/kg/die) for 6 weeks. The experimental groups were compared with a group of intact rats. In vitro contractility studies were performed on noradrenaline (NA) pre-contracted distal vaginal strips, isolated from each experimental group, stimulated with increasing doses of ADO (1 μM - 1 mM), with or without NO-synthase inhibitor L-NAME (100 μM) pretreatment, or treated with ADORA2A selective agonist CGS21680 (10 Pm-100 μM). Messenger ribonucleic acid (mRNA) isolated from vaginal tissue was analyzed by semi-quantitative Reverse Transcriptase-Polymerase Chain Reaction. Results The results showed that OVX resulted in a decreased responsiveness to ADO, completely restored by T supplementation, with or without letrozole. In contrast, E2 did not affect the hyporesponsiveness to ADO in OVX rats. Likewise, ADO induced dose-dependent relaxation was significantly reduced by in vitro L-NAME (100 μM) only in the OVX animals treated with T or T+L. Accordingly, vagina expressed all ADO receptors (ADORA1, ADORA2A, ADORA2B, ADORA3) mRNAs, with pro-relaxant ADORA2A (ADO receptor coupled to adenylyl cyclase activation) being significantly modulated by hormonal treatments: OVX significantly reduced ADORA2A mRNA expression, an effect counteracted by either T or T+L administration, but not by E2. These data are supported by the observed T-induced upregulation of cyclic adenosine monophosphate (cAMP)-dependent genes, where T, but not E2, treatment upregulated the mRNA expression of the ubiquitous isoform of ADCY (ADCY4), protein kinase A (PKA) catalytic subunit b (PKAcb), PKA regulatory subunit 2b (PKAr2b) and cAMP responsive element modulator (CREM). Finally, as further demonstration of the functional activity of ADO signaling in distal vagina, stimulation by increasing doses of CGS21680, a selective agonist of ADORA2A receptor, enhanced the relaxant effect induced by T and T+L treatments but not by E2. Conclusions Our data demonstrated for the first time a novel ADO-mediated relaxant mechanism in vagina and suggested that T positively modulates the responsiveness to ADO stimulation, thus confirming this process as a major player in the relaxant mechanism regulation of muscle contractility in vagina. Disclosure No.

A divergent protein kinase A regulatory subunit essential for morphogenesis of the human pathogen Leishmania.

Parasitic protozoa of the genus Leishmania cycle between the phagolysosome of mammalian macrophages, where they reside as rounded intracellular amastigotes, and the midgut of female sand flies, which they colonize as elongated extracellular promastigotes. Previous studies indicated that protein kinase A (PKA) plays an important role in the initial steps of promastigote differentiation into amastigotes. Here, we describe a novel regulatory subunit of PKA (which we have named PKAR3) that is unique to Leishmania and most (but not all) other Kinetoplastidae. PKAR3 is localized to subpellicular microtubules (SPMT) in the cell cortex, where it recruits a specific catalytic subunit (PKAC3). Promastigotes of pkar3 or pkac3 null mutants lose their elongated shape and become rounded but remain flagellated. Truncation of an N-terminal formin homology (FH)-like domain of PKAR3 results in its detachment from the SPMT, also leading to rounded promastigotes. Thus, the tethering of PKAC3 via PKAR3 at the cell cortex is essential for maintenance of the elongated shape of promastigotes. This role of PKAR3 is reminiscent of PKARIβ and PKARIIβ binding to microtubules of mammalian neurons, which is essential for the elongation of dendrites and axons, respectively. Interestingly, PKAR3 binds nucleoside analogs, but not cAMP, with a high affinity similar to the PKAR1 isoform of Trypanosoma. We propose that these early-diverged protists have re-purposed PKA for a novel signaling pathway that spatiotemporally controls microtubule remodeling and cell shape.

14-3-3 interaction with phosphodiesterase 8A sustains PKA signaling and downregulates the MAPK pathway

The cAMP/PKA and mitogen-activated protein kinase (MAPK) signaling cascade control many cellular processes and are highly regulated for optimal cellular responses upon external stimuli. Phosphodiesterase 8A (PDE8A) is an important regulator that inhibits signaling via cAMP-dependent PKA by hydrolyzing intracellular cAMP pool. Conversely, PDE8A activates the MAPK pathway by protecting CRAF/Raf1 kinase from PKA-mediated inhibitory phosphorylation at Ser259 residue, a binding site of scaffold protein 14-3-3. It still remains enigmatic as to how the cross-talk involving PDE8A regulation influences cAMP/PKA and MAPK signaling pathways. Here, we report that PDE8A interacts with 14-3-3ζ in both yeast and mammalian system, and this interaction is enhanced upon the activation of PKA, which phosphorylates PDE8A's Ser359 residue. Biophysical characterization of phospho-Ser359 peptide with 14-3-3ζ protein further supports their interaction. Strikingly, 14-3-3ζ reduces the catalytic activity of PDE8A, which upregulates the cAMP/PKA pathway while the MAPK pathway is downregulated. Moreover, 14-3-3ζ in complex with PDE8A and cAMP-bound regulatory subunit of PKA, RIα, delays the deactivation of PKA signaling. Our results define 14-3-3ζ as a molecular switch that operates signaling between cAMP/PKA and MAPK by associating with PDE8A.

Neuronal ER-plasma membrane junctions couple excitation to Ca2+-activated PKA signaling

Junctions between the endoplasmic reticulum (ER) and the plasma membrane (PM) are specialized membrane contacts ubiquitous in eukaryotic cells. Concentration of intracellular signaling machinery near ER-PM junctions allows these domains to serve critical roles in lipid and Ca2+ signaling and homeostasis. Subcellular compartmentalization of protein kinase A (PKA) signaling also regulates essential cellular functions, however, no specific association between PKA and ER-PM junctional domains is known. Here, we show that in brain neurons type I PKA is directed to Kv2.1 channel-dependent ER-PM junctional domains via SPHKAP, a type I PKA-specific anchoring protein. SPHKAP association with type I PKA regulatory subunit RI and ER-resident VAP proteins results in the concentration of type I PKA between stacked ER cisternae associated with ER-PM junctions. This ER-associated PKA signalosome enables reciprocal regulation between PKA and Ca2+ signaling machinery to support Ca2+ influx and excitation-transcription coupling. These data reveal that neuronal ER-PM junctions support a receptor-independent form of PKA signaling driven by membrane depolarization and intracellular Ca2+, allowing conversion of information encoded in electrical signals into biochemical changes universally recognized throughout the cell.

PKA regulatory subunit Bcy1 couples growth, lipid metabolism, and fermentation during anaerobic xylose growth in Saccharomyces cerevisiae.

Organisms have evolved elaborate physiological pathways that regulate growth, proliferation, metabolism, and stress response. These pathways must be properly coordinated to elicit the appropriate response to an ever-changing environment. While individual pathways have been well studied in a variety of model systems, there remains much to uncover about how pathways are integrated to produce systemic changes in a cell, especially in dynamic conditions. We previously showed that deletion of Protein Kinase A (PKA) regulatory subunit BCY1 can decouple growth and metabolism in Saccharomyces cerevisiae engineered for anaerobic xylose fermentation, allowing for robust fermentation in the absence of division. This provides an opportunity to understand how PKA signaling normally coordinates these processes. Here, we integrated transcriptomic, lipidomic, and phospho-proteomic responses upon a glucose to xylose shift across a series of strains with different genetic mutations promoting either coupled or decoupled xylose-dependent growth and metabolism. Together, results suggested that defects in lipid homeostasis limit growth in the bcy1Δ strain despite robust metabolism. To further understand this mechanism, we performed adaptive laboratory evolutions to re-evolve coupled growth and metabolism in the bcy1Δ parental strain. The evolved strain harbored mutations in PKA subunit TPK1 and lipid regulator OPI1, among other genes, and evolved changes in lipid profiles and gene expression. Deletion of the evolved opi1 gene partially reverted the strain's phenotype to the bcy1Δ parent, with reduced growth and robust xylose fermentation. We suggest several models for how cells coordinate growth, metabolism, and other responses in budding yeast and how restructuring these processes enables anaerobic xylose utilization.

Upregulated TGF-β1 contributes to hyperglycaemia in type 2 diabetes by potentiating glucagon signalling.

Glucagon-stimulated hepatic gluconeogenesis contributes to endogenous glucose production during fasting. Recent studies suggest that TGF-β is able to promote hepatic gluconeogenesis in mice. However, the physiological relevance of serum TGF-β levels to human glucose metabolism and the mechanism by which TGF-β enhances gluconeogenesis remain largely unknown. As enhanced gluconeogenesis is a signature feature of type 2 diabetes, elucidating the molecular mechanisms underlying TGF-β-promoted hepatic gluconeogenesis would allow us to better understand the process of normal glucose production and the pathophysiology of this process in type 2 diabetes. This study aimed to investigate the contribution of upregulated TGF-β1 in human type 2 diabetes and the molecular mechanism underlying the action of TGF-β1 in glucose metabolism. Serum levels of TGF-β1 were measured by ELISA in 74 control participants with normal glucose tolerance and 75 participants with type 2 diabetes. Human liver tissue was collected from participants without obesity and with or without type 2 diabetes for the measurement of TGF-β1 and glucagon signalling. To investigate the role of Smad3, a key signalling molecule downstream of the TGF-β1 receptor, in mediating the effect of TGF-β1 on glucagon signalling, we generated Smad3 knockout mice. Glucose levels in Smad3 knockout mice were measured during prolonged fasting and a glucagon tolerance test. Mouse primary hepatocytes were isolated from Smad3 knockout and wild-type (WT) mice to investigate the underlying molecular mechanisms. Smad3 phosphorylation was detected by western blotting, levels of cAMP were detected by ELISA and levels of protein kinase A (PKA)/cAMP response element-binding protein (CREB) phosphorylation were detected by western blotting. The dissociation of PKA subunits was measured by immunoprecipitation. We observed higher levels of serum TGF-β1 in participants without obesity and with type 2 diabetes than in healthy control participants, which was positively correlated with HbA1c and fasting blood glucose levels. In addition, hyperactivation of the CREB and Smad3 signalling pathways was observed in the liver of participants with type 2 diabetes. Treating WT mouse primary hepatocytes with TGF-β1 greatly potentiated glucagon-stimulated PKA/CREB phosphorylation and hepatic gluconeogenesis. Mechanistically, TGF-β1 treatment induced the binding of Smad3 to the regulatory subunit of PKA (PKA-R), which prevented the association of PKA-R with the catalytic subunit of PKA (PKA-C) and led to the potentiation of glucagon-stimulated PKA signalling and gluconeogenesis. The hepatic TGF-β1/Smad3 pathway sensitises the effect of glucagon/PKA signalling on gluconeogenesis and synergistically promotes hepatic glucose production. Reducing serum levels of TGF-β1 and/or preventing hyperactivation of TGF-β1 signalling could be a novel approach for alleviating hyperglycaemia in type 2 diabetes.

5-adenosine monophosphate promotes signal adaptation in PKA signaling.

Protein Kinase A (PKA) plays a central role in several cellular processes and its activity is tightly regulated by 2nd messenger 3,5-cyclic adenosine monophosphate (cAMP)-signaling. While the role of cAMP in PKA activation is well established, the significance of its hydrolysis end-product, 5-adenosine monophosphate (AMP) is poorly understood. AMP is generated upon hydrolysis of cAMP bound to the PKA regulatory subunits (RIα) catalyzed by phosphodiesterases. All components of the cAMP-PKA signaling pathway that include PDEs are compartmentalized into signalosomal units. Our research has shown that PDEs in proximity with PKA RIα in signalosomes lead to formation of a signal termination complex of PDEs and RIα, that facilitates cAMP substrate channeling from the PKA binding site to the PDE catalytic site. In contrast to the long held view that AMP diffused away from the PKA signalosome following cAMP hydrolysis, AMP is a regulatory end-product of each round of PKA activation and termination. Here we report high resolution structures of the AMP bound conformations of RIα and PDE8A catalytic domain (PDE8c) by cryo-EM. We have further captured AMP-bound end-state signal termination complex of PDE8c with RIα, by orthogonal electron microscopy screening, amide hydrogen/deuterium exchange mass spectrometry. Our results describe an undiscovered active role of AMP in signal termination and adaptation in PKA signaling.

Oncogenic PKA signaling increases c-MYC protein expression through multiple targetable mechanisms.

Genetic alterations that activate protein kinase A (PKA) are found in many tumor types. Yet, their downstream oncogenic signaling mechanisms are poorly understood. We used global phosphoproteomics and kinase activity profiling to map conserved signaling outputs driven by a range of genetic changes that activate PKA in human cancer. Two signaling networks were identified downstream of PKA: RAS/MAPK components and an Aurora Kinase A (AURKA)/glycogen synthase kinase (GSK3) sub-network with activity toward MYC oncoproteins. Findings were validated in two PKA-dependent cancer models: a novel, patient-derived fibrolamellar carcinoma (FLC) line that expresses a DNAJ-PKAc fusion and a PKA-addicted melanoma model with a mutant type I PKA regulatory subunit. We identify PKA signals that can influence both de novo translation and stability of the proto-oncogene c-MYC. However, the primary mechanism of PKA effects on MYC in our cell models was translation and could be blocked with the eIF4A inhibitor zotatifin. This compound dramatically reduced c-MYC expression and inhibited FLC cell line growth in vitro. Thus, targeting PKA effects on translation is a potential treatment strategy for FLC and other PKA-driven cancers.

Analysis of A-kinase Anchoring Protein 13 (AKAP13) in Peri-Ovulatory Oocyte Maturation [A328

INTRODUCTION: Arrest of oocytes at prophase I of meiosis is crucial for fertility and is maintained by Protein Kinase A (PKA). PKA activity is regulated by A-kinase anchoring proteins (AKAPs) that bind the regulatory subunit of PKA to control enzymatic activity. AKAP13 is expressed in oocytes and granulosa cells of humans and mice. Female mice haploinsufficient for Akap13 showed reduced litter sizes. To determine if AKAP13 in oocytes contributes to female reproductive function, we used an oocyte-specific conditional knockout (cKO) to delete Akap13 in the final stages of oocyte growth. METHODS: Oocytes were collected from mice harboring an oocyte-specific conditional knockout of Akap13 mice, with Cre recombinase expression driven by the Zp3 promoter (Zp3-Cre+, Akap13flox/flox). Akap13 DNA and mRNA were assessed by polymerase chain reaction (PCR) and quantitative PCR (qPCR), respectively. Controls were oocytes harvested from Akap13+/flox mice lacking Cre. Oocytes from control or cKO mice were examined for morphology, meiotic resumption, spindle organization, and spindle positioning at metaphase II. RESULTS: Cre-mediated excision of Akap13 DNA in oocytes was confirmed using PCR. Oocyte mRNA levels of Akap13 in cKO mice were reduced by 90% compared to controls. Live-cell imaging revealed no differences in germinal vessel breakdown, polar body emission, or completion of meiosis I in cKO oocytes as compared to controls. Staining of fixed oocytes revealed that metaphase II spindles and chromosome alignment appeared normal in in vitro-matured cKO oocytes. CONCLUSION: These studies suggest that impairment of female fertility in Akap13 haploinsufficient mice is not caused by impairment of oocyte meiotic maturation.

Using Optical Tweezers to Monitor Allosteric Signals Through Changes in Folding Energy Landscapes.

Signaling proteins are composed of conserved protein interaction domains that serve as allosteric regulatory elements of enzymatic or binding activities. The ubiquitous, structurally conserved cyclic nucleotide binding (CNB) domain is found covalently linked to proteins with diverse folds that perform multiple biological functions. Given that the structures of cAMP-bound CNB domains in different proteins are very similar, it remains a challenge to determine how this domain allosterically regulates such diverse protein functions and folds. Instead of a structural perspective, we focus our attention on the energy landscapes underlying the CNB domains and their responses to cAMP binding. We show that optical tweezers is an ideal tool to investigate how cAMP binding coupled to interdomain interactions remodel the energy landscape of the regulatory subunit of protein kinase A (PKA), which harbors two CNB domains. We mechanically manipulate and probe the unfolding and refolding behavior of the CNB domains as isolated structures or selectively as part of the PKA regulatory subunit, and in the presence and absence of cAMP. Optical tweezers allows us to dissect the changes in the energy landscape associated with cAMP binding, and to examine the allosteric interdomain interactions triggered by the cyclic nucleotide. This single molecule approach can be used to study other modular, multidomain signaling proteins found in nature.

Protein Kinase A in Human Retina: Differential Localization of Cβ, Cα, RIIα, and RIIβ in Photoreceptors Highlights Non-redundancy of Protein Kinase A Subunits.

Protein kinase A (PKA) signaling is essential for numerous processes but the subcellular localization of specific PKA regulatory (R) and catalytic (C) subunits has yet to be explored comprehensively. Additionally, the localization of the Cβ subunit has never been spatially mapped in any tissue even though ∼50% of PKA signaling in neuronal tissues is thought to be mediated by Cβ. Here we used human retina with its highly specialized neurons as a window into PKA signaling in the brain and characterized localization of PKA Cα, Cβ, RIIα, and RIIβ subunits. We found that each subunit presented a distinct localization pattern. Cα and Cβ were localized in all cell layers (photoreceptors, interneurons, retinal ganglion cells), while RIIα and RIIβ were selectively enriched in photoreceptor cells where both showed distinct patterns of co-localization with Cα but not Cβ. Only Cα was observed in photoreceptor outer segments and at the base of the connecting cilium. Cβ in turn, was highly enriched in mitochondria and was especially prominent in the ellipsoid of cone cells. Further investigation of Cβ using RNA BaseScope technology showed that two Cβ splice variants (Cβ4 and Cβ4ab) likely code for the mitochondrial Cβ proteins. Overall, our data indicates that PKA Cα, Cβ, RIIα, and RIIβ subunits are differentially localized and are likely functionally non-redundant in the human retina. Furthermore, Cβ is potentially important for mitochondrial-associated neurodegenerative diseases previously linked to PKA dysfunction.

AKAP79 enables calcineurin to directly suppress protein kinase A activity.

Interplay between the second messengers cAMP and Ca2+ is a hallmark of dynamic cellular processes. A common motif is the opposition of the Ca2+-sensitive phosphatase calcineurin and the major cAMP receptor, protein kinase A (PKA). Calcineurin dephosphorylates sites primed by PKA to bring about changes including synaptic long-term depression (LTD). AKAP79 supports signaling of this type by anchoring PKA and calcineurin in tandem. In this study, we discovered that AKAP79 increases the rate of calcineurin dephosphorylation of type II PKA regulatory subunits by an order of magnitude. Fluorescent PKA activity reporter assays, supported by kinetic modeling, show how AKAP79-enhanced calcineurin activity enables suppression of PKA without altering cAMP levels by increasing PKA catalytic subunit capture rate. Experiments with hippocampal neurons indicate that this mechanism contributes toward LTD. This non-canonical mode of PKA regulation may underlie many other cellular processes.

PRKAR2A deficiency protects mice from experimental colitis by increasing IFN-stimulated gene expression and modulating the intestinal microbiota

Protein kinase A (PKA) plays an important role in regulating inflammation via its catalytic subunits. Recently, PKA regulatory subunits have been reported to directly modulate some signaling pathways and alleviate inflammation. However, the role of PKA regulatory subunits in colonic inflammation remains unclear. Therefore, we conducted this study to investigate the role of the PKA regulatory subunit PRKAR2A in colitis. We observed that PRKAR2A deficiency protected mice from dextran sulfate sodium (DSS)-induced experimental colitis. Our experiments revealed that the intestinal epithelial cell-specific deletion of Prkar2a contributed to this protection. Mechanistically, the loss of PRKAR2A in Prkar2a−/− mice resulted in an increased IFN-stimulated gene (ISG) expression and altered gut microbiota. Inhibition of ISGs partially reversed the protective effects against DSS-induced colitis in Prkar2a−/− mice. Antibiotic treatment and cross-fostering experiments demonstrated that the protection against DSS-induced colitis in Prkar2a−/− mice was largely dependent on the gut microflora. Altogether, our work demonstrates a previously unidentified function of PRKAR2A in promoting DSS-induced colitis.

Mitochondrial A Kinase Anchoring Proteins in Cylic AMP compartmentation

Cyclic AMP is the basis of sympathetic control of cardiac contractility. Upon activation of the beta-adrenergic receptors numerous proteins in the excitation-contraction machinery are phosphorylated by the cAMP effector protein kinase A (PKA). In addition to this, cAMP is also utilized by other systems to modulate gene transcription, apoptosis, and cell survival. To preserve the fidelity of receptor-specific responses, precise spatial and temporal control of cAMP occurs and this has been experimentally observed. This phenomenon of cAMP compartmentation relies on various factors limiting cAMP mobility. One of these involves the buffering effect of PKA. Binding and unbinding to the regulatory subunits of PKA slow cAMP as it attempts to move throughout the cytoplasm. Previous work indicates that regulatory subunits anchored to the outer membrane of mitochondria by A-kinase anchoring proteins (AKAPs) play a particularly important role in this process. At least seven different mitochondrial AKAPs have been identified in various cell types. These include AKAPs capable of interacting with type I and/or type II PKA regulatory subunits. The purpose of this study was to determine which of those AKAPs are associated with mitochondria in adult rat ventricular myocytes. Quantitative PCR detected evidence for expression of OPA1, D‑AKAP1, D‑AKAP2, and ACBD3, while message levels for Rab32, SKIP, and WAVE1 were barely measurable. Western blot analysis determined that D‑AKAP1, D‑AKAP2, ACBD3 are expressed in mitochondrial fractions of these cells, along with type I and type II PKA regulatory subunits. The mitochondrial association of these proteins was confirmed by immunocytochemistry. Our results also show low expression levels of OPA1 and Rab32 in the mitochondrial fraction. These results suggest that D-AKAP1, D‑AKAP2, and ACBD3 are involved in anchoring type I and type II PKA regulatory subunits to mitochondria in adult ventricular myocytes. In addition to playing a role in PKA-dependent regulation of mitochondrial function, they may play an essential role in cAMP compartmentation by buffering its movement throughout the cell.

Role of the PKA regulatory subunit RIα in the contractile tone of the aorta

Protein kinase A (PKA) is a key enzyme activated by binding of cAMP. PKA induces smooth muscle relaxation and vasodilatation. The PKA-RIα knock-in heterozygous mice, [R368X]/[+] (KI), leading to the generation of a truncated RIα and the repression of PKA activation (Le Stunff et al., 2017, J Bone Miner Res ) may help decipher the contribution of PKA-RIα in vascular system which has remained elusive. Our aim is to document the expression of PKA-RIα in mouse aorta and to decipher its role in arterial tone by using wild-type (WT) and KI mice. Aortas were harvested from adult male WT and KI mice. PKA subunits expressions were studied by immunoblot. Vascular reactivity was tested by mounting the aorta on a wire-myograph. The internal circumference (IC) was recorded. Concentration-contractile responses were performed for K + (20–120 mM), U46619 (1 nM–3 μM) and phenylephrine (PE, 1 nM–3 μM). Vasorelaxant responses to acetylcholine (ACh, 1 nM–10 μM) and to L858051 (adenylate cyclase activator, 10 nM–30 μM) were studied in aorta contracted with PE. All groups included 5–6 mice. In WT aorta, the anti-PKA-RIα antibody yielded a signal at 49 kDa, as expected. In KI mice, mutant allele expression was confirmed by an additional signal at 47 kDa. RIIα and Cα expressions were similar in both strains. IC was 20% smaller in KI mice than in WT. The contractile response to K + and PE were similar in both groups, whereas the response to U46619 was stronger in mutant aorta with a higher E max . ACh and L858051 relaxed aortas similarly in both groups. These preliminary data show that PKA-RIα is expressed in mouse aorta. The smaller IC of aorta in [R368X]/[+] mice is consistent with their smaller size (Le Stunff et al.). The stronger constriction to U46619 in mutant mice may be explained by reduced PKA activity compared to WT. The similar relaxation to L858051 in mutant mice maybe due to redundancy of PKA-RIα in mouse aorta.

Role of A-kinase anchoring proteins in cyclic-AMP-mediated Schwann cell proliferation

Proliferation of Schwann cells during peripheral nerve development is stimulated by the heregulin/neuregulin family of growth factors expressed by neurons. However, for neonatal rat Schwann cells growing in culture, heregulins produce only a weak mitogenic response. Supplementing heregulin with forskolin, an agent that elevates cyclic AMP levels, produces a dramatic increase in the proliferation of cultured Schwann cells. The mechanisms underlying this synergistic effect required for Schwann cell proliferation in vivo is not well established. Characterizing the A-kinase anchoring proteins (AKAPs) in Schwann cells might help identify substrates tethered to and phosphorylated by the cAMP-dependent protein kinase A (PKA). Using an RII overlay assay that detects AKAPs that are bound to the type II regulatory subunits of PKA, we identified AKAP150 in Schwann cells. Western blot analysis revealed that additional AKAPs, specifically AKAP95, and yotiao were also present. Disruption of PKA/AKAP interaction with Ht-31 peptide resulted in an increase in luciferase-conjugated cyclin D3 promoter activity. Transfection with sequence-specific AKAP siRNAs for AKAP150 and AKAP95 produced a marked reduction in cell proliferation. Immunoblot analysis revealed that knock down of AKAP95 protein caused a significant decrease in expression of the cell cycle regulatory proteins cyclin D2, cyclin D3 and the cell survival signal Akt/Protein Kinase B (Akt/PKB). Morphological characterization of Schwann cell AKAPs indicated the presence of nuclear (AKAP95), cytoplasm-associated (AKAP150) and perinuclear (yotiao) A-kinase anchoring proteins. These results indicate a role for AKAP95 and AKAP150 in the synergistic response of Schwann cells to treatment with heregulin and forskolin.

Nutrient sensing and cAMP signaling in yeast: G-protein coupled receptor versus transceptor activation of PKA.

A major signal transduction pathway regulating cell growth and many associated physiological properties as a function of nutrient availability in the yeast Saccharomyces cerevisiae is the protein kinase A (PKA) pathway. Glucose activation of PKA is mediated by G-protein coupled receptor (GPCR) Gpr1, and secondary messenger cAMP. Other nutrients, including nitrogen, phosphate and sulfate, activate PKA in accordingly-starved cells through nutrient transceptors, but apparently without cAMP signaling. We have now used an optimized EPAC-based fluorescence resonance energy transfer (FRET) sensor to precisely monitor in vivo cAMP levels after nutrient addition. We show that GPCR-mediated glucose activation of PKA is correlated with a rapid transient increase in the cAMP level in vivo, whereas nutrient transceptor-mediated activation by nitrogen, phosphate or sulfate, is not associated with any significant increase in cAMP in vivo. We also demonstrate direct physical interaction between the Gap1 amino acid transceptor and the catalytic subunits of PKA, Tpk1, 2 and 3. In addition, we reveal a conserved consensus motif in the nutrient transceptors that is also present in Bcy1, the regulatory subunit of PKA. This suggests that nutrient transceptor activation of PKA may be mediated by direct release of bound PKA catalytic subunits, triggered by the conformational changes occurring during transport of the substrate by the transceptor. Our results support a model in which nutrient transceptors are evolutionary ancestors of GPCRs, employing a more primitive direct signaling mechanism compared to the indirect cAMP second-messenger signaling mechanism used by GPCRs for activation of PKA.

Exercise Training-Enhanced Lipolytic Potency to Catecholamine Depends on the Time of the Day

Exercise training is well known to enhance adipocyte lipolysis in response to hormone challenge. However, the existence of a relationship between the timing of exercise training and its effect on adipocyte lipolysis is unknown. To clarify this issue, Wistar rats were run on a treadmill for 9 weeks in either the early part (E-EX) or late part of the active phase (L-EX). L-EX rats exhibited greater isoproterenol-stimulated lipolysis expressed as fold induction over basal lipolysis, with greater protein expression levels of hormone-sensitive lipase (HSL) phosphorylated at Ser 660 compared to E-EX rats. Furthermore, we discovered that Brain and muscle Arnt-like (BMAL)1 protein can associate directly with several protein kinase A (PKA) regulatory units (RIα, RIβ, and RIIβ) of protein kinase, its anchoring protein (AKAP)150, and HSL, and that the association of BMAL1 with the regulatory subunits of PKA, AKAP150, and HSL was greater in L-EX than in E-EX rats. In contrast, comparison between E-EX and their counterpart sedentary control rats showed a greater co-immunoprecipitation only between BMAL1 and ATGL. Thus, both E-EX and L-EX showed an enhanced lipolytic response to isoproterenol, but the mechanisms underlying exercise training-enhanced lipolytic response to isoproterenol were different in each group.

Abstract MP166: PRKAR1A Deficiency Abrogates Cardiac Hypertrophy Through Inhibition of Mitochondrial Fission

Protein kinase A (PKA) is pivotal for cardiac function of human heart, and its dysregulation is involved with various cardiac pathologies. PKA regulatory subunit 1α (R1α, encoded by PRKAR1A gene) controls PKA kinase activity by sequestering the PKA catalytic subunits. Patients with PRKAR1A mutations are often diagnosed with Carney complex (CNC) and may die prematurely from cardiac complications such as heart failure. However, it remains unknown whether PRKAR1A deficiency interferes with normal heart growth during postnatal development. Here, we show that left ventricular mass is reduced in young CNC patients with PRKAR1A mutations or deletions. To investigate the impact of PRKAR1A deficiency on heart growth, we generated cardiac-specific PRKAR1A heterozygous knockout mice. Ablation of the PRKAR1A gene in mice increased cardiac PKA activity, reduced heart weight to body weight ratio and cardiomyocyte size without altering contractile function. Cardiomyocyte hypertrophy in response to activation of the α1-adrenergic receptor, was completely abolished by silencing of PRKAR1A . Mechanistically, depletion of PRKAR1A provoked PKA-dependent phosphorylation of the mitochondrial fission protein Drp1 at S637, resulting in impaired mitochondrial fission and diminished cardiomyocyte hypertrophy. In conclusion, PRKAR1A deficiency abrogates cardiac hypertrophy during postnatal development, likely through inhibiting Drp1-mediated mitochondrial fission. Our study provides novel mechanistic insights regarding the cardiac mortality associated with CNC.

A-kinase anchoring protein 8L interacts with mTORC1 and promotes cell growth

mTOR complex 1 (mTORC1) senses nutrients to mediate anabolic processes within the cell. Exactly how mTORC1 promotes cell growth remains unclear. Here, we identified a novel mTORC1-interacting protein called protein kinase A anchoring protein 8L (AKAP8L). Using biochemical assays, we found that the N-terminal region of AKAP8L binds to mTORC1 in the cytoplasm. Importantly, loss of AKAP8L decreased mTORC1-mediated processes such as translation, cell growth, and cell proliferation. AKAPs anchor protein kinase A (PKA) through PKA regulatory subunits, and we show that AKAP8L can anchor PKA through regulatory subunit Iα. Reintroducing full-length AKAP8L into cells restored mTORC1-regulated processes, whereas reintroduction of AKAP8L missing the N-terminal region that confers the interaction with mTORC1 did not. Our results suggest a multifaceted role for AKAPs in the cell. We conclude that mTORC1 appears to regulate cell growth, perhaps in part through AKAP8L.