Birgitte Lygren

AKAP complex regulates Ca2+ re‐uptake into heart sarcoplasmic reticulum

The β‐adrenergic receptor/cyclic AMP/protein kinase A (PKA) signalling pathway regulates heart rate and contractility. Here, we identified a supramolecular complex consisting of the sarcoplasmic reticulum Ca2+‐ATPase (SERCA2), its negative regulator phospholamban (PLN), the A‐kinase anchoring protein AKAP18δ and PKA. We show that AKAP18δ acts as a scaffold that coordinates PKA phosphorylation of PLN and the adrenergic effect on Ca2+ re‐uptake. Inhibition of the compartmentalization of this cAMP signalling complex by specific molecular disruptors interferes with the phosphorylation of PLN. This prevents the subsequent release of PLN from SERCA2, thereby affecting the Ca2+ re‐uptake into the sarcoplasmic reticulum induced by adrenergic stimuli.

An entirely specific type I A-kinase anchoring protein that can sequester two molecules of protein kinase A at mitochondria

A-kinase anchoring proteins (AKAPs) tether the cAMP-dependent protein kinase (PKA) to intracellular sites where they preferentially phosphorylate target substrates. Most AKAPs exhibit nanomolar affinity for the regulatory (RII) subunit of the type II PKA holoenzyme, whereas dual-specificity anchoring proteins also bind the type I (RI) regulatory subunit of PKA with 10–100-fold lower affinity. A range of cellular, biochemical, biophysical, and genetic approaches comprehensively establish that sphingosine kinase interacting protein (SKIP) is a truly type I-specific AKAP. Mapping studies located anchoring sites between residues 925–949 and 1,140–1,175 of SKIP that bind RI with dissociation constants of 73 and 774 nM, respectively. Molecular modeling and site-directed mutagenesis approaches identify Phe 929 and Tyr 1,151 as RI-selective binding determinants in each anchoring site. SKIP complexes exist in different states of RI-occupancy as single-molecule pull-down photobleaching experiments show that 41 ± 10% of SKIP sequesters two YFP-RI dimers, whereas 59 ± 10% of the anchoring protein binds a single YFP-RI dimer. Imaging, proteomic analysis, and subcellular fractionation experiments reveal that SKIP is enriched at the inner mitochondrial membrane where it associates with a prominent PKA substrate, the coiled-coil helix protein ChChd3.

Dual Specificity A-kinase Anchoring Proteins (AKAPs) Contain an Additional Binding Region That Enhances Targeting of Protein Kinase A Type I

A-kinase anchoring proteins (AKAPs) target protein kinase A (PKA) to a variety of subcellular locations. Conventional AKAPs contain a 14-18-amino acid sequence that forms an amphipathic helix that binds with high affinity to the regulatory (R) subunit of PKA type II. More recently, a group of dual specificity AKAPs has been classified on the basis of their ability to bind the PKA type I and the PKA type II isozymes. In this study we show that dual specificity AKAPs contain an additional PKA binding determinant called the RI Specifier Region (RISR). A variety of protein interaction assays and immunoprecipitation and immunolocalization experiments indicates that the RISR augments RI binding in vitro and inside cells. Cellular delivery of the RISR peptide uncouples RI anchoring to Ezrin leading to release of T cell inhibition by cAMP. Likewise, expression of mutant Ezrin forms where RI binding has been abrogated by substitution of the RISR sequence prevents cAMP-mediated inhibition of T cell function. Thus, we propose that the RISR acts in synergy with the amphipathic helix in dual specificity anchoring proteins to enhance anchoring of PKA type I.

Positional cloning identifies a novel cyclophilin as a candidate amplified oncogene in 1q21

Gains of 1q21-q23 have been associated with metastasis and chemotherapy response, particularly in bladder cancer, hepatocellular carcinomas and sarcomas. By positional cloning of amplified genes by yeast artificial chromosome-mediated cDNA capture using magnetic beads, we have identified three candidate genes (COAS1, -2 and -3) in the amplified region in sarcomas. COAS1 and -2 showed higher amplification levels than COAS3. Most notably, amplification was very common in osteosarcomas, where in particular COAS2 was highly expressed. COAS1 has multiple repeats and shows no homology to previously described genes, whereas COAS2 is a novel member of the cyclosporin-binding peptidyl-prolyl isomerase family, very similar to cyclophilin A. COAS2 was overexpressed almost exclusively in aggressive metastatic or chemotherapy resistant tumours. Although COAS2 was generally more amplified than COAS1, it was not expressed in well-differentiated liposarcomas, where amplification of this region is very common. All three genes were found to be amplified and over-expressed also in breast carcinomas. The complex nature of the 1q21-23 amplicons and close proximity of the genes make unequivocal determination of the gene responsible difficult. Quite likely, the different genes may give selective advantages to different subsets of tumours.

A PKA-ezrin-Cx43 signaling complex controls gap junction communication and thereby trophoblast cell fusion.

ABSTRACT Cell fusion occurs as part of the differentiation of some cell types, including myotubes in muscle and osteoclasts in remodeling bone. In the human placenta, mononuclear cytotrophoblasts in a human chorionic gonadotropin (hCG)-driven process fuse to form multinucleated syncytia that allow the exchange of nutrients and gases between the maternal and fetal circulation. Experiments in which protein kinase A (PKA) is displaced from A-kinase anchoring proteins (AKAPs), or in which specific AKAPs are depleted by siRNA-mediated knockdown, point to ezrin as a scaffold required for hCG-, cAMP- and PKA-mediated regulation of the fusion process. By a variety of immunoprecipitation and immunolocalization experiments, we show that ezrin directs PKA to a molecular complex of connexin 43 (Cx43, also known as GJA1) and zona occludens-1 (ZO-1, also known as TJP1). A combination of knockdown experiments and reconstitution with ezrin or Cx43 with or without the ability to bind to its interaction partner or to PKA demonstrate that ezrin-mediated coordination of the localization of PKA and Cx43 is necessary for discrete control of Cx43 phosphorylation and hCG-stimulated gap junction communication that triggers cell fusion in cytotrophoblasts.

The adaptor protein EBP50 is important for localization of the protein kinase A-Ezrin complex in T-cells and the immunomodulating effect of cAMP.

We recently reported that the dual-specificity AKAP (A-kinaseanchoring protein) Ezrin targets type I PKA (protein kinase A) to the vicinity of the TCR (T-cell receptor) in T-cells and, together with PAG (phosphoprotein associated with glycosphingolipid-enriched membrane microdomains) and EBP50 [ERM (Ezrin/Radixin/Moesin)-binding phosphoprotein 50], forms a scaffold that positions PKA close to its substrate, Csk (C-terminal Src kinase). This complex is important for controlling the activation state of T-cells. Ezrin binds the adaptor protein EBP50, which again contacts PAG. In the present study, we show that Ezrin and EBP50 interact with high affinity (KD=58+/-7 nM). A peptide corresponding to the EB (Ezrin-binding) region in EBP50 (EBP50pep) was used to further characterize the binding kinetics and compete the Ezrin-EBP50 interaction by various methods in vitro. Importantly, loading T-cells with EBP50pep delocalized Ezrin, but not EBP50. Furthermore, disruption of this complex interfered with cAMP modulation of T-cell activation, which is seen as a reversal of cAMP-mediated inhibition of IL-2 (interleukin 2) production, demonstrating an important role of EBP50 in this complex. In summary, both the biochemical and functional data indicate that targeting the Ezrin-EBP interaction could be a novel and potent strategy for immunomodulation.

Diastolic dysfunction in alveolar hypoxia: a role for interleukin-18-mediated increase in protein phosphatase 2A

AIMS Chronic obstructive pulmonary disease with alveolar hypoxia is associated with diastolic dysfunction in the right and left ventricle (LV). LV diastolic dysfunction is not caused by increased afterload, and we recently showed that reduced phosphorylation of phospholamban at serine (Ser) 16 may explain the reduced relaxation of the myocardium. Here, we study the mechanisms leading to the hypoxia-induced reduction in phosphorylation of phospholamban at Ser16. METHODS AND RESULTS In C57Bl/6j mice exposed to 10% oxygen, signalling molecules were measured in cardiac tissue, sarcoplasmic reticulum (SR)-enriched membrane preparations, and serum. Cardiomyocytes isolated from neonatal mice were exposed to interleukin (IL)-18 for 24 h. The beta-adrenergic pathway in the myocardium was not altered by alveolar hypoxia, as assessed by measurements of beta-adrenergic receptor levels, adenylyl cyclase activity, and subunits of cyclic AMP-dependent protein kinase. However, alveolar hypoxia led to a significantly higher amount (124%) and activity (234%) of protein phosphatase (PP) 2A in SR-enriched membrane preparations from LV compared with control. Serum levels of an array of cytokines were assayed, and a pronounced increase in IL-18 was observed. In isolated cardiomyocytes, treatment with IL-18 increased the amount and activity of PP2A, and reduced phosphorylation of phospholamban at Ser16 to 54% of control. CONCLUSION Our results indicate that the diastolic dysfunction observed in alveolar hypoxia might be caused by increased circulating IL-18, thereby inducing an increase in PP2A and a reduction in phosphorylation of phospholamban at Ser16.

Design of proteolytically stable RI-anchoring disruptor peptidomimetics for in vivo studies of anchored type I protein kinase A-mediated signalling.

We have reported previously the design of a RIAD (RI-anchoring disruptor) peptide that specifically displaces PKA (protein kinase A) type I from the AKAP (A-kinase-anchoring protein) ezrin, which is present in the immunological synapse of T-cells. This increases immune reactivity by reducing the threshold for activation and may prove a feasible approach for improving immune function in patients with cAMP-mediated T-cell dysfunction. However, the use of RIAD in biological systems is restricted by its susceptibility to enzymatic cleavage and, consequently, its short half-life in presence of the ubiquitous serum peptidases. In the present study, carefully selected non-natural amino acids were employed in the design of RIAD analogues with improved stability. The resulting peptidomimetics demonstrated up to 50-fold increased half-lives in serum compared with RIAD, while maintaining similar or improved specificity and potency with respect to disruption of PKA type I-AKAP interactions.

The potential use of AKAP18δ as a drug target in heart failure patients

Background: The adrenaline–β-adrenoreceptor–cAMP–protein kinase A signaling pathway regulates heart rate and contractility. Although changes in contractility are associated with cardiovascular disease, surprisingly few drugs are available that modulate the cardiac myocyte cAMP system. β-blocking agents reduce cAMP levels only by 50%. Objective: Compounds that interfere with the pathway at other levels are wanted as they may provide new tools for treatment of heart failure used alone or together with β-blockers and may make therapy more potent and/or more targeted and avoid side effects. Methods: Original findings and strategies for targeting protein–protein interactions are reviewed. Results/conclusion: We have shown that A-kinase anchoring protein (AKAP)18δ is important for organizing the molecular machinery that mediates adrenergic control of calcium reabsorption into sarcoplasmic reticulum. Calcium reabsorption is essential for relaxation and filling of the heart and is the rate-limiting step for making the heart beat faster in response to adrenaline or noradrenaline. We conclude that targeting AKAP18δ may have application in manipulating calcium reabsorption and protecting the heart from adrenergic pacing at the level of specific signaling events in heart failure patients.

Both El Tor and classical cholera toxin bind blood group determinants

Cholera is a disease which shows a clear blood group profile, with blood group O individuals experiencing the most severe symptoms. For a long time, the cholera toxin has been suspected to be the main culprit of this blood group dependence. Here, we show that both El Tor and classical cholera toxin B-pentamers do indeed bind blood group determinants (with equal affinities), using Surface Plasmon Resonance and NMR spectroscopy. Together with previous structural data, this confirms our earlier hypothesis as to the molecular basis of cholera blood group dependence, with an interesting twist: the shorter blood group H-determinant characteristic of blood group O individuals binds with similar binding affinity compared to the A-determinant, however, with different kinetics.